High-resolution imaging apparatus and method

Abstract

This invention relates to high-resolution imaging of samples using imaging mass spectrometry (IMS), and imaging of biological samples by imaging mass cytometry (IMC®) in which labeled atoms are detected by IMS. LA-ICP-MS (a form of IMS in which the sample is ablated by a laser, the ablated material is ionized in an inductively coupled plasma, and then the ions are detected by mass spectrometry) is used for the analysis of various materials, including mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological materials. However, conventional LA-ICP-MS systems and methods may not provide high resolution. [Solution] This specification describes methods and systems for high-resolution IMS and IMC (use of immersion lenses in an ablation laser and combined use with electron microscopy observation, and laser ionization after sample sputtering by ion scanning).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 686,341, filed on June 18, 2018, entitled "HIGH RESOLUTION IMAGING APPARATUS AND METHOD", and U.S. Provisional Patent Application No. 62 / 792,759, filed on January 15, 2019, entitled "HIGH RESOLUTION IMAGING APPARATUS AND METHOD", both of which are hereby incorporated by reference in their entirety.

[0002] The present invention relates to high - resolution imaging of samples using imaging mass spectrometry (IMS) and imaging of biological samples by imaging mass cytometry (IMC™).

Background Art

[0003] LA - ICP - MS (a form of IMS where the sample is ablated by a laser, the ablated material is ionized in an inductively coupled plasma, and the ions are detected by mass spectrometry) has been used for the analysis of various substances, such as mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological materials.

[0004] Regarding imaging at the cellular resolution, imaging of biological samples by IMC has been previously reported. In recent years, detailed imaging at the intracellular resolution has also been reported.

[0005] Despite these recent advancements, there is a need for further devices and techniques to increase the resolution of IMC to the sub - micrometer scale. Two main challenges in increasing the resolution of IMC to the sub - micrometer scale are as follows: 1) Limiting the sampling spot area to a size of about 200 nm or less, and 2) Ensure that the amount of analyte in the ablated material generates a sufficient signal-to-noise ratio. [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide subsequent improved apparatus and techniques that overcome these two challenges in order to provide high-resolution imaging of a sample. [Means for solving the problem]

[0007] Generally, the analytical instruments disclosed herein comprise two broadly characterized systems for performing imaging elemental mass spectrometry.

[0008] The first is a sampling and ionization system. This system includes a sample chamber, which is a component in which the sample is placed when it is subjected to analysis. The sample chamber comprises a stage that holds the sample (typically, the sample is placed on a sample carrier, e.g., a microscope slide, e.g., a tissue section, a monolayer of cells, or individual cells, e.g., a cell suspension dropped onto a microscope slide, where the slide is placed on the stage). The sampling and ionization system functions to remove material from the sample in the sample chamber, either as part of a process to remove material from the sample, or via a separate ionization system downstream of the sampling system, to be converted into ions (the removed material is referred to herein as sample material). Hard ionization techniques are used to generate elemental ions.

[0009] Next, the ionized material is analyzed by a second system, which is a detector system. The detector system can take different forms depending on the specific characteristics of the ionized sample material, such as a mass detector in an analyzer based on mass spectrometry.

[0010] This invention provides improvements to current IMS and IMC apparatus and methods by applying various apparatuses and techniques that take advantage of the benefits of analyzing tissue sections less than 100 nm thick. The use of such ultrathin sections increases opportunities for additional analytical techniques. The inventors have discovered that it is possible to use a lens (e.g., an immersion lens) that maintains a particularly high refractive index around the ablation spot, focusing the laser used for laser ablation to a particularly small ablation spot. The inventors have determined that when ultrathin sections are analyzed, such a lens can be used to focus the laser radiation without damaging areas of the sample outside the region targeted for ablation. This is because the high refractive index of the lens material ensures that the laser is focused through the lens to a narrow focal point leading to the ablative spot. Thus, this narrow focal point is smaller than possible without high refractive index materials, and since the size of the focal point determines the lateral resolution, this invention provides apparatuses and techniques that offer improved lateral resolution. Furthermore, the focal point of the lens is not simply two-dimensional; it is a three-dimensional volume of the focused radiation. Therefore, not only the diameter of the ablative spot but also its depth (i.e., the focal point of the radiation) is important. If the focal length is set so that the focal point of the radiation is within the substrate rather than on the surface, this can lead to unpredictable destruction and fragmentation of the sample. This problem arises particularly from the fact that, in order to use the immersion lens most effectively, the sample is best ablated in a way that guides the laser radiation through the sample carrier.

[0011] Similarly, the inventors have found that when thin sections are used, for example, it is possible to perform electron microscopy on a sample that will also be analyzed by IMS or IMC before analyzing the sample by IMC. Thus, high-resolution structural images can be obtained by electron microscopy, for example, transmission electron microscopy, and these high-resolution images can then be used to refine the resolution of image data obtained by IMS or IMC to a resolution that exceeds the resolution achievable by ablation using laser radiation (due to the much shorter wavelength of electrons compared to photons). Alternatively, or further, electron microscopy images can be associated with IMC images of the same region of the sample, for example, by correlating the localization of protein targets imaged by IMC with intracellular (e.g., nanoscale) structures identified by electron microscopy. As will be described in more detail below, electron microscopy imaging may be performed "offline" in a separate instrument, or the components of the electron microscope may be incorporated into the IMS or IMC instrument.

[0012] Furthermore, the inventors have developed another technique for high-resolution imaging based on a combination of charged particle (e.g., ions or electrons) impacts on a sample, combined with laser ionization of the sample material after sputtering the sample material from the sample or during sampling. Since the charged particles can be focused more tightly while achieving material removal from the sample, this method can also achieve higher resolution than conventional laser ablation-based techniques. Ion optics may be used to guide the charged particles toward the sample and / or to guide the ionized sample toward a mass spectrometer (e.g., TOF or magnetic sector detector). The ion optics may be similar to those used in electron microscopes or ion microscopes. The ion optics may include ion scanning optics (e.g., for scanning charged particles across the sample), ion focusing optics (e.g., for determining spot size or for focusing sample ions toward a detector), and ion accelerating optics (for determining the energy of charged particles impacting the sample or for accelerating ions toward a detector). An ion optical system may comprise one or more charged surfaces (e.g., plates) of appropriate shape, charge, and / or orientation.

[0013] Therefore, in the operation of one apparatus according to the present invention, a sample is taken into the apparatus, sampled, and an ionized material is generated using a laser system equipped with an optical system, in which laser radiation is guided onto the sample through an immersion lens (sampling may generate a vaporized / specific material, which is then ionized by the ionization system), and the ions of the sample material are passed through a detector system.

[0014] Therefore, in the operation of another apparatus according to the present invention, a sample is taken into the apparatus and imaged using an electron microscope (e.g., a transmission electron microscope), and then sampled and ionized material is produced using a sampling and ionization system (sampling may produce ions or uncharged vapor / specific material (or uncharged vapor / specific material may be formed by a charge that neutralizes any ions formed at sampling), in the latter case the uncharged vapor / specific material is subsequently ionized by a separate ionization system), and the ions of the sample material are passed through a detector system.

[0015] Finally, in the operation of the additional apparatus of the present invention, the sample is taken into the apparatus and sampled, and a material is generated using a sputtering-based sampling system (charged particle-based sampling system), and after sputtering the sample material from the sample, or at the time of sampling, the sample material is ionized by a laser post-ionization system, and the ions of the sample material are passed through a detector system.

[0016] The detector system of the present invention can detect many ions, most of which are ions of atoms that naturally constitute the sample. In some cases, for example, when analyzing a biological sample, the natural elemental composition of the sample may not provide suitable information. This is because typically, all proteins and nucleic acids are composed of the same major constituent atoms, and therefore it is possible to distinguish between regions containing proteins / nucleic acids and regions that do not contain such proteinaceous or nucleic acid material, but it is not possible to distinguish between a particular protein and all other proteins. However, certain properties of a sample can be determined by labeling the sample with atoms that are not present in the material being analyzed under normal conditions, or at least not present in substantial amounts (e.g., certain transition metal atoms such as rare earth metals; see the section on labeling below for details). Similar to IHC and FISH, detectable labels can be conjugated to specific targets on or in the sample (such as cell or tissue samples fixed on a slide), particularly by using SBPs such as antibodies, nucleic acids, or lectins that target molecules on or in the sample. To detect ionized labels, the detector system is used to detect ions from atoms that naturally exist in the sample. By associating the detected signals with known locations in the sample that produced these signals, it is possible to generate an image of the atoms present at each location, both in terms of the natural elemental composition and any labeled atoms. In embodiments where the natural elemental composition of the sample is depleted before detection, the image may consist only of labeled atoms. This technique allows for the simultaneous analysis of multiple labels (also known as multiplexing), which is a significant advantage when analyzing biological samples, and the apparatus and methods disclosed herein are accelerated by the application of laser scanning systems.

[0017] Therefore, the present invention relates to an apparatus for analyzing biological samples, Sample stage and Laser source and A focusing optical system equipped with an objective lens, comprising a focusing optical system adapted to guide a radiation beam from a laser source toward a position on a sample stage, The apparatus further comprises an immersion medium placed between the objective lens and the sample stage.

[0018] The present invention also, Staining a sample using a contrast agent for electron microscopy, This invention provides a method for preparing a biological sample for analysis, which includes labeling the sample with labeled atoms.

[0019] The present invention also, Imaging a sample using an electron microscope, The process involves guiding a radiation beam emitted from a laser source towards a specific location on the sample to generate an ablated plume of the sample material. Ionizing the ablated plume of the sample material, and This invention provides a method for analyzing biological samples, including the detection of sample ions from a sample material.

[0020] The present invention also, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The present invention provides an apparatus for analyzing a biological sample, comprising a first laser source and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage.

[0021] The present invention also provides a method for analyzing a biological sample, comprising passing a charged particle beam toward a position on the sample to sputter material from the sample, irradiating the sputtered sample material with laser radiation pulses to generate a plume of material containing sample ions, and detecting the sample ions by mass spectrometry.

[0022] The present invention also provides a method for analyzing a biological sample, comprising passing a charged particle beam toward a position on the sample to bring the sample to a sample ignition state, irradiating the sample with a laser radiation pulse to generate a plume of sample material containing sample ions from that position, and detecting the sample ions by mass spectrometry. [Brief explanation of the drawing]

[0023] [Figure 1] This is a schematic diagram of the optical system from the previous setup. [Figure 2] This is a schematic diagram of the optical system from a different, previous device configuration. [Figure 3] This is a schematic diagram of the optical system arrangement of an exemplary embodiment of the present invention. [Figure 4] This is a schematic diagram of an optical system arrangement in a further exemplary embodiment of the present invention. [Figure 5a] This is a schematic diagram of an optical system arrangement of another exemplary embodiment of the present invention, showing the path of a laser beam through a hemispherical solid immersion lens. [Figure 5b] This is a schematic diagram of an optical system arrangement in another exemplary embodiment of the present invention, showing the path of a laser beam through a Weierstrass solid immersion lens. [Figure 6] This is a schematic diagram of the optical system configuration of an apparatus for analyzing biological samples using laser post-ionization. [Figure 7] This is a schematic diagram of the optical system configuration of an apparatus for analyzing biological samples using laser pumping of the sample. [Figure 8] This is a schematic diagram of the optical system configuration of an apparatus for analyzing biological samples using laser pumping of the sample via a sample carrier. [Figure 9] This is a schematic diagram of the optical system configuration of an apparatus for analyzing biological samples using laser post-ionization of the sample via a sample carrier. [Figure 10] This is a schematic diagram of the optical system configuration of an apparatus for analyzing biological samples, comprising at least two laser sources: one for laser post-ionization and the other for laser pumping of the sample. [Figure 11] The image shows the detection of various osmium isotopes in a sample prepared according to an exemplary method of the present invention. [Figure 12] Figure 11 shows an image illustrating the detection of various osmium isotopes in a sample prepared according to the exemplary method of the present invention used. Figure 12 shows a region of 2100 × 10⁸⁷ micrometers with a process size of 250 nm. [Figure 13] This is a schematic diagram of a two-pulse sampling and ionization system. [Figure 14] This is a schematic diagram of a two-pulse sampling and ionization system in which the first and second pulses are focused onto the sample from the same side. [Figure 15] This is a schematic diagram of a two-pulse sampling and ionization system in which the sample is placed on a material that is at least semi-transparent to the first and second pulses. [Figure 16] This is a schematic diagram of a two-pulse sampling and ionization system in which the objective lens has an aperture that allows ions generated after ablation to pass through. [Figure 17] This is a schematic diagram of a 3-pulse sampling and ionization system. [Modes for carrying out the invention]

[0024] Therefore, various types of analytical instruments can be used in the implementation of this disclosure, many of which, for example, instruments equipped with immersion lenses, instruments equipped with electron microscopes (or their components), and instruments for performing secondary neutral mass spectrometry are described in detail below.

[0025] Mass detection-based analytical instrument 1. Sampling and ionization systems a. Laser ablation sampling and ionization system Laser ablation analyzers typically comprise three components. The first is a laser ablation sampling system for generating plumes of vaporized and particulate material from the sample for analysis. The sample must be ionized (and atomized) before atoms in the plume of ablated sample material (including any detectable labeled atoms described below) can be detected by the detector system, i.e., the mass spectrometer component (MS component; the third component). Thus, the instrument comprises a second component, an ionization system, which ionizes atoms to form elemental ions in order to enable detection by the MS component based on the mass / charge ratio (some ionization of the sample material may occur at the time of ablation, but space charge effects result in almost immediate neutralization of the charge). The laser ablation sampling system is connected to the ionization system by a transport conduit.

[0026] Laser ablation sampling system In summary, the components of a laser ablation sampling system include a laser source that emits a laser radiation beam directed onto a sample. The sample is placed on a stage within a chamber (sample chamber) in the laser ablation sampling system. Since the stage is typically a translational stage, the sample can move relative to the laser radiation beam, thereby allowing different locations on the sample to be sampled for analysis (for example, locations further apart from each other than those that can be ablated as a result of relative movement within the laser beam (in this specification, the term laser beam may be used without distinction from the term laser radiation) may be induced by the laser scanning system described herein). As will be described in more detail below, a gas flows through the sample chamber, and the gas flow carries away a plume of aerosolized material generated when the laser source ablates the sample, and an image of the sample is analyzed and constructed based on its elemental composition (including labeled atoms such as labeled atoms from elemental tags). As will be described in more detail below, in an alternative mode of operation, the laser system of the laser ablation sampling system can also be used to desorb material from the sample.

[0027] In particular, biological samples (cells, tissue sections, etc.) are often heterogeneous (although heterogeneous samples are known in other application areas of this disclosure, i.e., samples of non-biological nature). A heterogeneous sample is one that contains regions composed of different materials, and therefore, some regions of the sample may ablate at a lower threshold fluence than other regions at a given wavelength. Factors that affect the ablation threshold are the absorption coefficient of the material and the mechanical strength of the material. In biological tissues, the absorption coefficient has a dominant influence, as it can vary by several orders of magnitude depending on the laser radiation wavelength. For example, in biological samples, when using nanosecond laser pulses, regions containing proteinaceous material absorb relatively easily in the wavelength range of 200-230 nm, while regions mainly containing DNA absorb relatively easily in the wavelength range of 260-280 nm.

[0028] It is possible to perform laser ablation with a fluence close to the ablation threshold of the sample material. Ablation using this method often improves aerosol formation, which in turn can help improve the quality of the data after analysis. In many cases, a Gaussian beam is used to obtain the smallest crater and maximize the resolution of the resulting image. A cross-section of a Gaussian beam records an energy density distribution with a Gaussian distribution. In this case, the beam fluence changes with distance from the center. As a result, the diameter of the ablation spot size is determined by two parameters, namely (i) the Gaussian beam waist (1 / e 2 (ii) is a function of the ratio of the applied fluence to the threshold fluence.

[0029] Therefore, maintaining a constant ablation diameter is useful to ensure a consistent removal of a reproducible amount of material by each ablative laser pulse, and consequently to maximize the quality of imaging data. This, in turn, means adjusting the ratio between the energy supplied to the target by the laser pulse and the ablation threshold energy of the material being ablated. This requirement presents a problem when ablating heterogeneous samples where the threshold ablation energy varies across the sample, such as biological tissues where the ratio of DNA to protein material changes, or geological samples where it varies with the specific composition of minerals within a region of the sample. To address this, laser radiation of multiple wavelengths can be focused at the same ablation location on a single sample to more effectively ablate the sample based on the composition of the sample at that location.

[0030] Laser system for laser ablation sampling system A laser system can be configured to produce laser radiation of one or more wavelengths (i.e., two or more). Typically, the wavelength of the laser radiation described refers to the wavelength with the highest intensity ("peak" wavelength). If the system produces various wavelengths, they can be used for various purposes, for example, to target various materials in a sample (as used herein, to target means that the selected wavelength is well absorbed by the material).

[0031] When multiple wavelengths are used, at least two of the two or more wavelengths of the laser radiation may be distinct wavelengths. Therefore, if the first laser source emits radiation of a first wavelength distinct from radiation of a second wavelength, it means that the first laser source in pulses of the first wavelength either does not produce radiation of the second wavelength, or produces very low levels of radiation of the second wavelength, e.g., less than 10% of the intensity at the first wavelength, e.g., less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Typically, laser radiation of various wavelengths is produced by harmonic generation or other nonlinear frequency conversion processes, and then, when a particular wavelength is referred to herein, a person skilled in the art will understand that there will be some variation in the specified wavelength of the spectrum produced by the laser. For example, a reference to X nm encompasses lasers that produce spectra in the range of X ± 10 nm, such as X ± 5 nm, e.g., X ± 3 nm.

[0032] Focusing optical system and objective lens As a common configuration issue, optical components can be used to guide the laser radiation beam to a focused spot. Figure 1 is a schematic diagram of the optical system of a previous apparatus setup. Here, a laser source (e.g., a pulsed laser source with an optional pulse picker) 101 emits a laser radiation beam, which is guided through an energy control module 102 and then an optical system 103. The radiation beam is then guided towards the sample by a beam / irradiation composite optical system 104, through a focusing optical system and objective lens 105. The sample is on a sample stage 107 in a sample chamber 106. The sample stage 107 (e.g., a glass slide) may be mounted on a three-axis (i.e., x, y, z) translational stage 108 in the sample chamber 106. The setup in Figure 1 also includes a camera 111 for observing the sample using the same focusing optical system and objective lens 105. The irradiation source 109 emits visible light, which is then guided to the sample via the irradiation / inspection splitting optical system 110, the beam / irradiation combined optical system 104, and the focusing optical system 105.

[0033] Figure 2 shows an alternative arrangement to the previous apparatus configuration. Here, the sample 215 is positioned on the sample stage 207, opposite the objective lens 205, and the laser radiation ablates the sample 215 through the sample stage 207 or the sample chamber. In some cases, the sample chamber is held under vacuum or partial vacuum. Lines in the ray diagram are shown to represent possible paths for the laser beam 216 from the objective lens 205 through the glass slide 207.

[0034] As described above, one of the main challenges in achieving spatial resolutions of less than 200 nm, e.g., less than 150 nm, e.g., less than 100 nm, with conventional IMC and IMS is limiting the laser spot size to less than 200 nm (e.g., less than 150 nm or less than 100 nm). In systems with a numerical aperture (NA) greater than 0.7, the full width at half maximum of the laser spot size is

number

[0035] However, the numerical aperture of a lens is expressed as NA = n × sinθ, where n is the refractive index of the medium between the lens and the sample stages 107, 207, and θ is half the angle of reception of the objective lens. Therefore, since the refractive index of vacuum is 1.0 and the refractive index of air is approximately 1.0, in a typical IMC and IMS, the maximum theoretical numerical aperture of the objective lens (e.g., objective lenses 105, 205 in the previous instrument configuration shown in Figures 1 and 2) is limited to 1.0.

[0036] Immersion lens This invention overcomes the limitations of conventional IMC and IMS by utilizing an immersion medium. The immersion medium has a refractive index greater than 1.0 and is placed between the objective lens and the sample stage. Thus, the apparatus of this invention achieves a numerical aperture greater than 1.0, and therefore the laser spot size is less than 200 nm, less than 150 nm, or less than 100 nm. Accordingly, this invention provides an apparatus for imaging mass cytometry having a spatial resolution of 200 nm or more, 150 nm or more, or 100 nm or more.

[0037] Therefore, the present invention is a laser sampling system, Sample stage and Laser source and A focusing optical system equipped with an objective lens, comprising a focusing optical system adapted to guide radiation from a laser source toward a position on a sample stage, The laser sampling system further comprises an immersion medium placed between the objective lens and the sample stage.

[0038] Therefore, the present invention relates to an apparatus for analyzing biological samples, Sample stage and Laser source and A focusing optical system equipped with an objective lens, comprising a focusing optical system adapted to guide radiation from a laser source toward a position on a sample stage, The apparatus further comprises an immersion medium placed between the objective lens and the sample stage.

[0039] Typically, the apparatus also includes a detector based on mass spectrometry.

[0040] Therefore, during operation, the sample stage holds the sample, and typically the sample rests on a sample carrier, the same stage holding the sample carrier. Laser radiation is then guided to the sample through the instrument's optics, via the objective lens and immersion medium, where the radiation ablates the material from the sample.

[0041] To achieve optimal focusing conditions for the laser, the immersion medium of the present invention has a refractive index greater than 1.00, for example, 1.33 or higher, 1.50 or higher, 2.00 or higher, or 2.50 or higher.

[0042] Furthermore, as will be further described herein, in order to reconstruct a single layer image of the thickness of (or less than) living cells, or to read thicker specimens layer by layer and generate a 3D image, the specimen preferably has a thickness of 100 micrometers or less, for example, 10 micrometers or less, 5 micrometers or less, 2 micrometers or less, or 100 nm or less, or 50 nm or less, or 30 nm or less. In some embodiments described in further detail herein, the combination of the objective lens and the immersion medium is referred to as the immersion lens.

[0043] In some embodiments, the above apparatus or sampling system, which includes an immersion medium, is equipped with a medium- or high-NA objective lens, such as one with an NA of 0.7 or higher, 0.8 or higher, or 0.9 or higher. Such a system typically also includes a mass detector, such as a TOF mass spectrometry detector.

[0044] immersion medium In some embodiments of the present invention, the immersion medium is a liquid immersion medium, and Figure 3 is a schematic diagram of an optical system configuration in an exemplary embodiment of the present invention. This includes elements common to the configurations shown in Figures 1 and 2. A focusing optical system with an objective lens 305 directs a radiation beam 316 from a laser source (source not shown) to a position on a sample stage 307, and the liquid immersion medium 312 is positioned between the objective lens 305 and the sample stage 307. Here, a biological sample 315 is positioned on the sample stage 307, opposite the liquid immersion medium 312. The ray diagram shows how the liquid immersion medium 312 provides tighter focusing conditions than the conventional configuration shown in Figure 2. Suitable liquids for immersion media include water with a refractive index n of 1.333, oils such as glycerin (n=1.4695), paraffin oil (n=1.480), cedarwood oil (n=1.515), and synthetic oil (n=1.515), or anisole (n=1.5178), bromonaphthalene (n=1.6585), and methylene iodide (n=1.740). Commercially available immersion oils are also available, some of which have properties particularly advantageous for the applications of the present invention. For example, low-fluorescence or non-fluorescence immersion oils such as Olympus Low Auto Fescence Immersion Oil and Nikon are particularly useful for use with short-wavelength lasers because they provide an improved signal-to-noise ratio compared to general-purpose immersion oils. Other oils, such as Cargille Labs type NVH and OVH, which are used when the distance between the objective lens 305 and the sample stage 307 is large, have very high viscosity.

[0045] Commercially available oil immersion objective lenses can achieve a maximum numerical aperture of approximately 1.49 (close to the refractive index of the immersion oil), allowing a 515nm laser beam to be focused to an FWHM focal diameter of approximately 160nm.

[0046] When using an immersion medium, the sample must be positioned on the opposite side of the sample carrier from the liquid medium (as shown in Figure 3) so that the carrier gas can collect the ablated material. Therefore, ablation techniques through a sample carrier must be applied here. This has the additional advantage of a relatively small achievable working distance for the ablated material collection hardware and does not require bending the transport conduit between the sample chamber and the detector. This also reduces transient time and therefore increases the achievable ablation rate within a spot per second.

[0047] Immersion lenses (e.g., objective-in-water lenses or objective-in-oil lenses) are commercially available from Olympus, ThorLabs, and Leica.

[0048] solid immersion medium In some embodiments of the present invention, the present invention provides an apparatus in which the immersion medium is a solid immersion medium. Figure 4 is a schematic diagram of an optical system arrangement in a further exemplary embodiment of the present invention, in which the solid immersion 413 is positioned between the objective lens 405 and the sample stage 407. Other components in Figure 4 correspond to components in Figure 3.

[0049] Similar to liquid immersion media, the refractive index n of solid immersion lenses is greater than that of air. Suitable materials for solid immersion lenses are glasses such as S-LAH79™ glass, which has a refractive index of 2.0 when operating at a wavelength of approximately 520 nm. Suitable alternative materials for the solid immersion media of this invention are diamond or fused silica. At 266 nm, diamond is well-suited for optical applications because it has a refractive index of >2.5 in UV, providing an opportunity to focus 266 nm light to a spot size of 100 nm or less. Fused silica may be even more attractive from a cost standpoint. Fused silica is practical as both a sample substrate and a solid immersion lens material. However, the refractive index of fused silica in UV is only about 1.5, so the spot size is proportionally larger.

[0050] There are two standard optical schemes for solid immersion media: hemispherical immersion lenses and Weierstrass immersion lenses.

[0051] Hemispherical solid immersion lens: Figure 5a) shows the shape of a hemispherical solid immersion lens. A hemispherical solid immersion lens can increase the numerical aperture of the optical system by the refractive index n of the lens material.

[0052] Weierstrass solid immersion lenses: Figure 5b) shows the shape of a Weierstrass solid immersion lens. A Weierstrass solid immersion lens is a top-cut sphere with a height of (1 + 1 / n)r from the glass slide 507, where r is the radius of the spherical surface of the lens. The Weierstrass lens provides an optical system with a numerical aperture of n 2 Because it can be increased by this much, the numerical aperture of the optical system can be increased even further than with a hemispherical lens.

[0053] Accordingly, the present invention provides an apparatus in which the solid immersion medium is a hemispherical solid immersion lens or a Weierstrass solid immersion lens. As shown in Figures 3 and 4, when biological samples 315, 415 are placed on sample stages 307, 407, the biological samples may be placed on the side of the sample stage opposite to the solid immersion material. The stage on which the sample is placed can be made from a material with the same refractive index as the solid immersion lens, and the solid immersion lens can be made thinner by an amount equal to the thickness of the substrate in order to maintain the focal position.

[0054] Combination of immersion lens and biological sample The present invention, comprising an immersion medium positioned between the objective lens and the sample stage, offers further advantages when used to analyze biological samples prepared according to other methods of the present invention described herein (page 112). In particular, the present invention offers further advantages when the biological sample has a thickness of 100 micrometers or less, for example, 10 micrometers or less, or 100 nm or less, or 50 nm or less, or 30 nm or less.

[0055] For example, using the apparatus of the present invention, which includes an immersion medium between the objective lens and the sample stage, biological samples with a thickness of 100 nm or less, for example, 50 nm or less, or 30 nm or less, can be analyzed. Since the apparatus of the present invention provides an apparatus for imaging mass cytometry having a spatial resolution of 200 nm or more (preferably 100 nm or less), the laser spot size is 200 nm or less (preferably 100 nm or less), and therefore the ablation depth is typically 200 nm or less (preferably 100 nm or less). Thus, when analyzing a biological sample with a thickness of 200 nm or less using the present invention, ablation will occur throughout the entire sample (or, if the laser spot size is 100 nm or less, preferably 100 nm or less). In this way, the present invention provides the possibility of reconstructing a single-layer image of the thickness of biological cells by utilizing the apparatus with a series of sections of biological cells.

[0056] Alternatively, the apparatus of the present invention, which includes an immersion medium between the objective lens and the sample stage, can be used to analyze biological samples of 100 micrometers or less, for example, 10 micrometers or less, 5 micrometers or less, or 2 micrometers or less. As described above, the sharp focusing of the laser beam by the immersion medium results in a very short depth of field. Therefore, the present invention provides an opportunity to read relatively thick specimens layer by layer and generate 3D images. Those skilled in the art will understand that this analysis of relatively thick specimens becomes easier the more homogeneous the specimen is. In non-homogeneous tissue specimens, distortion of the laser light as the laser passes through the tissue can result in a larger spot size at the focus than expected.

[0057] Therefore, the present invention provides an imaging mass cytometer or imaging mass spectrometer equipped with a biological sample, wherein the biological sample has a thickness of less than 100 nm, for example, less than 50 nm or less than 30 nm. The present invention also provides an imaging mass cytometer or imaging mass spectrometer equipped with a biological sample, wherein the biological sample has a thickness of less than 100 nm, for example, less than 50 nm or less than 30 nm, and the imaging mass cytometer or imaging mass spectrometer is equipped with a solid immersion lens. The present invention also provides an imaging mass cytometer or imaging mass spectrometer equipped with a biological sample, wherein the biological sample has a thickness of less than 100 nm, for example, less than 50 nm or less than 30 nm, and the imaging mass cytometer or imaging mass spectrometer is equipped with a solid immersion lens.

[0058] Substitutes for immersion lenses combined with thin biological samples: As described above, in conventional IMC and IMS, short-wavelength lasers such as 213 nm deep ultraviolet lasers, or focusing optical systems with n NA greater than 0.6, for example greater than 0.7, for example greater than 0.8, such as high NA (greater than 0.9), are used to reduce the size of the laser spot and, consequently, improve the resolution to 100 nm.

[0059] High NA objective lenses can be refractive and / or reflective optical components. For example, aspherical mirrors can be used to enable reflective objective lenses to have a numerical aperture of up to 0.99 (Inagawa et al., Scientific Reports 5, 12833 (2015)). Reflective-refracting mirror systems can also be used. Furthermore, short-wavelength lasers (below 300 nm) can be provided by using sixth-harmonic generation from extreme UV light such as a 266 nm laser, or 213 nm, 193 nm solid-state lasers, or ND:Yag, ArF, F2 lasers, or 13.5 nm and 7 nm wavelengths (CO2 laser + Sn plasma).

[0060] Therefore, the present invention relates to an apparatus for analyzing biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a biological sample, Laser source and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage, The present invention provides an apparatus in which the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9, for example, at least 0.9.

[0061] Therefore, the present invention relates to an apparatus for analyzing biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a biological sample, Laser source and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage, The present invention provides a device in which the laser source has a wavelength of 300 nm or less.

[0062] Therefore, the present invention relates to an apparatus for analyzing biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a biological sample, A laser source having a wavelength of 300 nm or less, A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage, The present invention provides an apparatus in which the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9, for example, at least 0.9.

[0063] As further described herein, the ablated material may be ionized or reionized near the surface of the sample by laser ionization of the ablation plume that has expanded beyond a significant charge neutralization point (after ionization / reionization). In such systems and methods, a gas-fluid element may not be necessary because it is not necessary to deliver the plume to an ICP for ionization. Instead, an ion optics system positioned near the sample may directly guide the ionized labeled atoms to a mass spectrometer (e.g., a TOF or magnetic sector mass spectrometer). As described herein, in certain cases, a small ablation spot size may reduce the neutralization and space charge effects of the ions generated during ablation, facilitating ionization and guiding the ions to the mass spectrometer. In such cases, a second pulse for reionization is not required. The sample stage and the optics immediately surrounding it (e.g., high NA lenses and / or immersion media) may be operated in a vacuum or at low pressure (e.g., to reduce collision-induced charge), as further described herein.

[0064] As those skilled in the art will understand, the apparatus may include a focusing optical system having an objective lens with an numerical aperture of at least 0.9 and / or a laser source having a wavelength of 300 nm or less. Furthermore, high numerical apertures or short wavelengths may be used as an alternative to or in addition to immersion lenses as described herein, while the other components of the system remain the same as in the case of an immersion lens system.

[0065] Scanning system In certain embodiments, ablation of a sample may be performed by a scanning system. A radiation source, such as a laser beam or a charged particle beam (such as an ion beam or electron beam), may be scanned over an entire portion of the sample to generate a single transient (e.g., a single instance of the ablated material). The transient may be an ablation plume delivered to an ICP for atomization and ionization before detection by MS. Alternatively, the transient may be ionized by laser radiation on or near the surface of the sample, as described herein. Scanning with an ion beam or electron beam (e.g., using an ion optics system) may allow for high-resolution ablation of a small region of interest (such as a single organelle) of the sample. For example, the region of interest may be 100,000 nm. 2 Less than 50,000 nm 2 Less than or 20,000 nm 2 Less than 10,000 nm 2 It may have an area less than [a certain value]. Alternatively, the region of interest may be larger, such as a single cell or a group of cells.

[0066] In certain embodiments, a laser scanning system guides laser radiation onto the sample to be ablated. Because the laser scanner can re-guide the laser focal point on the sample much faster than moving the sample stage relative to a stationary laser beam (due to the much smaller or nonexistent inertia of the operating components of the scanning system), ablation of distinct spots on the sample can be performed even more rapidly. This faster speed allows for the ablation of very large areas and recording them as single pixels, or the speed of laser spot movement can be simply converted into, for example, an increase in pixel acquisition speed, or a combination of both. In addition, rapid changes in the position of the spot to which the laser radiation pulse can be guided can enable ablation of arbitrary patterns; for example, bursts of laser radiation pulses / shots in rapid succession guided to a position on the sample by the laser scanner system can ablate entire cells of heterogeneous shape, then ionize them, detect them as a single cloud of material, and thus enable single-cell analysis (see the "Sample Chamber of Laser Ablation Sampling Systems" section on page 33 onwards). Similar rapid burst techniques can be applied to methods that use desorption to remove sample material from a sample carrier, i.e., cell LIFTing (Laser-Induced Forward Transfer).

[0067] Therefore, the present invention is (i) A sampling and ionization system for removing material from a sample and ionizing the material to form elemental ions, comprising a laser scanning system and a sample stage. (ii) To provide an apparatus for analyzing samples such as biological samples, which is equipped with a detector for receiving elemental ions from the sampling and ionization system described above and for detecting the elemental ions described above.

[0068] Similarly, this combination of features can be combined with any of the other embodiments described herein, as will be well understood by those skilled in the art. The use of a scanning system to increase the acquisition rate offers more advantages than other strategies for increasing the rate at which a sample is imaged. For example, using a well-fitted apparatus, a 100 μm × 100 μm area can be ablated with a single laser pulse. However, such ablation presents many problems. Ablating a large area of ​​a sample at once with a single laser pulse results in the ablated material being broken down into large clumps that fly first at near-sonic speeds, rather than small particles, the material not being rapidly transported away from the sample in the carrier gas flow (as will be described in more detail below), the large clumps taking longer to be taken up than small clumps (extending the washout time in the sample chamber), failing to be taken up, simply flying away from the sample, or randomly scattering onto another part of the sample. When large clumps of material scatter from the sample, information about the clumps of material in the form of detectable atoms, such as labeled atoms, is lost. When a clump of material lands on another part of the sample, information is lost from the ablated region, and furthermore, any detectable atoms present within the clump of material may interfere with the signal that would be obtained from the other part of the sample. Differences in biomaterials within the ablated spot (e.g., cartilage versus muscle) can also affect how the product is broken down, so even with a larger ablation spot size, sample fractionation may worsen, and some types of material may be incorporated into the gas flow to a lower degree than others. In addition, as described herein, many applications prefer small spot sizes on the order of micrometers rather than hundreds of micrometers, and switching between laser spot sizes that differ by several orders of magnitude (e.g., 100 μm versus 1 μm) also presents technical challenges.For example, a laser capable of ablation with a spot size of 1 μm may not have enough energy in a single laser pulse to ablate a region with a spot size of 100 μm. Therefore, a sophisticated optical system is required to facilitate the transition between 1 μm and 100 μm without significantly losing laser beam energy or the sharpness of the ablation spot.

[0069] Therefore, 100 μm 2 Instead of ablating a single spot, the entire region can be ablated by rasterizing it using a 100x100 (i.e., 10,000) spot with a diameter of 1 μm. The relatively small spot size of the ablation is, naturally, not significantly affected by the above problems. Inevitably, the particles themselves generated by the relatively small ablation spot are much smaller in size. Furthermore, smaller spots result in shorter washout times from the sample chamber for the relatively small particles resulting from the ablation, and they become clearer. If it is desired that each of the relatively small spots be resolved separately, this results in the ability to acquire data more quickly because the transients from each ablative laser pulse do not overlap (or overlap to an acceptable degree, as described below) when detected by the detector.

[0070] However, moving the sample stage along the row in 1 μm increments, and then moving it down the row, is relatively slow due to inertia, as mentioned above. Therefore, by not moving the sample stage, or moving the sample stage relatively infrequently or at a constant speed, and rasterizing the entire area using a laser scanner system, the speed at which the sample can be ablated is not limited, even if the speed of the sample stage is relatively slow.

[0071] Therefore, in order to enable rapid scanning, the laser scanning system must be able to quickly switch the position on the sample where the laser radiation is directed. The time it takes to switch the ablation position of the laser radiation is called the response time of the laser scanning system. Thus, in some embodiments of the present invention, the response time of the laser sampling system is faster than 1 ms, faster than 500 μs, faster than 250 μs, faster than 100 μs, faster than 50 μs, faster than 10 μs, faster than 5 μs, faster than 1 μs, faster than 500 ns, faster than 250 ns, faster than 100 ns, faster than 50 ns, faster than 10 ns, or about 1 ns.

[0072] A laser scanning system can guide a laser beam in at least one direction relative to the sample stage on which the sample is placed during ablation. In some cases, the laser scanning system can guide the laser radiation in two directions relative to the sample stage. For example, the sample stage may be used to move the sample stepwise in the X-axis direction, and the laser may be swept across the sample in the Y-axis direction (see Figures 7-9 for diagrams of relative movement). When using a spot size of 1 μm, the movement in the X-axis direction may be in 1 μm increments. Using the laser scanning system at a given position on the X-axis, the laser can be guided to a series of positions 1 μm apart in the Y-axis direction. Since the speed at which the laser scanning system can guide the laser radiation to different positions on the Y-axis is much faster than the speed at which the stage can move stepwise in the X-axis direction, a significant increase in ablation speed is achieved in this simple example of scanner operation.

[0073] In some cases, the laser scanning system directs the laser beam in both the X and Y axes. Therefore, in this case, a more sophisticated ablation pattern can be generated. For example, if the laser scanning system can direct laser radiation in both the X and Y axes, the sample stage can move at a constant speed in the X-axis direction (this eliminates inefficiencies associated with the inertia of the sample stage during movement across each row, other than acceleration / deceleration at the start / end of each row), while the laser scanning system directs the laser radiation pulses up and down the column on the sample, compensating for the movement of the sample stage. To achieve this movement, a triangular wave control signal can be applied to the scanner in the X direction and a sawtooth wave signal in the Y direction. Alternatively, as those skilled in the art will understand, depending on the processing algorithm used, it may be desirable to apply a sawtooth drive signal to the scanner in the Y direction. As an additional alternative, one of the scanner components may be slightly pre-rotated to pre-compensate for the inclined scanning pattern. In some embodiments, the controller of the laser scanning system directs the beam to the laser scanner system in a figure-eight pattern as the sample stage moves.

[0074] Therefore, if the laser used in the laser sampling system has a sufficiently high repetition rate (as described below), the (re)induction of laser radiation onto different positions on the sample is significantly accelerated, and it becomes possible to ablate a large area of the sample much faster. For example, if only less than 5 pulses per second can be directed to different positions on the sample, it would take more than 2 days to test a 1 mm×1 mm area using ablation with a 1 μm spot size. At a speed of 200 Hz, this is about 80 minutes, and the analysis time is further shortened as the pulse frequency increases further. However, the sample is often much larger. An average microscope slide on which tissue sections can be placed can be 25×75 mm. This would take about 110 days to ablate at a speed of 200 Hz. However, when using a laser scanning system, for example, moving the sample stage at a constant speed (1 mm / s) along the X-axis and moving the laser beam back and forth in the Y-axis direction by the laser scanning system, the time can be dramatically shortened. The laser scanning system can scan the position of the laser focus at a speed that matches the speed of the stage movement, in this case 500 Hz. This generates a 1 μm spacing between adjacent lines in the raster pattern at this speed. Then, depending on the maximum laser repetition rate, the degree of deflection of the laser radiation by the laser scanning system is selected to match. Here, a laser repetition rate of 100 kHz is required to generate a peak-to-peak amplitude of 100 microns. This allows the device to process 0.1 mm 2 / s compared to a maximum of 0.0004 mm 2 / s of current devices. Compared to the 110-day figure above, the laser scanning system described in this paragraph takes only about 5 hours to process the slide.

[0075] Another application is the shaping of arbitrary ablation regions. Using high repetition rate lasers, a nanosecond laser can deliver bursts of closely spaced laser pulses simultaneously with the delivery of a single pulse. By rapidly adjusting the X and Y positions of the ablation spots during the bursts of laser pulses, ablation craters of any shape and size (up to the diffraction limit of light) can be generated. For example, the n and n+1 positions in the burst may be at a distance equal to 10 times the diameter of the laser spot (based on the centers of the ablation spots of the nth and (n+1)th spots), e.g., less than 8 times, less than 5 times, less than 2.5 times, less than 2 times, less than 1.5 times, about 1 time, or less than 1 time of the diameter of the spot size. Specific methods for using this technique are described in the following Methods section on page 32.

[0076] Therefore, in some embodiments, the laser scanning system includes a positioner for imparting a first relative movement of the laser beam emitted by the laser with respect to a sample stage (e.g., the Y-axis with respect to the surface of the sample).

[0077] In some embodiments, the positioner of the laser scanning system can impart a second relative movement of the laser beam relative to the sample stage, where the first and second relative movements are not parallel but, for example, orthogonal (e.g., the first movement direction is in the Y-axis relative to the sample surface and the second movement direction is in the X-axis relative to the sample surface).

[0078] In some embodiments, the laser scanning system further comprises a second positioner capable of imparting a second relative movement of the laser beam with respect to a sample stage, wherein the first and second relative movements are not parallel, but rather orthogonal (for example, the first direction of movement is in the Y-axis relative to the surface of the sample, and the second direction of movement is in the X-axis relative to the surface of the sample).

[0079] Any component capable of rapidly guiding laser radiation to various positions on a sample can be used as a positioner within a laser scanning system. Various types of positioners, described below, are commercially available, each with its own advantages and limitations, and can be selected by those skilled in the art to suit the specific application in which the device will be used. In some embodiments of the present invention, multiple positioners described below can be combined within a single laser scanning system, as described below. Positioners can generally be classified into those that introduce relative movement to the laser beam by relying on movable parts (examples include galvanometer mirrors, piezoelectric mirrors, MEMS mirrors, polygon scanners, etc.) and those that do not (examples include such acousto-optical and electro-optical devices). The types of positioners listed above function to deflect the laser radiation beam in a controllable manner to various angles, resulting in translation of the ablation spot. A laser scanning system may comprise a single positioner or a positioner and a second positioner. The descriptions of "positioner" and "second positioner" in a laser scanning system do not define the order in which the laser radiation pulses collide with the positioners on the path from the laser source to the sample.

[0080] A galvanometer motor mounted on a shaft with mirrors attached can be used to deflect laser radiation to various positions on a sample. Movement can be achieved using a fixed magnet and a movable coil, or a fixed coil and a movable magnet. The arrangement of a fixed coil and a movable magnet results in even faster response times. Typically, sensors that detect the position of the shaft and mirrors are present within the motor, thereby providing feedback to the motor's controller. One galvanometer mirror can guide the laser beam within one axis; therefore, using a pair of galvanometer mirrors, this technique can be used to guide the beam in both the X and Y axes.

[0081] One advantage of galvanometer mirrors is that they allow for large deflection angles (e.g., much larger than those of solid-state deflectors), which reduces the frequency of sample stage movement. However, since the moving parts of the motor and mirror have mass and are affected by inertia, the acceleration time of the parts must be matched to the sampling method. Typically, non-resonant galvanometer mirrors are used. As those skilled in the art will understand, resonant galvanometer mirrors can be used, but devices that use only resonant parts, such as positioners in laser scanning systems, cannot perform arbitrary (also known as random access) scanning patterns. Because galvanometer mirror deflectors are based on mirrors, they can degrade the quality of the laser radiation beam and increase the ablation spot size, and therefore, as those skilled in the art will again understand, they are most applicable in situations where such effects on the beam are acceptable.

[0082] In devices based on galvanometer mirrors, positioning errors can easily occur due to sensor noise or tracking errors. Therefore, in some embodiments, each mirror is associated with a position sensor that feeds the mirror's position back to the galvanometer to refine the mirror's position. In some cases, the position information is relayed to another component, such as an AOD or EOD, in series with the galvanometer mirror, thereby correcting mirror positioning errors.

[0083] Galvano mirror systems and components are commercially available from various manufacturers, including Thorlabs (NJ, USA), Laser2000 (UK), ScanLab (Germany), and Cambridge Technology (MA, USA).

[0084] In embodiments comprising only a galvanometer-based positioner, the speeds at which an ablative laser pulse can be guided to the sample may be 200 Hz to 1 MHz, 200 Hz to 100 kHz, 200 Hz to 50 kHz, 200 Hz to 10 kHz, 1 kHz to 1 MHz, 5 kHz to 1 MHz, 10 kHz to 1 MHz, 50 kHz to 1 MHz, 100 kHz to 1 MHz, 1 kHz to 100 kHz, or 10 kHz to 100 kHz.

[0085] Therefore, in some embodiments of the present invention, the laser scanner system comprises one or more positioners, which are galvano mirrors such as a galvano mirror array.

[0086] Figure 1 is a schematic diagram of the optical system of a previous apparatus configuration. Here, a laser source (e.g., a pulsed laser source with an optionally incorporated pulse picker) 101 emits a laser radiation beam, which is guided through an energy control module 102 and then an optical system 103. The optical system 103 may include a beam shaping optical system. The radiation beam is then guided toward the sample by a beam / irradiation combined optical system 104, via a focusing optical system and an objective lens 105. The sample may be on a support such as a glass side 107 positioned on a three-axis (i.e., x, y, z) translational stage 108 in a sample chamber 106. The configuration in Figure 1 also includes a camera 111 for observing the sample using the same focusing optical system and objective lens 105. An irradiation source 109 emits visible light, which is guided toward the sample by an irradiation / inspection splitting optical system 110, via the beam / irradiation combined optical system 104 and the focusing optical system 105.

[0087] In more advanced configurations, the optical system (e.g., optical system 103) may further include a positioner (e.g., a movable mirror or scanner as described herein) to enable scanning of the laser across the sample. For example, a positioner such as a galvanometer mirror, piezoelectric mirror, MEMS mirror, or polygon scanner deflects the laser radiation beam before it is shaped by the beam shaping optical system of optical system 203. For example, a single mirror in a galvanometer-based apparatus allows scanning of the beam in one direction, e.g., along the Y-axis relative to the sample. The deflection introduced by the positioner is transmitted throughout the optical system, resulting in ablation at various locations on the sample depending on the mirror's position. The positioner may be coupled (e.g., by a controller) with a position on the sample stage to determine a specific location on the sample ablated by the laser radiation beam. The controller may also be connected to a laser source to coordinate the generation of laser pulses (resulting in pulses being generated by the laser source when the positioner is at a defined position, rather than while the positioner is moving between positions).

[0088] However, instead of a single mirror positioner, a pair of mirror positioners may be used to induce deflection in the laser radiation beam. As described elsewhere in this specification, the pair of mirrors can be arranged to provide scanning in two orthogonal directions (X and Y), thereby compensating for the movement of the sample on the sample stage.

[0089] Similarly, a piezoelectric actuator on a shaft to which a mirror is mounted can be used as a positioner to deflect laser radiation to various positions on a sample. In this case as well, as a mirror positioner based on the movement of a mass-bearing component, inertia is inherent, and therefore there is a time overhead inherent in the movement of the mirror by this component. Thus, it will be understood by those skilled in the art that this positioner is applied to specific embodiments where nanosecond response time is not essential for the laser scanning system. Similarly, because piezoelectric mirror positioners are based on mirrors, they can degrade the quality of the laser radiation beam and increase the ablation spot size, and therefore it will again be understood by those skilled in the art that they are most applicable in situations where such effects on the beam are acceptable.

[0090] In piezoelectric mirrors based on a tilt-tip mirror configuration, the guidance of laser radiation to a sample in the X and Y axes is provided by a single component.

[0091] Piezoelectric mirrors are commercially available from suppliers such as Physik Instrumente (Germany).

[0092] Therefore, in some embodiments of the present invention, the laser scanner system comprises piezoelectric mirrors such as a piezoelectric mirror array or a tilt-tip mirror.

[0093] In embodiments comprising only a piezoelectric mirror-based positioner, such as a piezoelectric mirror array or a tilt-tip mirror, the speeds at which an ablative laser pulse can be guided onto the sample may be 200 Hz to 1 MHz, 200 Hz to 100 kHz, 200 Hz to 50 kHz, 200 Hz to 10 kHz, 1 kHz to 1 MHz, 5 kHz to 1 MHz, 10 kHz to 1 MHz, 50 kHz to 1 MHz, 100 kHz to 1 MHz, 1 kHz to 100 kHz, or 10 kHz to 100 kHz. A third type of positioner that relies on the physical transport of a surface guiding the laser radiation onto the sample is a MEMS (micro-electromechanical system) mirror. Micromirrors within this component can be actuated by electrostatic, electromechanical, and piezoelectric effects. Many advantages of this type of component stem from their small size, e.g., light weight, ease of positioning within the device, and low power consumption. However, since the deflection of the laser radiation is ultimately based on the movement of components within the component, the components are subject to inertia. To reiterate, because MEMS mirror positioners are based on mirrors, they degrade the quality of the laser radiation beam and increase the ablation spot size. Therefore, a person skilled in the art will understand that such scanner components are applicable only in situations where such effects on the laser radiation are acceptable.

[0094] MEMS mirrors are commercially available from suppliers such as Mirrorcle Technologies (CA, USA), Hamamatsu (Japan), and Preciseley Microtechnology Corporation (Canada).

[0095] Therefore, in some embodiments of the present invention, the laser scanner system includes a MEMS mirror.

[0096] In embodiments comprising only a positioner based on a MEMS mirror, the speeds at which an ablative laser pulse can be guided onto the sample may be 200 Hz to 1 MHz, 200 Hz to 100 kHz, 200 Hz to 50 kHz, 200 Hz to 10 kHz, 1 kHz to 1 MHz, 5 kHz to 1 MHz, 10 kHz to 1 MHz, 50 kHz to 1 MHz, 100 kHz to 1 MHz, 1 kHz to 100 kHz, or 10 kHz to 100 kHz. Another type of positioner that relies on the physical transport of a surface guiding the laser radiation onto the sample is a polygon scanner. Here, a reflective polygon mirror or polyhedron rotates on a mechanical axis, and each time a flat facet of the polygon crosses the incident beam, an angularly deflected scanning beam is generated. A polygon scanner is a one-dimensional scanner and can guide the laser beam along a scanning line (thus requiring a secondary positioner to introduce a second relative movement of the laser beam relative to the sample, or requiring the sample to be moved on a sample stage). For example, in contrast to the reciprocating motion of a scanner based on a galvanometer, at the end of one line of raster scanning, the beam is returned to the starting position of the scanning line. The polygon can be regular or irregular, depending on the application. The spot size depends on the size and flatness of the facets, and the scan line length / scan angle depends on the number of facets. Very high rotational speeds can be achieved, resulting in high scanning speeds. However, this type of positioner has drawbacks in that it suffers from reduced positioning / feedback accuracy due to manufacturing tolerances of the facets and axial wobble, as well as potential wavefront distortion from the mirror surface. Therefore, a person skilled in the art will understand that in this case too, such scanner components are applicable to situations where such effects on laser radiation are acceptable.

[0097] Polygon scanners are commercially available from companies such as Precision Laser Scanning (AZ, USA), II-VI (PA, USA), and Nidec Copal Electronics Corp (Japan).

[0098] In embodiments comprising only a polygon scanner-based positioner, the speeds at which an ablative laser pulse can be guided to the sample may be 200Hz-10MHz, 200Hz-1MHz, 200Hz-100kHz, 200Hz-50kHz, 200Hz-10kHz, 1kHz-10MHz, 5kHz-10MHz, 10kHz-10MHz, 50kHz-10MHz, 100kHz-10MHz, 1kHz-1MHz, 10kHz-1MHz, or 100kHz-1MHz. Unlike the above types of laser scanner system components, EODs are solid components, i.e., they do not have moving parts. Therefore, they have a very fast response time on the order of 1 ns because they are not subject to mechanical inertia when deflecting laser radiation. Also, they are not affected by wear like mechanical parts. EODs are formed from an optically transparent material (e.g., a crystal) which has a refractive index that changes in response to an electric field applied across it, and this refractive index is controlled by applying a voltage to the medium. The refraction of laser radiation is caused by the introduction of a phase lag across the beam's cross-section. When the refractive index changes linearly with respect to the electric field, this effect is called the Pockels effect. When it changes quadratically with respect to the electric field strength, it is called the Kerr effect. The Kerr effect is usually much weaker than the Pockels effect. Two typical configurations are EODs based on refraction at the interface of an optical prism, and EODs based on refraction due to a refractive index gradient perpendicular to the direction of laser radiation propagation. To position the electric field across the EOD, electrodes are coupled to opposing sides of an optically transparent material that acts as a medium. When one pair of opposing electrodes are coupled, a one-dimensional scanning EOD is produced. When a second pair of electrodes is coupled orthogonally to the first pair of electrodes, a two-dimensional (X,Y) scanner is produced.

[0099] For example, although the deflection angle of an EOD is smaller than that of a galvanometer mirror, the angle can be increased when necessary for a given device setting by arranging multiple EODs sequentially. Exemplary materials for the refractive medium in an EOD include potassium tantalate niobate KTN(KTa x Nb 1-x O3), LiTaO3、 LiNbO3, BaTiO3, SrTiO3, SBN(Sr 1-x Ba x This includes Nb2O6, and KTiOPO4, which exhibits a relatively large deflection angle at the same electric field strength as KTN.

[0100] The angular accuracy of EOD is high and depends mainly on the precision of the driver connected to the electrode. Furthermore, as mentioned above, the response time of EOD is very fast, faster than that of AOD, which is described below (due to the fact that the (changing) electric field in the crystal is established at the speed of light in the material, not the speed of sound in the material; see the explanation in Roemer and Bechtold, 2014, Physics Procedia 56:29-39).

[0101] Accordingly, in some embodiments of the present invention, the laser scanner system includes an EOD. In some embodiments, the EOD has two sets of electrodes connected perpendicular to the refractive medium.

[0102] In embodiments comprising an EOD-based positioner, the speeds at which an ablative laser pulse can be guided to the sample may be 200 Hz to 100 MHz, 200 Hz to 10 MHz, 200 Hz to 1 MHz, 200 Hz to 100 kHz, 200 Hz to 50 kHz, 200 Hz to 10 kHz, 1 kHz to 100 MHz, 5 kHz to 100 MHz, 100 kHz to 100 MHz, 1 MHz to 100 MHz, 1 MHz to 100 MHz, 1 kHz to 10 MHz, 1 kHz to 10 MHz, or 100 kHz to 10 MHz. Positioners of this class are also solid components. The deflection of the component is based on sound waves propagating through an optically transparent material to induce a periodically changing refractive index. The change in refractive index is caused by compression and dilution (i.e., a change in density) of the material due to the sound waves propagating through the material. The periodically changing refractive index acts like an optical grating, causing the laser beam to diffract as it passes through the material.

[0103] Acousto-optical diodes (AODs) are created by coupling transducers (typically piezoelectric elements) to an acousto-optic crystal (e.g., TeO2). The transducers, driven by an electric amplifier, introduce sound waves into the refractive medium. At the opposite end, the crystal is typically skew-cut and fitted with acoustic-absorbing material to avoid reflection of sound waves into the crystal. As the wave propagates through the crystal in one direction, this forms a one-dimensional scanner. Two-dimensional scanners can be created by arranging two AODs in series or orthogonally, or by coupling two transducers on orthogonal crystal planes.

[0104] Regarding EODs, while the deflection angle of AODs is lower than that of galvanometer mirrors, their angular accuracy is higher compared to scanners based on such mirrors, and the frequency driving the crystal is digitally controlled, generally resolving to 1 Hz. Roemer and Bechtold (2014) note that drift, common to scanners based on galvanometer mirrors as well as temperature dependence compared to analog controllers, is not typically a problem encountered by AODs.

[0105] Examples of materials for use as refractive media in AOD include tellurium dioxide, fused silica, crystalline quartz, sapphire, AMTIR, GaP, GaAs, InP, SF6, lithium niobate, PbMoO4, arsenic trisulfide, tellurite glass, lead silicate, and Ge. 55 As 12 S 33 It contains mercury(I) chloride and lead(II) bromide.

[0106] To change the deflection angle, the frequency of the sound introduced into the crystal must be changed, and since it takes a finite amount of time for the sound wave to fill the crystal (depending on the propagation speed of the sound wave within the crystal and the size of the crystal), this means that there is a certain degree of delay. Nevertheless, the response time is relatively fast compared to laser system positioners based on moving parts.

[0107] An additional feature of AODs that can be used in specific cases is that the acoustic power applied to the crystal determines how much of the laser radiation is diffracted relative to the zero-order (i.e., non-diffracting) beam. The non-diffracting beam is typically directed towards a beam dump. Therefore, AODs can be used to rapidly and effectively control (or modulate) the intensity and power of the deflected beam.

[0108] Since the diffraction efficiency of an AOD is typically nonlinear, the diffraction efficiency versus output curve can be mapped for various input frequencies. The mapped efficiency curves for each frequency can then be recorded as equations or in a lookup table and used in conjunction with the apparatus and methods disclosed herein.

[0109] Therefore, in some embodiments of the present invention, the laser scanner system includes an AOD.

[0110] Instead of a rotating mirror, a solid positioner (e.g., AOD or EOD) may be used to induce deflection in the laser radiation beam rather than a mirror-based positioner. As described elsewhere in this specification, a solid scanner can scan in two orthogonal directions (X and Y) by attaching orthogonal electrodes to an EOD medium or by arranging two AODs orthogonally in series.

[0111] In embodiments equipped with an AOD-based positioner, the speeds at which the ablative laser pulse can be guided to the sample may be 200Hz~100MHz, 200Hz~10MHz, 200Hz~1MHz, 200Hz~100kHz, 200Hz~50kHz, 200Hz~10kHz, 1kHz~100MHz, 5kHz~100MHz, 10kHz~100MHz, 50kHz~100MHz, 100kHz~100MHz, 1MHz~100MHz, 10~100MHz, 1kHz~10MHz, 10kHz~10MHz, or 100kHz~10MHz. The previous paragraph described two types of laser scanning system positioners: mirror-based positioners with movable parts and solid-state positioners. The former is characterized by a large deflection angle, but its response time is relatively slow due to inertia. In contrast, the deflection angle range of a solid positioner is narrower, but the response time is much faster. Therefore, in some embodiments of the present invention, the laser scanning system includes both mirror-based and solid-state components in series. This arrangement takes advantage of the benefits of both, for example, the wide range provided by the mirror-based component, but with the inertia of the mirror-based component. See, for example, Matsumoto et al., 2013 (Journal of Laser Micro / Nanoengineering 8:315:320).

[0112] Therefore, a solid positioner (i.e., AOD or EOD) can be used to correct errors in, for example, mirror-based scanner components. In this case, to correct the positional errors of mirror-based scanner components, the position sensor associated with mirror position feedback to the solid component and the deflection angle introduced to the laser radiation beam by the solid component can be appropriately modified.

[0113] An example of a combined system is a galvanometer mirror and an AOD (the AOD can enable unidirectional or bidirectional deflection (by using two AODs in series, or by coupling two drivers to orthogonal planes of a single AOD crystal)). The system may also include two galvanometer mirrors to generate a two-dimensional scanning system in combination with the AOD (the AOD can enable unidirectional or bidirectional deflection (by using two AODs in series, or by coupling two drivers to orthogonal planes of a single AOD crystal)). In such a system, the speeds at which ablative laser pulses can be guided to the sample may be 200Hz-100MHz, 200Hz-10MHz, 200Hz-1MHz, 200Hz-100kHz, 200Hz-50kHz, 200Hz-10kHz, 1kHz-100MHz, 5kHz-100MHz, 10kHz-100MHz, 50kHz-100MHz, 100kHz-100MHz, 1MHz-100MHz, 10-100MHz, 1kHz-10MHz, 10kHz-10MHz, or 100kHz-10MHz. Alternatives to the combined system include galvanometer mirrors and EODs (EODs can enable unidirectional or bidirectional deflection by bonding two orthogonally positioned electrodes to a crystal). The system may also include two galvanometer mirrors to generate a two-dimensional scanning system in combination with an EOD (the EOD may allow unidirectional or bidirectional deflection by bonding two orthogonally positioned electrodes to a crystal). In such a system, the speeds at which an ablative laser pulse can be guided to the sample may be 200 Hz to 100 MHz, 200 Hz to 10 MHz, 200 Hz to 1 MHz, 200 Hz to 100 kHz, 200 Hz to 50 kHz, 200 Hz to 10 kHz, 1 kHz to 100 MHz, 5 kHz to 100 MHz, 100 kHz to 100 MHz, 1 MHz to 100 MHz, 1 MHz to 100 MHz, 1 kHz to 10 MHz, 1 kHz to 10 MHz, or 100 kHz to 10 MHz.The laser scanning system may include a scanner control module (such as a computer or programmed chip) that coordinates the movement of the positioner in the Y and / or X axes along with the movement of the sample stage in order to control the positioner of the laser scanning system. In some cases, appropriate patterns, such as pre- and post-rasterization, are pre-programmed into the chip. However, in other cases, the control module can apply inverse kinematics to determine the appropriate ablation pattern to follow. Inverse kinematics can be particularly useful, for example, when generating arbitrary ablation patterns to plot the best ablation course between multiple and / or irregularly shaped cells to be ablated. The scanner control module may also coordinate the emission of laser radiation pulses, for example, by adjusting the operation of a pulse picker.

[0114] A positioner can, in some cases, cause dispersion of the laser radiation beam it directs. Therefore, in some embodiments of the apparatus described herein, the laser scanning system includes at least one dispersion compensator between the positioner and / or a second positioner and the sample, which is adapted to compensate for the dispersion caused by the positioner. If the positioner is an AOD and / or the second positioner is an AOD, the dispersion compensator is (i) a diffraction grating having a line spacing suitable for compensating for the dispersion caused by the positioner and / or the second positioner, (ii) a prism suitable for compensating for the dispersion caused by the positioner and / or the second positioner (i.e., of appropriate material, thickness, and prism angle), (iii) a combination comprising the diffraction grating (i) and the prism (ii), and / or (iv) an additional acousto-optical device. If a first positioner causes dispersion and a second positioner causes dispersion, the laser scanning system may include a first dispersion compensator for compensating for any dispersion caused by the first positioner and a second dispersion compensator for compensating for any dispersion caused by the second positioner. International Publication No. 03 / 028940 describes how dispersion caused by an AOD positioner can be compensated for using another appropriately adapted AOD.

[0115] In some cases, moving the positioner to guide the laser radiation to a different location can cause the focal length of the radiation beam to change with respect to the sample position. This can be compensated for in various ways. For example, a movable focusing lens can be moved to maintain a constant or nearly constant spot size on the sample, regardless of the specific location on the sample to which the laser radiation is guided. Alternatively, an adjustable focusing lens (commercially available from Optotune) may be used. It is also possible to compensate for spot size variations by changing the height of the sample stage in the z-axis. All of these methods rely on moving parts, but introduce timing overhead into the system's operation. When an AOD is used with a Gaussian beam, the ablation spot size can be controlled by the output applied to the crystal in the AOD, allowing for rapid modulation of the primary beam intensity relative to the zero-order beam intensity.

[0116] In the alternative configurations shown in Figures 2 to 4, the components may be similar to those shown in Figure 1, except that the system operates to ablate the sample via a sample carrier. This configuration may be preferred, for example, when it is desired to impart additional kinetic energy to the sample material being ablated to assist in material clearance from areas close to the ablation spot. Alternatively, the configurations shown in Figures 2, 3, and / or 4 may allow for even smaller spot sizes, as described herein. In some embodiments, ablation via a sample carrier can be combined with a laser scanning optical system.

[0117] laser Generally, the selection of the wavelength and power of the laser used for sample ablation can follow the usual practices in cell analysis. The laser must have sufficient fluence to cause ablation to the desired depth without substantially ablating the sample carrier. (0.1–5 J / cm²) 2 For example, 3-4 J / cm 2 Or approximately 3.5 J / cm2 A laser fluence of is typically preferred, and the laser can ideally generate pulses having this fluence at a speed of 200 Hz or higher. In some cases, a single laser pulse from such a laser should be sufficient to ablate cellular material for analysis, such that the laser pulse frequency matches the frequency at which the ablation plume is generated. Generally, for a laser useful for imaging biological samples, the laser should generate pulses with a duration of less than 100 ns (preferably less than 1 ns), which can be focused to a specific spot size, for example, as described herein.

[0118] For example, the frequencies used for ablation with a laser system are within the ranges of 200Hz to 100MHz, 200Hz to 10MHz, 200Hz to 1MHz, 200Hz to 100kHz, 500Hz to 50kHz, or 1kHz to 10kHz.

[0119] At these frequencies, the instrumentation must be able to analyze the ablated material quickly enough to avoid substantial signal overlap between consecutive ablations, if it is desired to resolve each ablated plume individually (which may not always be desirable when firing bursts of pulses at the sample, as described below). The signal overlap between consecutive plumes is preferably <10%, more preferably <5%, and ideally <2%. The time required for plume analysis depends on the sample chamber washout time (see the sample chamber section below), the time it takes for the plume aerosol to pass through the laser ionization system, and the time required for analysis of the ionized material. As will be described in more detail below, each laser pulse can be correlated to a pixel on a later constructed image of the sample.

[0120] In some embodiments, the laser source is a laser having a nanosecond pulse duration, or an ultrafast laser (with a pulse duration of 1 ps (10) -12The system features a femtosecond laser (or faster), for example. The ultrafast pulse duration offers many advantages, as it limits thermal diffusion from the ablated zone, thereby providing a more accurate and reliable ablation crater, and minimizes the scattering of debris from each ablation event. Femtosecond lasers are particularly useful in the systems and apparatus described herein. In particular, femtosecond lasers are highly compatible with systems including laser scanning components, and their short pulse duration enables ablation techniques based on multiphoton events and electron seeding processes. Femtosecond lasers are particularly well-suited to this application due to their three attributes. The first attribute is the high repetition rate of typical configurations on femtosecond lasers, which is generally in the range of 1 MHz to 100 MHz. In contrast, nanosecond lasers are generally limited to pulse speeds of less than 100 kHz. The second attribute is the nonlinear ablation mechanism. Nonlinear ablation sharpens the definition of the ablation edge and enables ablation with even higher spatial resolution. Thirdly, nonlinear ablation thresholds also have low material specificity, which can make it easier to obtain a consistent size of ablation spots when testing heterogeneous materials such as microstructures.

[0121] In some cases, femtosecond lasers are used as laser sources. A femtosecond laser is a laser that emits light pulses with a duration of less than 1 ps. Passive mode-locking techniques are often used to generate such short pulses. Femtosecond lasers can be generated using various types of lasers. Typical durations of 30 fs to 30 ps can be achieved using passive mode-locked solid-state bulk lasers. Similarly, various diode-pumped lasers based on neodymium-doped or ytterbium-doped gain media, for example, operate in this form. Titanium-sapphire lasers with high dispersion compensation are also suitable for pulse durations of less than 10 fs, and in extreme cases, up to about 5 fs. The pulse repetition rate is almost always 10 MHz to 500 MHz, but there are also low-repetition-rate versions with repetition rates of several megahertz for even higher pulse energies (e.g., available from Lumentum (CA, USA), Radiantis (Spain), and Coherent (CA, USA)). This type of laser may be accompanied by an amplifier system to increase the pulse energy.

[0122] Furthermore, there are various types of ultrafast fiber lasers, which are also, in most cases, passively mode-locked and typically offer pulse durations of 50–500 fs and repetition rates of 10–100 MHz. Such lasers are commercially available, for example, from NKT Photonics (Denmark; formerly Fianium), Amplitude Systems (France), and Laser-Femto (CA, USA). The pulse energy of this type of laser can often be increased by amplifiers in the form of integrated fiber amplifiers.

[0123] Some mode-locked diode lasers can generate pulses with femtosecond durations. Directly at the laser output, pulse durations are typically around several hundred femtoseconds (e.g., available from Coherent (CA, USA)).

[0124] Femtosecond lasers are particularly well-suited for use with immersion lenses, such as oil immersion lenses (as described above). While oil immersion lenses can achieve a laser spot diameter of approximately 160 nm, ablation using a femtosecond laser is a multiphoton process, so when used with a femtosecond laser, the effective spot diameter decreases by √m, where m is the order of the nonlinear process. Thus, it is possible to achieve an effective spot diameter of less than 100 nm. Any possible configuration of the apparatus including the immersion lens described above can achieve an ablation spot size of 100 nm when used with a femtosecond laser, and therefore, the present invention provides an apparatus and method that improves spatial resolution tenfold compared to conventional IMC and IMS. Since femtosecond lasers generally have wavelengths in the near-infrared region (700-1300 nm), the wavelength must be shortened using frequency conversion techniques in order to achieve an ablation spot diameter of 100 nm. Of these, the easiest and best known is second-harmonic generation (SHG), which can efficiently convert near-infrared laser beams to visible wavelengths and can use commercially available, off-the-shelf microscope optics.

[0125] In some cases, picosecond lasers are used. Many of the types of lasers already described in the previous paragraph can also be adapted to produce pulses with durations in the picosecond range. The most common light sources are active or passive mode-locked solid bulk lasers, e.g., passive mode-locked Nd-doped YAG, glass, or vanadate lasers. Similarly, picosecond mode-locked lasers and laser diodes are commercially available (e.g., NKT Photonics (Denmark), EKSPLA (Lithuania)).

[0126] Nanosecond pulse duration lasers (gain switching and Q switching) can also find usefulness in certain instrument configurations (Coherent (CA, USA), Thorlabs (NJ, USA)). Nanosecond UV lasers are particularly well suited to the solid immersion lenses mentioned above. In particular, a 266 nm wavelength nanosecond UV laser can be used with diamond and fused silica solid immersion lenses to focus light to spot sizes of 100 nm or less. As those skilled in the art will understand, laser ablation using UV lasers is generally a thermal process, meaning that the area around the laser spot size is affected by the heat from the ablation process. This sets a limit on the achievable ablation spot size, and as a result, those skilled in the art will understand that while UV lasers are applicable in certain situations, other lasers may be more useful in others.

[0127] Alternatively, an externally modulated continuous-wave laser may be used to generate pulses with a duration of less than nanoseconds.

[0128] Typically, the laser beams used for ablation in the laser systems described herein have a spot size of 100 μm or less at the sampling position, e.g., 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less, e.g., approximately 3 μm or less, approximately 2 μm or less, approximately 1 μm or less, approximately 500 nm or less, approximately 250 nm or less. As described above in section 8, by applying an immersion medium between the objective lens and the sample, even smaller spot sizes such as 200 nm or less, 150 nm or less, or 100 nm or less can be achieved. The distance referred to as the spot size corresponds to the longest internal dimension of the beam, for example, the beam diameter in the case of a circular beam, the length of the diagonal between opposite corners in the case of a square beam, and the length of the longest diagonal in the case of a quadrilateral (as described above, the diameter of a circular beam with a Gaussian distribution is such that the fluence is 1 / e of the peak fluence). 2(Defined as the distance between points reduced by up to twice the initial value). As an alternative to a Gaussian beam, beam shaping and beam masking can be used to provide a desired ablation spot. For example, in some applications, a square ablation spot with a top-hat energy distribution (i.e., a beam with a nearly uniform fluence, as opposed to a Gaussian energy distribution) may be useful. This arrangement reduces the dependence of the ablation spot size on the ratio between the fluence at the peak of the Gaussian energy distribution and the threshold fluence. Ablation close to the threshold fluence provides the generation of a more reliable ablation crater and controls debris generation. Thus, the laser system may include beam masking and / or beam shaping components, such as diffractive optics placed on the Gaussian beam, to reshape the beam and generate a laser focus of uniform or nearly uniform fluence, such as a fluence that varies by less than ±25% across the entire beam, e.g., less than ±20%, ±15%, ±10%, or less than ±5%. In some cases, the laser beam has a square cross-sectional shape. In some cases, the beam has a top-hat energy distribution. As described above, in the context of the present invention, the distance referred to as the spot size is the longest internal dimension of the beam at the sampling position, i.e., the spot size is the lateral dimension of the beam, and therefore, for example, the spot size of a circular beam is the diameter. However, those skilled in the art will understand that, in the context of the present invention, the focal point of an objective lens is a three-dimensional volume, and the axial dimension of the focused spot size is generally longer than the lateral dimension, so in some cases, the axial dimension of the focal point may be longer than the distance referred to as the "spot size". If desired by the user, beam shaping and masking can also be used to enable a device that can switch from a high-resolution spot size achievable with an immersion lens to an even larger spot size.In addition to high-resolution imaging under specific conditions, larger spots can be used when projection specifications require it (for example, when lower resolution is preferable, or when it is necessary to sample and analyze a wider area of ​​tissue within a given time).

[0129] When used for the analysis of biological samples, the spot size of the laser beam used to analyze individual cells depends on the size and spacing of the cells. For example, if cells are densely packed together (e.g., within a tissue section), one or more laser sources in the laser system may have spot sizes no larger than these cells. This size depends on the specific cells in the sample, but generally, the laser spot has a diameter of less than 4 μm, e.g., less than or equal to about 3 μm, less than or equal to about 2 μm, less than or equal to about 1 μm, less than or equal to about 500 nm, or less than or equal to about 250 nm. To analyze a given cell at intracellular resolution, the system uses a laser spot size no larger than these cells, and more specifically, a laser spot size that can ablate the material at intracellular resolution. The smaller the spot size, the higher the resolution of the resulting image. Therefore, when high-resolution intracellular imaging is required, the techniques described herein can be used. In some cases, for example, if cells are spread on a slide and there is spacing between them (e.g., after analysis by electron microscopy, so that the internal structure of the cells is determined separately from the elemental analysis of the cells), single-cell analysis can be performed using a spot size larger than the size of the cells. Here, since the additional ablated region around the target cell does not contain additional cells, a larger spot size can be used, enabling single-cell characterization. Therefore, the specific spot size used can be appropriately selected depending on the size of the cells being analyzed. In biological samples, since it is rare for all cells to be the same size, if intracellular resolution imaging is desired, the ablation spot size must be smaller than the smallest cell, provided that a constant spot size is maintained throughout the ablation procedure. Small spot sizes can be achieved by focusing the laser beam. A laser spot diameter of 1 μm corresponds to a laser focal point of 1 μm (i.e., the diameter of the laser beam at the beam's focal point), but the laser focal point can vary by more than +20% due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and the variation in total laser energy relative to the ablation threshold energy.Suitable objective lenses for focusing a laser beam include reflective objective lenses, such as Schwarzschild-Cassegrain (inverse Cassegrain) objective lenses. Refractive objective lenses can also be used, as well as combinations of reflective and refractive objective lenses. The required focus can also be achieved using a single aspherical lens. A laser beam can also be focused using a solid immersion lens or a diffractive optical system. Another means of controlling the spot size of the laser, which can be used alone or in combination with the objective lenses described above, is to pass the beam through an aperture before focusing. Different beam diameters can be achieved by passing the beam through apertures of different diameters from an array of diameters. In some cases, for example, if the aperture is a diaphragm, there is a single aperture of variable size. In some cases, the diaphragm is an iris diaphragm. Variation in spot size can also be achieved by dithering of the optical system. One or more lenses and one or more apertures are placed between the laser and the sample stage.

[0130] For completeness, as is well known in the art, the standard lasers for LA at intracellular resolution are excimer or exciplex lasers. Using an argon fluoride laser (λ=193nm) yields suitable results. Pulse durations of 10–15 ns with these lasers can achieve appropriate ablation for specific applications.

[0131] Overall, the laser pulse frequency and intensity are selected in combination with the response characteristics of the MS detector to enable clear detection of individual laser ablation plumes. Combined with the use of a sample chamber with a small laser spot and short washout time, rapid, high-resolution imaging is achievable.

[0132] Laser ablation focus To maximize the efficiency of a laser ablation of material from a sample, the sample should be in a suitable position relative to the laser's focal point, for example, at the focal point, since the focal point is where the diameter of the laser beam is smallest and the energy is most concentrated. This can be achieved in various ways. The first method is to move the sample along the axis of the laser beam guided to the sample (i.e., up and down the path of the laser beam / towards and away from the laser source) to a desired point where the light has sufficient intensity to perform the desired ablation. Alternatively, or further, lenses can be used to shift the focal point of the laser beam, and as a result, its effective ability to ablate material at the sample's position can be used, for example, by reduction. One or more lenses are placed between the laser and the sample stage. A third method, which can be used alone or in combination with either or both of the above two methods, is to change the position of the laser.

[0133] To assist the system user in positioning the sample optimally for ablation, a camera can be guided to the stage holding the sample (as described in further detail below). Thus, this disclosure provides a laser ablation sampling system comprising a camera guided onto a sample stage. The image detected by the camera can be focused on the same point where the laser is focused. This can be achieved by using the same objective lens for both laser ablation and optical imaging. By matching the two focal points, the user can ensure that laser ablation is most effective when the optical image is in focus. Precise movement of the stage to focus on the sample can be achieved by using a piezoelectric activator, available from Physik Instrumente, Cedrat-technologies, Thorlabs, and other suppliers.

[0134] In another mode of operation, laser ablation is guided to the sample via a sample carrier. In this case, the sample support should be selected to be transparent (at least partially) to the frequency of the laser radiation used to ablate the sample. In certain situations, this mode of ablation can have advantages because it can impart additional kinetic energy to the plume of ablated material from the sample, driving the ablated material further away from the surface of the sample and thus facilitating its movement from the sample for analysis within the detector.

[0135] To achieve 3D imaging of a sample, the sample, or a defined area thereof, can be ablated to a first depth that does not completely penetrate the sample. Subsequently, the same area can be ablated again to a second depth, and then to third, fourth, and so on. In this way, a 3D image of the sample can be constructed. In some cases, it may be preferable to ablate the entire area to the first depth before proceeding to ablation at the second depth. Alternatively, repeated ablations may be performed at the same spot to different depths before proceeding to the next location within the area for ablation. In either case, imaging software can be used to deconvolve the MS-obtained signal against the position and depth of the sample. Similar to the workflow used for confocal imaging, thick tissue staining can be used, and the tissue is stabilized in a moist state (Clendenon et al., 2011. Microsc Microanal. 17:614-617).

[0136] Sample chamber of a laser ablation sampling system The sample is placed in a sample chamber when subjected to laser ablation. The sample chamber has a stage that holds the sample (typically, the sample rests on a sample carrier). When the material in the sample is ablated, it forms a plume, and the gas flow that passes through the sample chamber from the gas inlet to the gas outlet carries away the plume of aerosolized material containing the labeled atoms that were at the ablated locations. The gas carries the material to an ionization system, which ionizes the material to enable detection by a detector. Since atoms containing the labeled atoms in the sample can be distinguished by the detector, their detection reveals the presence or absence of multiple targets in the plume, and thus determines which targets were present at the ablated locations on the sample. Thus, the sample chamber plays a dual role: it contains the solid sample to be analyzed and it is the starting point for the transfer of the aerosolized material to the ionization and detection systems. This means that the gas flow passing through the chamber can influence how the ablated plume of material diffuses as it passes through the system. A measure of how the ablated plume diffuses is the washout time of the sample chamber. This number is a measure of the time it takes for the ablated material from the sample to be removed from the sample chamber by the gas flowing through it.

[0137] Thus, the spatial resolution of the signal generated from laser ablation (i.e., when ablation is used not only for removal but also for imaging, as described below) depends on factors including (i) the laser spot size and the rate of plume generation relative to the movement of the sample relative to the laser when the signal is integrated over the entire ablated area, and (ii) the rate at which the plume can be analyzed relative to the rate of plume generation in order to avoid signal overlap from the continuous plume as described above. Therefore, if individual analysis of plumes is desired, the possibility of plume overlap can be minimized if the plume can be analyzed in the shortest possible time (and as a result, plumes can be generated more frequently).

[0138] Therefore, sample chambers with short washout times (e.g., 100 ms or less) are advantageous for use with the apparatus and methods disclosed herein. Sample chambers with long washout times limit the rate at which images can be generated or result in overlap between signals arising from continuous sample spots (e.g., Kindness et al. (2003; Clin Chem 49:1916-23), where the signal duration exceeded 10 seconds). Thus, aerosol washout time is a significant limiting factor for achieving high resolution without increasing the total scan time. Sample chambers with washout times ≤ 100 ms are known in the art. For example, Gurevich & Hergenroeder (2007; J. Anal. At. Spectrom., 22:1043-1050) disclose a sample chamber with a washout time of less than 100 ms. Wang et al. (2013; Anal. Chem. 85:10107-16) (see also International Publication No. 2014 / 146724) disclosed a sample chamber having a washout time of 30 ms or less, enabling high ablation frequencies (e.g., above 20 Hz) and thus rapid analysis. Another such sample chamber is disclosed in International Publication No. 2014 / 127034. The sample chamber of International Publication No. 2014 / 127034 comprises a sample capture cell configured to be positioned operably close to a target, the sample capture cell including a capture cavity having an opening formed on the surface of the capture cell, configured to receive target material emitted or generated from a laser ablation site through the opening, and a guide wall exposed within the capture cavity and configured to guide the flow of carrier gas within the capture cavity from inlet to outlet so that at least a portion of the target material received within the capture cavity can be transported to the outlet as a sample. According to International Publication No. 2014 / 127034, the volume of the capture cavity in the sample chamber is 1 cm³. 3 It is less than 0.005 cm. 3It may be less than 25 ms. In some cases, the sample chamber may have a washout time of 25 ms or less, for example, 20 ms, 10 ms or less, 5 ms or less, 2 ms or less, 1 ms or less, 500 μs or less, 200 μs or less, 100 μs or less, 50 μs or less, or 25 μs or less. For example, the sample chamber may have a washout time of 10 μs or more. Typically, the sample chamber has a washout time of 5 ms or less.

[0139] For the sake of perfection, in some cases, plumes from the sample may be generated more frequently than the washout time of the sample chamber, resulting in a correspondingly blurred image (for example, if the highest possible resolution is not considered necessary for the particular analysis being performed).

[0140] A sample chamber typically includes a translation stage that holds the sample (and sample carrier) and moves the sample relative to the laser radiation beam. When an operating mode is used that requires guiding the laser radiation to the sample via the sample carrier, the stage holding the sample carrier should also be transparent to the laser radiation being used.

[0141] Therefore, the sample may be positioned on the side of the sample carrier (e.g., a glass slide) facing the laser radiation as it is directed onto the sample, resulting in the ablation plume being emitted on the same side as the laser radiation being directed onto the sample and subsequently captured. Alternatively, the sample may be positioned on the side of the sample carrier opposite to the laser radiation as it is directed onto the sample (i.e., the laser radiation passes through the sample carrier before reaching the sample), and the ablation plume is emitted on the opposite side of the laser radiation and subsequently captured. In the case of immersion media such as solid immersion lenses or liquid immersion lenses, directing the laser radiation to the sample through the sample carrier is particularly useful.

[0142] One feature of a sample chamber, particularly used when specific portions within various distinct regions of a sample are to be ablated, is a wide range of movement that allows the sample to move in the x and y (i.e., horizontal) directions relative to the laser (the laser beam is guided to the sample in the z-axis), where the x and y axes are perpendicular to each other. By moving the stage within the sample chamber and keeping the laser position fixed within the instrument's laser ablation sampling system, a more reliable and accurate relative position can be achieved. The wider the range of movement, the further apart the distinct ablation regions can be from each other. The sample is moved relative to the laser by moving the stage on which the sample is placed. Thus, the sample stage can have a range of movement of at least 10 mm in the x and y axes within the sample chamber, e.g., 20 mm in the x and y axes, 30 mm in the x and y axes, 40 mm in the x and y axes, 50 mm in the x and y axes, e.g., 75 mm in the x and y axes. In some cases, the range of movement is such that the entire surface of a standard 25 mm × 75 mm microscope slide can be analyzed within the chamber. Needless to say, in addition to a wide range of movement, the movement must be precise to enable intracellular ablation. Therefore, the stage can be configured to move the sample in the x and y axes in increments of less than 10 μm, e.g., less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, 1 μm, or less than 1 μm, less than 500 nm, less than 200 nm, or less than 100 nm. For example, the stage can be configured to move the sample in increments of at least 50 nm. Precise stage movement may be in increments of approximately 1 μm, such as 1 μm ± 0.1 μm. Commercially available microscope stages, such as those from Thorlabs, Prior Scientific, and Applied Scientific Instrumentation, can be used. Alternatively, an electric stage can be constructed from components based on a positioner that provides the desired range of movement and preferably precise movement, such as the Smaract SLC-24 positioner. The speed of the sample stage movement can also affect the speed of the analysis.Therefore, the sample stage has an operating speed greater than 1 mm / s, for example, 10 mm / s, 50 mm / s, or 100 mm / s.

[0143] Naturally, if the sample stage within the sample chamber has a wide range of motion, the sample chamber must be appropriately sized to accommodate the movement of the stage. Therefore, the size of the sample chamber depends on the size of the sample involved, which in turn determines the size of the mobile sample stage. Exemplary sample chamber sizes include those with an internal chamber of 10 × 10 cm, 15 × 15 cm, or 20 × 20 cm. The chamber depth may be 3 cm, 4 cm, or 5 cm. Those skilled in the art will be able to select appropriate dimensions according to the teachings herein. For analyzing biological samples using a laser ablation sampler, the internal dimensions of the sample chamber must be larger than the range of motion of the sample stage, such as at least 5 mm, or even at least 10 mm. This is because if the chamber walls are too close to the edge of the stage, the flow of the carrier gas through the chamber, which extracts the ablated plume of material from the sample and carries it into the ionization system, can become turbulent. Turbulence disrupts the ablated plume, so instead of remaining as a dense cloud of ablated material, the plume begins to spread after being ablated and carried away to the instrument's ionization system. If the peaks of the ablated material are even broader, interference due to peak overlap will occur unless the ablation rate is slowed down to a rate no longer of experimental interest, ultimately resulting in spatially unresolved data, thus negatively impacting the data generated by the ionization and detection system.

[0144] As described above, the sample chamber comprises a gas inlet and a gas outlet for transporting material to the ionization system. However, the sample chamber may include additional ports that serve as inlets or outlets for guiding the flow of gas within the chamber and / or supplying a mixture of gases into the chamber, as determined by those skilled in the art for the specific ablation process being performed.

[0145] camera In addition to identifying the most effective positioning of the sample for laser ablation, various additional analyses and techniques become possible by including a camera (such as a charge-coupled image sensor (CCD) camera or an active pixel sensor camera) or other arbitrary photodetector in the laser ablation sampling system. A CCD is a means for detecting light and converting it into digital information that can be used to generate an image. A CCD image sensor has a series of capacitors that detect light, each capacitor representing a pixel on the determined image. These capacitors enable the conversion of incident photons into electric charge. These charges can then be read using the CCD, and the recorded charges can be converted into an image. An active pixel sensor (APS) is an image sensor consisting of an integrated circuit that includes an array of pixel sensors, each pixel including a photodetector and an active amplifier, such as a CMOS sensor.

[0146] A camera can be incorporated into any laser ablation sampling system described herein. The camera can be used to scan a sample to identify specific target cells or regions (e.g., specific cell morphologies), or to identify antigens or fluorescent probes specific to cells or structures. In certain embodiments, the fluorescent probe is a histochemical stain or antibody, which may also include a detectable metal tag. Once such cells are identified, a laser pulse can be directed to these specific cells to ablate the material for analysis, for example, in an automated process (the system identifies and ablates the target features / regions, e.g., cells) or a semi-automated process (a user of the system, e.g., a clinicopathologist, identifies the target features / regions, and then the system ablates in an automated manner). This significantly improves the speed at which analysis can be performed, as it eliminates the need to ablate the entire sample to analyze specific cells, allowing for the specific ablation of target cells. This contributes to the efficiency of methods for analyzing biological samples, in terms of the time required to perform ablation, particularly the time required to interpret the data from the ablation, and then construct images. Since constructing images from data is one of the relatively time-consuming parts of the imaging procedure, minimizing the data collected from relevant parts of the sample improves the overall speed of the analysis.

[0147] A camera may record images from the confocal microscope. A confocal microscope is a form of optical microscope that offers many advantages, including the ability to reduce interference from background information (light) away from the focal plane. This is achieved by eliminating out-of-focus light or glare. Using a confocal microscope, it is possible to evaluate unstained samples for the morphology of cells or to determine whether cells are individual cells or part of a cell cluster. Often, samples are specifically labeled with fluorescent markers (such as labeled antibodies or labeled nucleic acids). These fluorescent markers can be used to stain specific cell populations (e.g., those expressing specific genes and / or proteins) or specific morphological features on cells (such as the nucleus or mitochondria), and when illuminated with light of the appropriate wavelength, it becomes possible to specifically identify these regions of the sample. Therefore, some systems described herein may be equipped with a laser for exciting fluorophores in the label used to label the sample. Alternatively, an LED light source can be used to excite the fluorophores. Non-confocal (e.g., wide-field) fluorescence microscopes can also be used to identify specific regions of biological samples, but they have lower resolution than confocal microscopes.

[0148] Another imaging technique is two-photon excitation microscopy (also known as nonlinear or multiphoton microscopy). This technique typically uses near-infrared light to excite fluorophores. Two infrared photons are absorbed for each excitation event. Scattering within the tissue is minimized by infrared light. Furthermore, multiphoton absorption strongly suppresses background signals. The most commonly used fluorophores have excitation spectra in the 400-500 nm range, but the lasers used to excite two-photon fluorescence are in the near-infrared range. When a fluorophore absorbs two infrared photons simultaneously, it absorbs enough energy to be pulled into an excited state. The fluorophore then emits a single photon with a wavelength that depends on the type of fluorophore used, which can then be detected.

[0149] When a laser is used to excite fluorophores for fluorescence microscopy, this laser may be the same laser that generates the laser light used to ablate materials from biological samples, but used at a power level that is not sufficient to ablate the materials from the sample. In some cases, the fluorophores are excited by the wavelength of the light that the laser uses to ablate the sample. In other cases, different wavelengths may be used, for example, by generating different harmonics of the laser to obtain light of different wavelengths, or by utilizing different harmonics generated by the above-mentioned harmonic generation system, separate from the harmonics used to ablate the sample. For example, when using the 4th and / or 5th harmonics of an Nd:YAG laser, the fundamental wave, or the 2nd to 3rd harmonics, can be used for fluorescence microscopy.

[0150] As an example of a technique combining fluorescence and laser ablation, it is possible to label the nuclei of cells in a biological sample using antibodies or nucleic acids conjugated to the fluorescent portion. Therefore, by exciting the fluorescent label and then observing and recording the location of the fluorescence using a camera, it is possible to specifically guide the ablation laser to the nucleus or to regions that do not contain nuclear material. In the field of cell chemistry, special applications can be found when a sample is divided into a nuclear region and a cytoplasmic region. By using an image sensor (CCD detector or active pixel sensor, e.g., CMOS sensor) to correlate the location of the fluorescence with the x,y coordinates of the sample, and then using a control module (computer or programmed chip, etc.) to guide the ablation laser to that location, it is possible to fully automate the process of identifying desired features / regions and then ablating them. As part of this process, the first image captured by the image sensor may have a low objective lens magnification (low numerical aperture), allowing for the inspection of a large area of ​​the sample. Subsequently, by switching to an objective lens with a higher magnification, it is possible to focus on specific features of interest that have been determined to fluoresce by higher magnification optical imaging. These features, which are then recorded as fluorescing, can be ablated by a laser. Using a lens with a relatively low numerical aperture initially has the added advantage of increasing the depth of field, which means that features hidden within the sample can be detected with higher sensitivity than with screening using a lens with a relatively high numerical aperture from the start.

[0151] In methods and systems in which fluorescence imaging is used, the fluorescence emission path from the sample to the camera may include one or more lenses and / or one or more optical filters. The system is adapted to handle chromatic aberration associated with emission from fluorescent labels by including optical filters adapted to allow a selected spectral bandwidth from one or more of the fluorescent labels to pass through. Chromatic aberration is a result of the lens being unable to focus light of different wavelengths to the same focal point. Therefore, including optical filters reduces the background of the optical system and increases the resolution of the resulting optical image. Another way to minimize the amount of emitted light of undesirable wavelengths reaching the camera is to intentionally utilize the chromatic aberration of the lenses by using a set of lenses designed for the transmission and focusing of light of wavelengths transmitted by the optical filters, as in the system described in International Publication No. 2005 / 121864.

[0152] The precision of the optical image determines the accuracy with which the ablation laser can be guided to ablate the sample; therefore, relatively high-resolution optical images are advantageous in this combination of optical techniques and laser ablation sampling.

[0153] Accordingly, in some embodiments disclosed herein, the apparatus of the present invention is equipped with a camera. This camera can be used online to identify a feature / region of a sample, for example, a specific cell, and then, for example, to fire a burst of pulses onto the feature / region of interest to sample the sample material from the feature / region of interest by ablation. When a burst of pulses is directed onto the sample, the material detected in the resulting plume may be as a continuous event (the plume from each individual ablation forms substantially a single plume, which is then continued for detection). Each cloud of sample material formed from the aggregated plume from a location within the feature / region of interest can be analyzed together, but the sample material in the plumes from each different feature / region of interest remains distinct. That is, sufficient time is left between the ablation of different feature / regions of interest to allow sample material from the nth feature / region of interest before the ablation of the (n+1)th feature / region begins.

[0154] In an additional operating mode combining both fluorescence analysis and laser ablation sampling, instead of analyzing the fluorescence across the entire slide and then directing laser ablation to those locations, pulses from the laser can be emitted onto spots on the sample (at low energy to excite only the fluorescent portions within the sample, rather than ablating the sample itself). If fluorescence emission at a predicted wavelength is detected, the laser can be emitted at full energy at that spot, ablating the sample at that spot, and the resulting plume can be analyzed by the detector, as described below. This has the advantage of maintaining the rasterization mode of analysis, but allows for pulsed fluorescence and testing, and results can be obtained immediately from the fluorescence (rather than the time required to analyze and interpret ion data from the detector to determine if the region is of interest), and speed is improved because, in this case as well, only the gene loci important for analysis can be targeted. Therefore, when this strategy is applied to imaging biological samples containing multiple cells, the following steps can be performed: (i) labeling multiple different target molecules in the sample with one or more different labeled atoms and one or more fluorescent labels to provide a labeled sample; (ii) irradiating a known location in the sample with light to excite one or more fluorescent labels; (iii) observing and recording whether fluorescence is present at that location; (iv) if fluorescence is present, guiding laser ablation to that location to form a plume; (v) subjecting the plume to inductively coupled plasma mass spectrometry; and (vi) repeating steps (ii) to (v) for one or more additional known locations on the sample, thereby enabling the construction of an image of the ablated region of the sample by detecting the labeled atoms in the plume.

[0155] In some cases, the sample or sample carrier may be modified to include optically detectable portions (e.g., by an optical or fluorescence microscope) at specific locations. The fluorescence locations can then be used to positionally orient the sample within the apparatus. The use of such marker locations finds useful, for example, when the sample is visually "offline," i.e., may have been examined by part of an apparatus other than the apparatus of the present invention. Such optical images can be marked by a physician, for example, by the desired features / regions corresponding to specific cells before the optical image, with the desired features / regions highlighted, and the sample are transferred to the apparatus according to the present invention. Here, the apparatus of the present invention can use a camera to identify the corresponding fluorescence locations by referring to the marker locations in the annotated optical image and calculate an ablation plan for the laser pulse locations accordingly. Thus, in some embodiments, the present invention comprises an orientation controller module capable of performing the above steps.

[0156] In some cases, the selection of a desired feature / region may be performed using the apparatus of the present invention, based on an image of the sample captured by the camera of the apparatus of the present invention.

[0157] Electron microscope In some embodiments of the present invention, the apparatus also comprises components for performing electron microscopy.

[0158] At a general level, an electron microscope comprises an electron gun (e.g., having a tungsten filament cathode) and electrostatic / electromagnetic lenses and apertures that control the beam and direct it towards a sample in a sample chamber. Since the sample is held under vacuum, gas molecules cannot obstruct or diffract the electrons on their way from the electron gun to the sample. In a transmission electron microscope (TEM), electrons are deflected as they pass through the sample. The deflected electrons are then detected by a detector, such as a fluorescent screen, or possibly a high-resolution phosphor coupled to a CCD. Between the sample and the detector is an objective lens that controls the magnification of the deflected electrons on the detector.

[0159] TEM requires ultrathin sections that allow enough electrons to pass through the sample so that an image can be reconstructed from the deflected electrons that hit the detector. Typically, TEM samples are less than 100 nm thick when prepared using an ultramicrotome. Biological tissue specimens are chemically fixed, dehydrated, and embedded in polymer resin to stabilize them sufficiently to allow for ultrathin sectioning. Sections of biological specimens, organic polymers, and similar materials may require staining with heavy atom labeling to achieve the required image contrast, because unstained biological specimens rarely interact strongly with electrons in their natural unstained state, thus deflecting them to enable the recording of electron microscope images.

[0160] As mentioned above, using thin sections allows for electron microscopy on samples that are also analyzed by IMS or IMC. Therefore, high-resolution structural images can be obtained by electron microscopy, such as transmission electron microscopy, and these high-resolution images can then be used to refine the resolution of image data obtained by IMS or IMC to a resolution exceeding that achievable by ablation using laser radiation (due to the much shorter wavelength of electrons compared to photons). In some cases, both electron microscopy and elemental analysis by IMC or IMS are performed on a sample within a single instrument (electron microscopy is performed before IMC / IMS because IMC / IMS is a destructive process).

[0161] Accordingly, the present invention provides an imaging mass cytometer or imaging mass spectrometer as described herein, further comprising an electron microscope having the above-described components, for example, an electron source such as an electron gun. As will be understood by those skilled in the art, the specific arrangement of the components can be varied (e.g., the direction in which electrons are directed to the sample and the direction in which laser radiation is directed to the sample), and the common arrangement of the components can be achieved without undue burden. In some cases, the sample is not moved within the apparatus between the electron microscopy analysis and the subsequent ablation. As will be understood by those skilled in the art, electron microscopy is performed under vacuum, but ablation, as described in this section, is performed in the presence of a gas flow that captures particulate material in the plume generated by the ablation of the sample. Thus, after the completion of the electron microscopy stage of the analysis, and before elemental analysis is performed, the sample chamber can be returned to near atmospheric pressure.

[0162] In ICP and IMS apparatuses equipped with an electron microscope, the arrangement of components may be such that laser radiation for ablation is guided to the sample via a sample carrier, for example, as shown in Figure 3 or 4, and the sample carrier acts as part of the wall of the sample chamber, enabling the sample chamber to be maintained under vacuum for the purpose of electron microscopy. Accordingly, in some embodiments of this specification, the apparatus comprises an electron microscope and an immersion medium such as a liquid immersion lens or a solid immersion lens.

[0163] Laser ablation Laser ablation may be performed in the manner previously described, for example, in Giesen et al., 2014 and International Publication No. 2014169394, in light of the changes relating to this specification (for example, it is not essential to use ICP or a TOF MS detector to ionize the sample material). For example, as described herein, the method and system for ionization on or near the sample surface may use an ion optics system to directly transfer labeled atoms from the sample to a mass spectrometry detector (e.g., a TOF detector or a magnetic sector detector) without requiring a gas-fluid element to deliver the sample to the ICP. In some cases, the method and system may use non-laser forms of radiation (e.g., an electron beam or an ion beam) instead of, or in addition to, a laser.

[0164] In embodiments in which laser ablation is performed without sustained ionization of the ablated sample, the ablation plume may be transferred to an ICP-MS as described below.

[0165] transfer conduit The transport conduit forms a link between the laser ablation sampling system and the ionization system, allowing the plume of sample material generated by laser ablation of the sample to be transported from the laser ablation sampling system to the ionization system. Part (or all) of the transport conduit may be formed, for example, by perforating a suitable material to create a lumen (e.g., a lumen with a circular, rectangular, or other cross-section) for the plume to pass through. The transport conduit may have an inner diameter in the range of 0.2 mm to 3 mm. In some cases, the inner diameter of the transport conduit may vary along its length. For example, the transport conduit may be tapered at the end. The transport conduit may have a length in the range of 1 cm to 100 cm. In some cases, the length is 10 cm or less (e.g., 1 to 10 cm), 5 cm or less (e.g., 1 to 5 cm), or 3 cm or less (e.g., 0.1 to 3 cm). In some cases, the lumen of the transport conduit is straight along the entire or nearly entire distance from the ablation system to the ionization system. Furthermore, the lumen of the transport conduit may not be straight over its entire length, and its orientation may change. For example, the transport conduit may gradually rotate 90 degrees. This configuration allows the plume generated by the ablation of the sample in the laser ablation sampling system to initially move in the vertical plane while the axis of the transport conduit inlet is pointing straight up, and then move horizontally as it approaches the ionization system (e.g., an ICP torch, which is typically oriented horizontally to utilize convective cooling). The transport conduit may be straight for a distance of at least 0.1 centimeters, at least 0.5 centimeters, or at least 1 centimeter from the inlet opening through which the plume enters or through which the plume is formed. Generally speaking, the transport conduit is typically adapted to minimize the time it takes to transport material from the laser ablation sampling system to the ionization system.

[0166] Inlet of the transfer conduit containing the sample cone The transport conduit has an inlet within the laser ablation sampling system (in particular, within the sample chamber of the laser ablation sampling system; therefore, it also represents the main gas outlet of the sample chamber). The inlet of the transport conduit receives the ablated sample material from the sample in the laser ablation sampling system and transports it to the ionization system. In some cases, the laser ablation sampling system inlet is the source of any gas flow along the transport conduit to the ionization system. In some cases, the laser ablation sampling system inlet that receives material from the laser ablation sampling system is an opening in the wall of the conduit through which a second “transport” gas flows from a separate transport inlet (as disclosed, for example, in International Publication Nos. 2014146724 and International Publication Nos. 2014147260). In this case, the transport gas forms a significant proportion, and in many cases, the majority of the gas flow flows to the ionization system. The sample chamber of the laser ablation sampling system includes a gas inlet. When gas is flowed into the chamber through this inlet, a gas flow is generated from the chamber through the inlet of the transport conduit. This gas flow captures the plume of ablated material and brings it into a transport conduit (typically, the laser ablation sampling system inlet of the transport conduit is conical in shape and referred to herein as the sample cone) and into a conduit that passes over the sample chamber from the sample chamber. This conduit also has gas flowing into the conduit from a separate transport inlet (indicated by the transport flow arrow on the left side of the figure). The component that starts the transport conduit, which includes the transport inlet and the laser ablation sampling system inlet and carries the ablated sample material toward the ionization system, is also called a flow cell (as described in International Publication Nos. 2014146724 and International Publication Nos. 2014147260).

[0167] The transport flow serves at least three purposes: directing the plume entering the transport conduit towards the ionization system and preventing the plume material from contacting the side walls of the transport conduit; forming a "protective region" above the sample surface to ensure ablation takes place under a controlled atmosphere; and increasing the flow velocity in the transport conduit. Typically, the viscosity of the capture gas is lower than that of the transport gas. This helps to confine the plume of sample material in the capture gas at the center of the transport conduit and minimize the diffusion of the sample material plume downstream of the laser ablation sampling system (because the transport velocity is more constant and nearly flatter at the center of the flow). The gas may be, for example, argon, xenon, helium, nitrogen, or mixtures thereof, but is not limited to these. Argon is a common transport gas. Argon is particularly well suited to stopping the diffusion of the plume before it reaches the walls of the transport conduit (and also helps to improve instrument sensitivity in devices where the ionization system is an argon-based ICP). The capture gas is preferably helium; however, the capture gas may be replaced by or contain other gases, such as hydrogen, nitrogen, or water vapor. At 25°C, argon has a viscosity of 22.6 μPas, while helium has a viscosity of 19.8 μPas. In some cases, the capture gas is helium and the transport gas is argon.

[0168] As described in International Publication No. 2014169394, the use of a sample cone minimizes the distance between the target and the inlet of the laser ablation sampling system in the transport conduit. Because the distance between the sample and the point on the cone where the captured gas can flow is reduced, this results in improved capture of the sample material with less turbulence and reduces the spread of the plume of ablated sample material. Therefore, the inlet of the transport conduit is the opening at the tip of the sample cone, which protrudes into the sample chamber.

[0169] Any modification to the sample cone is to make it asymmetrical. When the cone is symmetrical, gas flow from all directions is neutralized at the center, so along the axis of the sample cone, the overall gas flow along the sample surface is zero. By making the cone asymmetrical, a non-zero velocity is created along the sample surface, which helps wash out the plume material from the sample chamber of the laser ablation sampling system.

[0170] In practice, any modification of the sample cone that causes a non-zero vector gas flow along the surface of the sample on the axis of the cone may be used. For example, an asymmetric cone may have a notch or a series of notches adapted to generate a non-zero vector gas flow along the surface of the sample on the axis of the cone. An asymmetric cone may have an orifice on the side of the cone adapted to generate a non-zero vector gas flow along the surface of the sample on the axis of the cone. This orifice unbalances the gas flow around the cone, thereby again generating a non-zero vector gas flow along the surface of the sample on the axis of the cone at the target. The side of the cone may have multiple orifices, and may include both one or more notches and one or more orifices. The edges of the notches and / or orifices are typically smoothed, rounded, or chamfered to prevent or minimize turbulence.

[0171] The different asymmetrical orientations of the cone are suitable for different situations, depending on the selection of the capture and transfer gas and its flow rate, and appropriately identifying the gas and flow rate combinations for each orientation is within the capabilities of a person skilled in the art.

[0172] Any of the above modifications may be present in a single asymmetrical sample cone, as used in the present invention. For example, the cone may be asymmetrically trimmed and formed from the halves of two different elliptical cones, or the cone may be asymmetrically trimmed and equipped with one or more orifices, etc.

[0173] Therefore, the sample cone is adapted to capture the plume of ablated material from the sample within the laser ablation sampling system. During use, the sample cone is positioned in operable proximity to the sample, for example, by manipulating the sample within the laser ablation sampling system on a movable sample carrier tray, as already described above. As described above, the plume of ablated sample material enters the transport conduit through an opening at the narrow end of the sample cone. The diameter of the opening may be a) adjustable, b) sized to prevent disturbance of the ablated plume as it enters the transport conduit, and / or c) approximately equal to the cross-sectional diameter of the ablated plume.

[0174] tapered conduit In tubes with relatively small inner diameters, gases at the same flow rate move at relatively high speeds. Therefore, by using tubes with relatively small inner diameters, plumes of ablated sample material carried in a gas flow can be transported more rapidly over a defined distance at a given flow rate (e.g., from the laser ablation sampling system to the ionization system within the transport conduit). One of the key factors in how quickly individual plumes can be analyzed is how much the plume diffuses between the generation of the plume by ablation and the detection of its component ions in the instrument's mass spectrometer components (detector transient). Therefore, by using a narrow transport conduit, the time between ablation and detection is shortened, thereby reducing diffusion because there is less time for diffusion to occur, and ultimately reducing the transient time of each ablation plume at the detector. A reduced transient time means that more plumes can be generated and analyzed per unit time, and therefore, higher quality and / or faster images can be produced.

[0175] The tapered structure may include a gradual change in the inner diameter of the transport conduit along the aforementioned portion of the transport conduit's length (i.e., the inner diameter of the tube was a cross-section that decreased along the tube from the end of the portion toward the inlet (located at the end of the laser ablation sampling system) toward the portion toward the outlet (located at the end of the ionization system)). Typically, the region of the conduit near where ablation occurs has a relatively wide inner diameter. A larger volume of the conduit before the tapered structure facilitates the containment of the material generated by ablation. When ablated particles are scattered from the ablated spot, they travel at high speed. Friction in the gas slows these particles down, but the plume can still spread on a scale of sub-millimeters to millimeters. Ensuring sufficient distance from the wall helps to suppress the plume near the center of the flow.

[0176] Because the wide inner diameter sections are simply short (about 1-2 mm), they do not contribute significantly to the overall transient time if the plume spends more time in the relatively long sections of the transport conduit with a relatively narrow inner diameter. Therefore, the relatively large inner diameter sections are used to capture the ablation products, and the relatively small inner diameter conduits are used to rapidly transport these particles to the ionization system.

[0177] The diameter of a narrow inner diameter section is limited by the diameter corresponding to the onset of turbulence. The Reynolds number can be calculated for round tubes and known flows. In general, Reynolds numbers above 4000 should be avoided as they indicate turbulence. Reynolds numbers above 2000 may also be desirable to avoid as they indicate transitional flow (between non-turbulent and turbulent). For a given mass flow rate of gas, the Reynolds number is inversely proportional to the diameter of the conduit. The inner diameter of a narrow inner diameter section of a transfer conduit is generally narrower than 2 mm, for example, narrower than 1.5 mm, narrower than 1.25 mm, and narrower than 1 mm, but a flow of 4 liters / min of helium in the conduit is greater than the diameter at which a Reynolds number above 4000 occurs.

[0178] Rough or angular edges in the transition between a constant-diameter section and a tapered structure of a transport conduit can cause turbulence in the gas flow and are typically avoided.

[0179] Sacrifice flow As flow rates increase, the risk of turbulence within the conduit increases, especially when the conduit has a small inner diameter (e.g., 1 mm). However, by using lighter gases such as helium or hydrogen instead of argon, which is conventionally used as the gas transport flow, high-speed transport (over 300 m / s) can be achieved in conduits with small inner diameters.

[0180] High-speed transfer presents problems insofar as it can cause a plume of ablated sample material to pass through the ionization system without achieving an acceptable level of ionization. The increased flow of low-temperature gas can lower the plasma temperature at the torch end, potentially reducing the level of ionization. If the sample material plume is not ionized to a satisfactory level, information from the ablated sample material is lost. This is because its components (including any labeled atoms / elemental tags) cannot be detected by the mass spectrometer. For example, because the sample passes through the plasma so rapidly at the torch end in the ICP ionization system, the plasma ions do not have enough time to act on the sample material and ionize it. This problem, caused by high flow rates and high-speed transfer in a narrow-bore transfer conduit, can be solved by introducing a flow sacrificial system at the outlet of the transfer conduit. The flow sacrificial system is adapted to receive the gas flow from the transfer conduit and allow only a portion of that flow (the central portion of the flow containing any plume of ablated sample material) to pass towards an injector leading to the ionization system. To facilitate the dispersion of gas from the transfer conduit within the flow sacrificial system, the outlet of the transfer conduit can be made to open in a trumpet shape.

[0181] Because the flow sacrificial system is located near the ionization system, the length of the tube (e.g., injector) from the flow sacrificial system to the ionization system is short (e.g., about 1 cm in length; compared to the length of the transport conduit, which is typically several tens of centimeters, e.g., about 50 cm). Therefore, the relatively slow portion of the entire transport system is much shorter, and the relatively low gas velocity in the tube from the flow sacrificial system to the ionization system does not significantly affect the total transport time.

[0182] In most configurations, significantly increasing the diameter of the tube (e.g., injector) passing from the flow sacrificial system to the ionization system is undesirable, or in some cases possible, as a way to reduce the gas velocity by volumetric flow. For example, if the ionization system is ICP, the conduit from the flow sacrificial system forms an injector tube at the center of the ICP torch. Using an injector with a relatively wide inner diameter degrades signal quality because the plume of ablated sample material cannot be accurately injected into the center of the plasma (the hottest part and therefore the most efficient ionization part of the plasma). Injectors with an inner diameter of 1 mm or even narrower (e.g., inner diameter of 800 μm or less, e.g., 600 μm or less, 500 μm or less, or 400 μm or less) are particularly preferred. Since other ionization techniques rely on materials being ionized in a relatively small volume in three-dimensional space (because the energy density required for ionization can only be achieved in small amounts), a conduit with a relatively wide inner diameter means that the majority of the sample material passing through the conduit is outside the zone where the energy density is sufficient to ionize the sample material. Therefore, narrow-diameter tubes leading from the flow sacrificial system to the ionization system are also used in apparatus with non-ICP ionization systems. As mentioned above, if the plume of the sample material is not ionized to a suitable level, information from the ablated sample material is lost. This is because its components (including any labeled atoms / elemental tags) cannot be detected by the mass spectrometer.

[0183] Pumping can be used to ensure a desired split ratio between the sacrificial flow and the flow flowing into the inlet of the ionization system. Therefore, in some cases, the flow sacrificial system includes a pump attached to the sacrificial outlet. A controlled restrictor can be added to the pump to control the sacrificial flow. In some cases, the flow sacrificial system also includes a mass flow controller adapted to control the restrictor.

[0184] When expensive gases are used, the gas pumped out from the sacrificial outlet can be purified using known gas purification methods and recycled back into the same system. Helium is particularly suitable as a transport gas, as mentioned above, but it is expensive. Therefore, it is advantageous to reduce the loss of helium in the system (i.e., when it flows into the ionization system and is ionized). Thus, in some cases, a gas purification system is connected to the sacrificial outlet of the flow sacrificial system.

[0185] Ionization system To generate elemental ions, it is necessary to use hard ionization techniques that can vaporize, atomize, and ionize the atomized sample.

[0186] Inductively coupled plasma torch Generally, inductively coupled plasma is used to ionize a material to be analyzed before sending it to a mass detector for analysis. Inductively coupled plasma is a plasma source energized by an electric current generated by electromagnetic induction. The inductively coupled plasma is maintained within a torch consisting of three concentric tubes, the innermost of which is known as the injector.

[0187] Induction coils, which provide the electromagnetic energy to sustain the plasma, are positioned around the output end of the torch. The alternating current electromagnetic field reverses its polarity millions of times per second. Argon gas is supplied between two outermost concentric tubes. Free electrons are introduced by the discharge, then accelerated by the alternating current electromagnetic field, colliding with argon atoms and ionizing them. In a steady state, the plasma consists mainly of argon atoms, with small amounts of free electrons and argon ions.

[0188] The gas flow between the two outermost tubes keeps the plasma away from the torch walls, thus retaining the ICP within the torch. A second flow of argon, introduced between the injector (central tube) and the intermediate tube, keeps the plasma away from the injector. A third flow of gas is introduced into the injector at the center of the torch. The sample to be analyzed is introduced into the plasma via the injector.

[0189] ICPs can be equipped with injectors with inner diameters of less than 2 mm and greater than 250 μm to introduce material from a sample into the plasma. The injector diameter refers to the inner diameter of the injector at the end closest to the plasma. As the injector extends away from the plasma, it can have different diameters, for example, wider diameters, and the difference in diameter is achieved by a gradual increase in diameter or by the injector tapering along its length. For example, the inner diameter of an injector can be 1.75 mm to 250 μm, e.g., 1.5 mm to 300 μm, 1.25 mm to 300 μm, 1 mm to 300 μm, 900 μm to 300 μm, 900 μm to 400 μm, e.g., approximately 850 μm. Using injectors with an inner diameter of less than 2 mm offers significant advantages compared to injectors with a larger diameter. One advantage of this feature is that the transient of the signal detected by the mass detector when the sample material plume is introduced into the plasma is reduced by the narrow injector (the sample material plume is a cloud of specific vaporized material removed from the sample by the laser ablation sampling system). Therefore, the time required to analyze the sample material plume from its introduction into the ICP for ionization until the resulting ions are detected by the mass detector is shortened. This reduction in the time required to analyze the sample material plume makes it possible to detect more sample material plumes within any given period. In addition, an injector with a relatively small inner diameter can introduce the sample material more precisely into the center of the inductively coupled plasma, where more efficient ionization occurs (in contrast to a relatively large diameter injector, which can introduce more sample material towards the periphery of the plasma where ionization is less efficient).

[0190] ICP torches (from Agilent, Varian, Nu Instruments, Spectro, Leeman Labs, PerkinElmer, Thermo Fisher, etc.) and injectors (e.g., Elemental Scientific and Meinhard) are available.

[0191] Other ionization technologies Electron ionization Electron ionization involves bombarding a gas-phase sample with an electron beam. An electron ionization chamber includes an electron source and an electron trap. Typical electron beam sources are rhenium or tungsten wires, usually operating at an energy of 70 electron volts. Electron beam sources for electron ionization are available from Markes International. The electron beam is guided towards the electron trap, and a magnetic field applied parallel to the direction of electron movement causes the electrons to move along a helical path. The gas-phase sample is guided through the electron ionization chamber and interacts with the electron beam to form ions. Electron ionization is typically considered a hard ionization method because it fragments the sample molecules. An example of a commercially available electron ionization system is the electron ionization chamber from Advanced Markus.

[0192] Optional additional components for laser ablation-based sampling and ionization systems Ion deflector A mass spectrometer detects ions when they collide with the surface of the detector. When an ion collides with the detector, electrons are emitted from the detector's surface. These electrons are multiplied as they pass through the detector (the initially emitted electrons further impact electrons within the detector, and these electrons then collide with the secondary plate, further amplifying the number of electrons). The number of electrons colliding with the detector's anode generates an electric current. The number of electrons colliding with the anode can be controlled by changing the voltage applied to the secondary plate. The current is an analog signal that can be converted into the number of ions colliding with the detector by an analog-to-digital converter. If the detector is operating within a linear range, the current can directly correlate with the number of ions. There is a limit to the amount of ions that can be detected at once (expressed as the number of detectable ions per second). Beyond this point, the number of electrons emitted by ions colliding with the detector no longer correlates with the number of ions. This thus imposes an upper limit on the detector's quantitative capability.

[0193] When ions collide with a detector, its surface becomes damaged by contamination. Over time, this irreversible contamination damage reduces the amount of electrons emitted from the detector surface when ions collide, ultimately leading to the need to replace the detector. This is known as "detector degradation" and is a well-known phenomenon in mass spectrometers (MS).

[0194] Therefore, the lifespan of the detector can be extended by avoiding introducing an overload of ions into the MS. As mentioned above, if the total number of ions colliding with the MS detector exceeds the detection limit, the signal becomes less quantitative than when the number of ions is below the limit, and thus becomes less useful as information. Therefore, it is desirable to keep the detection limit from being exceeded, as this will result in no useful data being generated and the deterioration of the detector over time will accelerate.

[0195] Analyzing large packets of ions by mass spectrometry presents a specific set of challenges not seen in conventional mass spectrometry. In particular, typical MS techniques involve introducing a low, constant level of material into the detector, which should not approach the detection limit or accelerate detector degradation over time. On the other hand, laser ablation-based techniques analyze ions from relatively large amounts of material—e.g., cell-sized patches of tissue samples, far larger than the small packets of ions typically analyzed in MS—within a very short time window within the MS. In practice, this intentionally nearly overloads the detector with packets of analyzed ions resulting from ablation or lifting. Between analytical events, the signal is at baseline (near-zero signal because ions from labeled atoms are intentionally not entering the MS from the sampling and ionization systems; some ions are inevitably detected because the MS is not a perfect vacuum).

[0196] Therefore, in the apparatus described herein, there is a high risk of accelerated detector degradation because ions from packets of ionized sample material labeled with a large number of detectable atoms may exceed the detection limit and damage the detector without providing useful data.

[0197] To address these issues, the instrument may be equipped with an ion deflector positioned between the sampling and ionization system and the detector system (mass spectrometer), which can be operated to control the entry of ions into the mass spectrometer. In one configuration, when the ion deflector is on, ions received from the sampling and ionization system are deflected (i.e., their path is altered so they do not reach the detector), while when the deflector is off, the ions are not deflected and reach the detector. How the ion deflector is deployed depends on the configuration of the sampling and ionization system and the instrument's MS. For example, if the entry point for ions into the MS does not directly coincide with the path of ions exiting the sampling and ionization system, by default, a well-placed ion deflector will be turned on to guide ions from the sampling and ionization system to the MS. When an event resulting from the ionization of a packet of ionized sample material is detected that could potentially overload the MS (see below), the ion deflector is switched off, and as a result, the remaining ionized material from the event is not deflected into the MS but instead simply collides with the internal surface of the system, thereby preserving the lifetime of the MS detector. The ion deflector, after preventing ions from damage events from entering the MS, is returned to its original state, thereby allowing ions from subsequent packets of the ionized sample material to enter the MS and be detected.

[0198] Alternatively, in configurations where (under normal operating conditions) there is no change in the direction of ions emerging from the sampling and ionization systems before they enter the MS, the ion deflector is turned off, and ions from the sampling and ionization systems pass through it and are analyzed by the MS. In this configuration, to prevent damage when a potential overload of the detector is detected, the ion deflector is turned on, diverting ions so that they do not enter the detector and thus preventing damage to the detector.

[0199] Ions entering the MS from the ionization of the sample material (the plume of material generated by laser ablation) do not all enter the MS simultaneously, but rather enter as peaks at frequencies that follow a probability distribution curve with respect to the maximum frequency: from the baseline, a small number of ions initially enter the MS and are detected, then the frequency of the ions increases to its maximum, and then the number decreases again, falling back to the baseline. Instead of a slow increase in the frequency of ions at the leading edge of the peak, the number of ions colliding with the detector increases very rapidly, allowing for the identification of events that could potentially damage the detector.

[0200] The flow of ions colliding with the detector of a TOF MS, specifically the type of detector described below, is not continuous during the analysis of ions in packets of ionized sample material. TOF includes a pulser that periodically emits ions into the flight chamber of the TOF MS in pulse groups. Emitting all known ions simultaneously enables time-of-flight mass determination. The time between pulses of ion emission for time-of-flight mass determination is known as the extraction or push in TOF MS. Pushes are on the order of microseconds. Therefore, signals from one or more packets of ions from the sampling and ionization systems encompass numerous pushes.

[0201] Therefore, if the ion count reading surges to a very high number within a single push from the baseline (i.e., the first portion of ions from a particular packet of ionized sample material), it can be predicted that the body of the ions resulting from the ionization of the sample material packet will be even larger and will exceed the detection limit. At this point, the ion deflector can be activated to ensure that the majority of the damaging ions are moved away from the detector (by activating or deactivating it, depending on the system configuration, as described above).

[0202] Suitable ion deflectors based on quadrupoles are available in the art (e.g., from Colutron Research Corporation and Dreebit GmbH).

[0203] The laser ablation-based sampling system of the present invention The components of a laser ablation-based sampling system can be combined depending on the analytical task being performed. Exemplary embodiments are shown below.

[0204] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, Sample stage and Laser source and Laser scanning system and The system comprises a focusing optical system which includes an objective lens (where the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9), and optionally further includes an immersion medium between the objective lens and the sample stage.

[0205] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, Sample stage and A laser source equipped with a femtosecond laser, Laser scanning system and The system comprises a focusing optical system which includes an objective lens (where the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9), and optionally further includes an immersion medium between the objective lens and the sample stage.

[0206] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, Laser source and Laser scanning system and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage (where the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9), and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0207] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A laser source equipped with a femtosecond laser, Laser scanning system and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage (where the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9), and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0208] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, Laser source and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage (where the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9), and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0209] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A laser source equipped with a femtosecond laser, A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from a laser source toward a second plane toward a position on a sample stage (where the objective lens has an numerical aperture of at least 0.7, at least 0.8, or at least 0.9), and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0210] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, Laser source and Laser scanning system and The system comprises a focusing optical system equipped with an objective lens, and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0211] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A laser source equipped with a femtosecond laser, Laser scanning system and The system comprises a focusing optical system equipped with an objective lens, and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0212] In some embodiments, a laser ablation-based sampling system for analyzing samples such as biological samples is, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A laser source equipped with a femtosecond laser, The system comprises a focusing optical system equipped with an objective lens, and optionally further comprising an immersion medium between the objective lens and the sample stage.

[0213] Each of the above laser ablation-based sampling systems is suitable for inclusion in the apparatus of the present invention for analyzing a sample, particularly one equipped with a mass detector (e.g., a TOF mass detector), and especially one equipped with an ICP ionization system.

[0214] b. Sampling and ionization systems based on sputtering Sputtering-based sampling systems and techniques provide alternative surface analysis techniques to the laser ablation-based systems and techniques described above. One such sputtering technique is secondary ion mass spectrometry (SIMS). SIMS involves sputtering material from a sample by colliding a focused ion beam with the sample. The sputtered material contains both ions and neutral atoms. In SIMS, the ions are then transferred to a mass detector in a vacuum, followed by capture by an immersion lens. The mass detector can be any of the mass detector systems described below. Similar sputtering can be achieved by inducing other charged particles on the sample, such as electrons.

[0215] SIMS is a useful surface analysis technique for several reasons. Firstly, it is very sensitive to low concentrations of analyte materials. Secondly, since the diffraction effect of primary ions can be ignored under most practical conditions, SIMS has virtually no diffraction limit. Therefore, SIMS has the potential to analyze materials on a scale of 10–30 nm.

[0216] However, ionization efficiency is very low in sputtered materials, and ionization is also highly dependent on the surface chemistry and specific elements being ionized, so the number of ions produced by SIMS is not always sufficient to provide a good signal-to-noise ratio. For example, the ionization efficiency is insufficient to detect a single copy of an antibody labeled with MaxPar reagent. Since a single antibody labeled with MaxPar mass tag carries approximately 100 atoms, a conventional SIMS workflow may require approximately 100 copies of the antibody to produce a signal larger than a few ions at the detector. Yet another drawback of SIMS is due to spectral interference from molecules within the same mass channel, as well as the formation of compound ions such as oxides and other species containing major tagging elements and abundant neutral atoms present in biological samples. Compound ions dilute the signal of elemental ions and further cause overlap with the mass channel of larger elements. Therefore, the resolution of imaging using SIMS can be limited by low detection sensitivity, partly due to low ionization efficiency.

[0217] This invention overcomes the limitations of SIMS by providing an improved method and apparatus for analyzing biological samples using laser-based secondary neutral mass spectrometry (SNMS).

[0218] The laser SNMS method and apparatus of the present invention involves impacting a sample with a focused beam of charged particles to sputter material from the sample. A laser is used to post-ionize the neutral sputtered material. These emitted ions (including any detectable ions from labeled atoms, as described below) can be detected by a detector system, such as a mass spectrometer (detectors are described in more detail below). Since most of the sputtered material is in a neutral state, and SNMS can ionize the sputtered material for analysis using a mass detector, SNMS provides a better quantitative estimate of the surface than SIMS. Furthermore, as mentioned above, one of the main challenges in improving the spatial resolution of conventional IMS and IMC is ensuring that the amount of analyte in the material being analyzed provides a sufficient signal-to-noise ratio. Therefore, since SNMS uses both neutral and ionized sputtered material, SNMS-based IMC and IMS provide improved resolution compared to SIMS-based IMC and IMS. For example, SNMS enables single-copy detection using antibodies tagged with MaxPar reagents. For example, if the post-ionization efficiency reaches 10%, 100 atoms per antibody generate 10 ions per antibody, and if these ions are delivered to the detector with good efficiency, reliable detection of each copy of the antibody is guaranteed.

[0219] A laser SNMS system typically comprises three components. The first component is a charged particle source for sputtering material from the sample to be analyzed (this charged particle source will be described in more detail below). The second component is a laser for post-ionizing the sputtered material. The third component is a detector component, such as a mass detector, for detecting the ionized material. In a laser SNMS, the laser and the charged particle source are typically pulsed. In the relevant system, the first component is a charged particle source guided to a location on the sample (this charged particle source will be described in more detail below). The second component is a laser for causing ablation and possibly ionization at the location where electrons have been preseeded by the charged particle. The third component is a detector component, such as a mass detector, for detecting the ionized material. In certain embodiments, the spot size of the charged particle impacting the sample is smaller than the spot size of the laser. Charged particles can ablate the sample at their location, after which a laser can ionize the sample near the sample surface (e.g., as shown in Figure 6). Charged particles can seed electrons at the sample location, after which a laser can ablate and ionize the electron-seed sample (e.g., as shown in Figure 7). The laser can ionize the sample within a few picoseconds (e.g., 10 to 100 ps) of the charged particles impacting the sample (e.g., within the charge ignition state). In certain embodiments, the spot size of the charged particle impacting the sample is smaller than the spot size of the laser, for example, less than half, less than one-fifth, less than one-tenth, less than one-twentieth, or less than one-hundredth of the laser spot size. For example, the laser may have a spot size (impacting the sample) of less than 10 micrometers and / or greater than 500 nanometers, e.g., 500 nanometers to 5 micrometers, 800 nm to 2 micrometers, or about 1 micrometer.Charged particles can provide a small spot size (e.g., within the range described herein) and enable ionization using minimal neutralization by laser radiation having a spot size of less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm, or less than 10 nm in diameter, for example.

[0220] Thus, the present invention provides a sample stage, and a charged particle source and a charged particle column for passing a charged particle beam to a position on the sample stage, and a first laser source and a first focusing optical system configured to direct a laser beam emitted by the first laser source towards the sample stage, and provides an apparatus for analyzing a sample such as a biological sample.

[0221] The apparatus typically includes a mass detector such as a TOF detector.

[0222] Thus, the present invention provides a sample stage, and a charged particle source and a charged particle column for passing a charged particle beam to a position on the sample stage, and a first laser source and a first focusing optical system configured to direct a laser beam emitted by the first laser source towards the sample stage, and provides a sampling and ionization system for analyzing a sample such as a biological sample.

[0223] The charged particle column of the present invention includes a suitable ion optical system arranged to focus charged particles to pass the beam to a position on the sample stage. Such a suitable ion optical system can include a mass filter, lenses and apertures, and deflection plates to shape the primary ion beam, as will be described in more detail below.

[0224] Figure 6 is a schematic diagram of the arrangement of an exemplary embodiment of the present invention. The energy source 40 emits radiation (e.g., a laser beam, primary ion beam, or electron beam, as further described below) that passes toward position 55 on the sample stage 20 by an optical system 80 (e.g., an optical or ion optical system, e.g., a charged particle column). The sample 30 is placed on the sample stage 20 so that charged particles pass toward position 55 and sputter the material 50 from the sample 30. The first laser source 60 emits a laser beam, and a focusing optical system (not shown) guides the laser beam toward the sample stage to ionize the sputtered material 50 and form a plume of material containing sample ions. The ions can then be transferred to a mass detector, e.g., a time-of-flight detector or a magnetic sector detector, or any other mass detector as further described below.

[0225] Accordingly, the present invention provides an apparatus or sampling and ionization system for analyzing a sample such as a biological sample, wherein a first focusing optical system is configured to synchronize laser beam pulses to ionize (referred to herein as post-ionization) a plume of material sputtered by charged particle pulses.

[0226] Therefore, the present invention is Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses and ionize a plume of material sputtered by charged particle pulses from a charged particle source.

[0227] In particular, the first focusing optical system directs and focuses the laser beam into a volume above the surface of the sample stage, and as a result, when material is sputtered from the sample on the sample stage, the plume of material emitted from the sample passes through the volume into which the radiation from the first laser source is focused, and the material can be ionized. This description applies to all apparatuses described below in this section that use a combination of features of a sample stage, a charged particle source, a first laser source, and a first focusing optical system that ionizes the plume. This description will not be repeated every time below for the sake of brevity.

[0228] The apparatus typically includes a mass detector, such as a TOF detector.

[0229] Therefore, the present invention is Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a sample stage, and configured to synchronize the laser beam pulses to ionize a plume of material sputtered by charged particle pulses from a charged particle source.

[0230] As described above, the first focusing optical system directs and focuses the laser beam into a volume above the position on the sample stage (more specifically, above the position on the sample on the sample stage), and as a result, when the material is sputtered from the sample on the sample stage, the plume of material emitted from the sample passes through the volume into which the radiation from the first laser source is focused, and the material can be ionized. This description applies to all systems described below in this section that use a combination of features of the sample stage, the charged particle source, the first laser source, and the first focusing optical system that ionizes the plume. This description will not be repeated every time below for the sake of brevity.

[0231] Figure 7 is a schematic diagram of the arrangement of a further exemplary embodiment of the present invention. Figure 7 includes elements common to Figure 6, and these elements share the same reference numerals. However, in the apparatus of the embodiment shown in Figure 7, the laser beam from the first laser source 60 is directed toward position 55 on the sample stage 20, which is led by an optical system 80 (e.g., a charged particle column). For example, a charged particle pulse passing toward position 55 on the sample forms an excited state with free electrons at position 55 on the sample, which is referred to herein as the “sample ignition state”. The laser beam from the first laser source irradiates position 55 on the sample immediately after the charged particle pulse reaches the position, so that the first laser source irradiates the sample in the ignition state. Because the sample is in the sample ignition state, the sample readily converts the laser light pulse from the laser beam into ablation and ionization energy. This state is referred to herein as the “sample energy pumping state”. This process forms a plume of material containing sample ions. Those skilled in the art will understand that in this embodiment, the energy of the laser pulse is less than the ablation threshold of the sample (as a result, the laser spot on the sample may be larger than the diameter of the location to which the charged particle is guided, so that the region at the same adjacent location to which the charged particle is guided will not be ablated in response to the laser pulse that puts that location into a "sample energy pumping state").

[0232] Accordingly, the present invention provides an apparatus for analyzing samples such as biological samples, wherein the first focusing optical system is configured to synchronize laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse. This configuration of the present invention overcomes the relatively slow sputtering of particles by the charged particle beam alone and thus can provide a more rapid method for analyzing biological samples.

[0233] Therefore, the present invention is Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source and a first focusing optical system configured to direct a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system being configured to synchronize laser beam pulses so as to reach a position on the sample stage immediately after a charged particle pulse, to provide an apparatus for analyzing a sample such as a biological sample.

[0234] In particular, the first focusing optical system directs and focuses the laser beam / laser radiation onto the same position on the sample on the sample stage that was previously targeted by charged particles. This description applies to any apparatus described below in this section that uses the combination of features of a sample stage, a charged particle source, a first laser source, and a first focusing optical system that irradiates the same position on the sample stage that was previously targeted by charged particles with pulses of radiation from the first laser source. This description will not be repeated every time for the sake of brevity hereinafter.

[0235] The apparatus typically includes a mass detector such as a TOF detector.

[0236] Therefore, the present invention a sample stage, a charged particle source and a charged particle column for passing a charged particle beam to a position on the sample stage, a first laser source and a first focusing optical system configured to direct a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system being configured to synchronize laser beam pulses so as to reach a position on the sample stage immediately after a charged particle pulse, to provide a sampling and ionization system for analyzing a sample such as a biological sample.

[0237] In particular, the first focusing optical system guides and focuses the laser beam / laser radiation to the same location on the sample on the sample stage that was previously targeted by the charged particles. This description applies to all systems described below in this section that use a combination of features of the sample stage, the charged particle source, the first laser source, and the first focusing optical system that causes pulses of radiation from the first laser source to irradiate the same location that was previously targeted by the charged particles. This description will not be repeated every time below for the sake of brevity. Figure 8 is a schematic diagram of the arrangement of a further exemplary embodiment of the present invention. Figure 8 includes elements common to Figures 6 and 7, and these elements share the same reference numerals. In this embodiment, the sample stage 20 is transparent (as with any sample carrier on which the sample is placed). Those skilled in the art will understand that a transparent sample stage, or a sample stage with a notched portion, may be used with any of the embodiments of sputtering-based sampling and ionization systems described herein. The transparent sample stage will be described in further detail herein.

[0238] In the embodiment shown in Figure 8, the charged particle source and charged particle column, as well as the first laser source and first focusing optical system, are configured to guide the charged particle beam and the laser beam toward the opposite side of the sample stage. In the embodiment shown in Figure 8, the charged particles pass toward position 55 on the sample, and the laser beam is also guided toward position 55 on the sample. Similar to the embodiment shown in Figure 7, in the embodiment of Figure 8, the pulsed charged particle beam brings about a sample ignition state, and the pulsed laser beam brings about an energy pumping state, generating a plume of material containing sample ions.

[0239] Figure 9 is a schematic diagram of the arrangement of a further exemplary embodiment of the present invention. Figure 9 includes elements common to Figures 6 to 8, and these elements share the same reference numerals. Similar to the embodiment shown in Figure 8, in this embodiment of the present invention, a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, are configured to direct the charged particle beam and the laser beam toward the opposite side of the sample stage. The sample 30 is placed on the sample stage 20 so that the charged particles pass toward position 55 and sputter the material 50 from the sample 30. The first laser source 60 emits a laser beam, and a focusing optical system (not shown) directs the laser beam toward the sample stage to ionize the sputtered material 50 and form a plume of material containing sample ions. The ions can then be transferred to a mass detector, e.g., a time-of-flight detector, or any other mass detector as described in further detail below.

[0240] Notably, in any of the embodiments described above, the charged particle beam may be scanned across the entire sample to analyze any region of interest, such as organelles, as previously stated.

[0241] Accordingly, the present invention provides an apparatus configured such that a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, are directed toward the same side of the sample stage. Examples of this type of apparatus are shown in Figures 6 and 7.

[0242] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The system comprises a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse. The present invention provides a device comprising a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, configured such that the charged particle beam and the laser beam are directed toward the same side of the sample stage.

[0243] The apparatus typically includes a mass detector, such as a TOF detector.

[0244] Therefore, the present invention relates to a sampling and ionization system for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The system comprises a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse. A sampling and ionization system is provided, comprising a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, configured such that the charged particle beam and the laser beam are directed toward the same side of the sample stage.

[0245] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a first surface of a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse.

[0246] The apparatus typically includes a mass detector, such as a TOF detector.

[0247] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a first surface of a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse.

[0248] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The first laser source and the first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses to ionize a plume of sputtered material by charged particle pulses from a charged particle source, The present invention provides a device comprising a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, configured such that the charged particle beam and the laser beam are directed toward the same side of the sample stage.

[0249] The apparatus typically includes a mass detector, such as a TOF detector.

[0250] Therefore, the present invention relates to a sampling and ionization system for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The first laser source and the first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses to ionize a plume of sputtered material by charged particle pulses from a charged particle source, A sampling and ionization system is provided, comprising a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, configured such that the charged particle beam and the laser beam are directed toward the same side of the sample stage.

[0251] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a first surface of a sample stage, the first focusing optical system configured to synchronize the laser beam pulses to ionize a plume of material sputtered by charged particle pulses from a charged particle source.

[0252] The apparatus typically includes a mass detector, such as a TOF detector.

[0253] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a first surface of a sample stage, and configured to synchronize the laser beam pulses to ionize a plume of material sputtered by charged particle pulses from a charged particle source.

[0254] Furthermore, the present invention provides a apparatus configured such that a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, are directed toward the opposite side of the sample stage. Examples of this type of apparatus are shown in Figures 8 and 9. The advantage of these types of configurations is that they minimize the mechanical complexity of combining the charged particle column and the focusing optical system. In certain embodiments, the charged particles are directed toward the sample via a support or substrate that is at least partially transparent to the charged particles, which can ablate the sample or seed electrons in the sample. In such embodiments, the sample may be thin, such as less than 200 nm, less than 100 nm, less than 50 nm, or less than 30 nm.

[0255] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The present invention provides a device comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse, wherein the charged particle source and charged particle column, as well as the first laser source and first focusing optical system, are configured to guide the charged particle beam and the laser beam toward the opposite side of the sample stage.

[0256] The apparatus typically includes a mass detector, such as a TOF detector.

[0257] Therefore, the present invention relates to a sampling and ionization system for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, The system comprises a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse. A sampling and ionization system is provided, comprising a charged particle source and a charged particle column, as well as a first laser source and a first focusing optical system, configured such that the charged particle beam and the laser beam are directed toward the opposite side of the sample stage.

[0258] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a second surface of a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse.

[0259] The apparatus typically includes a mass detector, such as a TOF detector.

[0260] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a first laser source and a first focusing optical system configured to guide a laser beam emitted by the first laser source toward a second surface of a sample stage, the first focusing optical system configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after a charged particle pulse.

[0261] As described elsewhere in this specification and repeated here for completeness, if a laser beam is guided through a sample stage to reach a sample on a first surface, the stage should be transparent to the laser radiation (as should the sample carrier for the sample), or the sample stage should contain a gap through which the laser radiation can pass (through the sample carrier) to reach the sample.

[0262] Furthermore, those skilled in the art will understand that the configurations shown in Figures 6 to 9 can be combined in various ways. Figure 10 shows a further exemplary embodiment of the present invention, which is a combination of the configurations shown in Figures 6 to 9, where again, elements common to Figures 6 to 9 are indicated by the same reference numerals.

[0263] The embodiment shown in Figure 10 includes a second laser source 61 and a second focusing optical system (not shown). Similar to the first laser source 60 and second focusing optical system in Figure 8, the second laser source 61 and second focusing optical system in this embodiment are configured to direct the second laser beam toward position 55 on the sample stage. Similar to the embodiments shown in Figures 7 and 8, the charged particle pulsed beam brings about a sample ignition state, and the pulsed laser beam from the second laser source 61 brings about an energy pumping state, generating a plume of material containing sample ions. In this embodiment of the present invention, the first laser source 60 and first focusing optical system are configured to ionize the plume of material sputtered by the charged particle pulse. Thus, the first laser source 60 ionizes any neutral material sputtered from a surface that has not been ionized by the energy pumping state. In this way, since both the first and second laser sources are configured to ionize the plume of material to generate sample ions, the present invention provides an apparatus that further improves the ionization probability and therefore increases the signal-to-noise ratio. Therefore, the present invention provides an apparatus for analyzing a sample with increased resolution.

[0264] Accordingly, the present invention provides an apparatus comprising a second laser source and a second focusing optical system, as described above in this section, wherein the second focusing optical system is configured to synchronize laser light pulses from the second laser source to ionize a plume of sputtered material with charged particle pulses. In certain embodiments, the first and second laser sources may comprise the same laser, and the control of the laser and / or optical system enables the two-stage laser radiation described herein.

[0265] Therefore, the present invention is Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a second laser source and a second focusing optical system configured to synchronize laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample.

[0266] The apparatus typically includes a mass detector, such as a TOF detector.

[0267] Therefore, the present invention is Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, the first focusing optical system configured to synchronize the laser beam pulses to ionize the plume of sputtered material by charged particle pulses from a charged particle source, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a second laser source and a second focusing optical system configured to synchronize laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from that position.

[0268] Accordingly, the present invention provides an apparatus comprising a first laser source and a first focusing optical system, and a second laser source and a second focusing optical system, configured such that the laser beam from the first laser source and the laser beam from the second laser source are directed toward the same side of the sample stage.

[0269] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse, The system comprises a second laser source and a second focusing optical system configured to synchronize laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample. The present invention provides a device comprising a charged particle source and a charged particle column, a first laser source and a first focusing optical system, and a second laser source and a second focusing optical system, configured such that the charged particle beam, the laser beam from the first laser source, and the laser beam from the second laser source are directed toward the same side of the sample stage.

[0270] The apparatus typically includes a mass detector, such as a TOF detector.

[0271] Therefore, the present invention relates to a sampling and ionization system for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse, The system comprises a second laser source and a second focusing optical system configured to synchronize laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample. A sampling and ionization system is provided, comprising a charged particle source and charged particle column, a first laser source and a first focusing optical system, and a second laser source and a second focusing optical system, configured such that the charged particle beam, the laser beam from the first laser source, and the laser beam from the second laser source are directed toward the same side of the sample stage.

[0272] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a first surface of a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the first surface of the sample stage immediately after the charged particle pulse, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a second laser source and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of a sample stage, the second focusing optical system configured to synchronize the laser beam pulses from the second laser source to ionize the plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample.

[0273] The apparatus typically includes a mass detector, such as a TOF detector.

[0274] Therefore, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a first surface of a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the first surface of the sample stage immediately after the charged particle pulse, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a second laser source and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of a sample stage, the second focusing optical system configured to synchronize the laser beam pulses from the second laser source to ionize the plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample.

[0275] Accordingly, the present invention provides an apparatus comprising a first laser source and a first focusing optical system, and a second laser source and a second focusing optical system, configured such that the laser beam from the first laser source and the laser beam from the second laser source are directed toward the opposite side of the sample stage.

[0276] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse, The system comprises a second laser source and a second focusing optical system configured to synchronize laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample. The present invention provides a device in which a charged particle source and a charged particle column, as well as a second laser source and a second focusing optical system, are configured to direct the charged particle beam and the laser beam from the second laser source toward the same side of the sample stage, and a first laser source and a first focusing optical system are configured to direct the laser beam from the first laser source toward the opposite side of the sample stage.

[0277] The apparatus typically includes a mass detector, such as a TOF detector.

[0278] Therefore, the present invention relates to a sampling and ionization system for analyzing samples such as biological samples, Sample stage and A charged particle source, and a charged particle column for passing the charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the sample stage immediately after the charged particle pulse, The system comprises a second laser source and a second focusing optical system configured to synchronize laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from a position on the sample. The present invention provides a sampling and ionization system comprising a charged particle source and a charged particle column, as well as a second laser source and a second focusing optical system, configured such that the charged particle beam and the laser beam from the second laser source are directed toward the same side of the sample stage, and a first laser source and a first focusing optical system, configured such that the laser beam from the first laser source is directed toward the opposite side of the sample stage.

[0279] For example, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a second surface of a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the first surface of the sample stage immediately after the charged particle pulse, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a second laser source and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of a sample stage, the second focusing optical system configured to synchronize the laser beam pulses from the second laser source to ionize the plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from that position.

[0280] The apparatus typically includes a mass detector, such as a TOF detector.

[0281] Therefore, the present invention is A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A charged particle source and a charged particle column for passing a charged particle beam to a position on the first surface of the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a second surface of a sample stage, wherein the first focusing optical system is configured to synchronize the laser beam pulses so that they reach a position on the first surface of the sample stage immediately after the charged particle pulse, The present invention provides a sampling and ionization system for analyzing samples such as biological samples, comprising a second laser source and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of a sample stage, the second focusing optical system configured to synchronize the laser beam pulses from the second laser source to ionize a plume of the sample material generated by the charged particle pulse and the laser beam pulse from the first laser source from that position.

[0282] The present invention also provides a method for analyzing biological samples using the apparatus described in this section. For example, the present invention provides: Passing a charged particle beam towards a specific location on the sample, The first laser beam pulse irradiates the sample to generate a plume of material containing sample ions, and The present invention provides a method that includes detecting the above-mentioned sample ions by mass spectrometry.

[0283] In some cases, the method includes passing a charged particle beam toward a position on the sample to sputter material from the sample, and the method includes irradiating the sputtered sample material with a first laser beam pulse, and thereby ionizing the sputtered material to generate sample ions. In some cases, passing a charged particle beam toward a position on the sample brings about a sample ignition state, and irradiating the sample at that position brings about a sample energy pumping state at that position on the sample.

[0284] In some methods, a charged particle beam passes from one side of the sample toward a position on the sample, and a first laser beam pulse irradiates the sample from the same side. In some embodiments, a charged particle beam passes from one side of the sample toward a position on the sample, and a first laser beam pulse irradiates the sample from the opposite side. In some cases, passing the charged particle beam toward a position on the sample further includes sputtering material from the sample, and the method includes irradiating the sputtered sample material with a second laser beam pulse and ionizing the sputtered material.

[0285] The first and second laser beam pulses irradiate the sample from the same side, or the first and second laser beam pulses irradiate the sample from the opposite side.

[0286] As those skilled in the art will understand, laser ionization of sputtered materials can be achieved by various ionization mechanisms, including single-photon ionization, resonant and non-resonant multiphoton ionization, avalanche ionization, and cold avalanche ionization. Furthermore, various ionization mechanisms, such as multiphoton ionization and avalanche ionization, can be operated simultaneously.

[0287] Single-photon ionization (SPI) is a process in which the absorption of a single photon is sufficient to overcome the ionization potential of a sputtered material. However, ionization by this means requires a complex system using very high-energy ultraviolet or vacuum ultraviolet lasers, such as excimer lasers, or nonlinear optical processes in a gas. Accordingly, the present invention provides a method and apparatus in which the first and / or second laser sources are high-energy ultraviolet or vacuum ultraviolet lasers.

[0288] Multiphoton ionization (MPI) involves the absorption of multiple photons to overcome the ionization potential, and absorption can be a non-resonant or resonant process. Multiphoton ionization requires a short, strong laser pulse. Lasers suitable for resonant systems include pulsed Nd:YAG lasers that pump two dye lasers, while lasers suitable for non-resonant laser systems include high-power excimer or Nd:YAg lasers. Accordingly, the present invention provides a method and apparatus in which the first and / or second laser source is a Nd:YAG laser, high-power excimer, or Nd:YAG laser that pumps two dye lasers.

[0289] Avalanche ionization (AI) is a process in which electrons collide with sputtered material, ionizing it and accelerating other sputtered material, generating additional electrons to collide with it, thereby creating a chain reaction. In this invention, the initial "seed" electrons may be the result of any such ionization mechanism arising from the application of a laser pulse, e.g., multiphoton ionization or single-photon ionization. Some models suggest that avalanche ionization is not significant when the laser pulse is shorter than 100 fs. However, various techniques have been demonstrated in which avalanche ionization persists with laser pulses shorter than 100 fs. This invention provides a method of utilizing these techniques to ionize sputtered material by avalanche ionization using laser pulses shorter than 100 fs.

[0290] In the first technique, an electric field applied to a sample by a strong ionization laser is used to lower the effective energy threshold at which collision ionization occurs, thereby enabling collision ionization to promote avalanche ionization even with short pulse lengths (less than 100 fs). This is the low-temperature avalanche ionization mechanism. A suitable laser may be, for example, a Ti:sapphire laser (800 nm, pulse length of 40-45 fs, energy of 75 nJ, e.g., the Octavius ​​Ti:sapphire laser available from Thorlabs), and it has been shown that avalanche ionization of fused silica can be achieved with an 800 nm laser pulse as low as 40 fs from a regenerative amplifier (Rajeev, Gertsvolf et al., 2009, PRL 201). Accordingly, the present invention provides a method and apparatus for ionizing a sputtered material by avalanche ionization, wherein the first and / or second laser source is a Ti:sapphire laser. In certain embodiments, the mechanisms of ignition (e.g., electron seeding) and subsequent plasma generation (e.g., avalanche ionization) may be similar in dielectrics and biological samples. In the case of biological samples, the sample form may be epoxy resin, which is a dielectric material, although it differs from silicon oxide.

[0291] In the second technique, free carriers are injected into the sample to promote exciton seed multiphoton ionization in combination with avalanche ionization. Free carriers can be injected into the dielectric from an extreme ultraviolet source generated by harmonic or attosecond pulse generation, and the sample can be ionized using a short laser pulse of 45 fs (800 nm laser) (Grojo, Gertsvolf et al., 2010, PR 81). Thus, the present invention provides a method and apparatus in which the first and / or second laser source is an extreme ultraviolet source.

[0292] Accordingly, the present invention provides a method and apparatus configured such that a first and / or second laser ionizes a sputtered material by avalanche ionization.

[0293] Thus, the present invention further addresses two major challenges in increasing the resolution of IMC to the submicrometer scale. First, avalanche ionization can be performed using laser pulses shorter than 100 fs, thus reducing the risk of damage to the area around the laser spot size due to heating effects. Thus, the present invention maintains a spot area of ​​approximately 200 nm or less. Second, the present invention improves the potential for multiphoton and avalanche ionization, and therefore improves the overall ionization rate. Thus, the total amount of analytes in the ablated material provides a sufficient signal-to-noise ratio.

[0294] Components of a sputtering-based sampling and ionization system Charged particle source As described above, in the present invention, a charged particle source and a charged particle column may be used to pass the charged particle beam to a position on the sample.

[0295] Primary ion beam Typically, the charged particle source used in secondary neutral mass spectrometry is a primary ion beam source. The primary ion can be any suitable ion for generating sputtering from the sample under analysis. Examples of primary ion sources are: oxygen ( 16 O - , 16 O2 + , 16 O2 - ),argon( 40 Ar + ), xenon (Xe + ), SF5 + or C 60 + A duoplasmatron that generates primary ions; 133 Cs + Surface ionization source that generates primary ions; and Ga + A liquid metal ion gun (LMIG) generates primary ions. Other primary ions include cluster ions, such as Au. n + (n=1-5), Bi n q+ (n=1-7, q=1 and 12), C 60 q+ This includes probes (q=1-3) and large Ar clusters (Muramoto, Brison, & Castner, 2012).

[0296] The choice of ion source depends on the type of ion bombardment being deployed (e.g., static or dynamic) and the sample being analyzed. Static ion sources use low-order ion beam currents (1 nA / cm²). 2 Static imaging typically involves the use of pulsed ion beams. Due to the low current, each ion collides with a fresh portion of the sample surface, removing only a single layer (2 nm) of particles. Therefore, static imaging is suitable for imaging and surface analysis (Gamble & Anderton, 2016). Dynamic imaging requires a high primary ion beam current (10 mA / cm²). 2This typically involves using a continuous primary ion beam, which rapidly removes surface particles. As a result, dynamic SIMS can be used for depth profiling. Furthermore, because a relatively large amount of material is removed from the sample surface, dynamic SIMS exhibits a better detection limit than static SIMS. Dynamic SIMS typically results in high image resolution (less than 100 nm) (Vickerman & Briggs, 2013).

[0297] Oxygen primary ions enhance the ionization of electronegatrates (Malherbe, Penen, Isaure, & Frank, 2016) and are used in the commercially available Cameca IMS 1280-HR, while cesium primary ions are used to investigate electronegatrates (Kiss, 2012) and are used in the commercially available Cameca NanoSIMS 50.

[0298] To rapidly analyze a sample, high-frequency sputtering, such as above 200 Hz (i.e., more than 200 packets of ions per second are introduced to the sample), is required. Generally, the frequency of primary ion pulse generation by the primary ion source is at least 400 Hz, for example, at least 500 Hz or at least 1 kHz. For example, in some embodiments, the frequency of the ion pulse is at least 10 kHz, at least 100 kHz, at least 1 MHz or at least 10 MHz. For example, the frequency of the ion pulse is in the range of 400 to 100 MHz, 1 kHz to 100 MHz, 10 kHz to 100 MHz, 100 kHz to 100 MHz or 1 MHz to 100 MHz.

[0299] Therefore, the present invention provides an apparatus in which the charged particle source is a primary ion beam.

[0300] electron beam Alternatively, in some embodiments of the present invention, the charged particle source is an electron beam. An electron beam having an energy of 2kV to 30kV may be particularly suitable for investigating a specimen having a thickness of 30nm.

[0301] A high-intensity pulsed electron beam is used to induce ablation / sputtering. If the pulsed electron current is insufficient for ablation, its effect can be used as an ignition event as described above, after which energy pumping can be performed with a laser pulse set to a brightness level below the level of ablation of the natural material but exceeding the level of energy pumping required for ablation of the already activated material. In the energy activation mode of ablation / sputtering, the electron energy can be reduced because the electrons function solely to inject charge carriers into other insulating materials.

[0302] In certain embodiments, an electron beam can be focused to a smaller spot (with a higher resolution than a laser beam). A relatively low-resolution laser can ablate and / or ignite a sample only at the spot emitted by the electron beam, even if the electron beam collides with the sample beyond the electron beam spot. For example, an electron beam can be focused to a spot of 200 nm, 100 nm, 50 nm, 30 nm, or less than 10 nm, and a laser can be focused to a spot of 200 nm, 300 nm, 500 nm, 800 nm, 1 μm, 2 μm or more that overlaps with the electron beam spot, or focused on the ablation plume emission by the electron beam.

[0303] To rapidly analyze a sample, high-frequency sputtering, such as above 200 Hz (i.e., more than 200 packets of electrons per second are introduced into the sample), is required. Generally, the frequency of electron pulse generation by the electron source is at least 400 Hz, for example, at least 500 Hz or at least 1 kHz. For example, in some embodiments, the frequency of the electron pulse is at least 10 kHz, at least 100 kHz, at least 1 MHz or at least 10 MHz. For example, the frequency of the electron pulse is in the range of 400 to 100 MHz, 1 kHz to 100 MHz, 10 kHz to 100 MHz, 100 kHz to 100 MHz, or 1 MHz to 100 MHz.

[0304] An advantage of using an electron beam as a charged particle source is that the entire apparatus can be constructed on a platform that includes an electron microscope. Therefore, the present invention provides an apparatus further comprising an electron microscope. Accordingly, the present invention provides an apparatus in which the charged particle source is an electron beam and the electron beam is the electron source within the electron microscope.

[0305] Charged particle column A charged particle column guides charged particles into a sample. The charged particle column comprises a mass filter for filtering impurities in the charged particle beam, lenses and apertures as needed to control the intensity and shape of the primary ion beam, and deflection plates for shaping the primary ion beam and optionally rasterizing the charged particle beam across the entire surface of the sample (Villacob, 2016). Ion lenses and other components for constructing the charged particle column are commercially available, for example, from Agilent. Therefore, the charged particle column of the present invention can provide a charged particle beam scanning system adapted to scan the charged particle beam across multiple positions on a sample stage.

[0306] Typically, the charged particle beams used in secondary ion generation herein have a spot size (i.e., the size of the charged particle beam when it impacts the sample) of 100 μm or less, for example, 20 μm or less, 5 μm or less, 1 μm or less, or 500 nm or less, 300 nm or less, 200 nm or less, 100 nm or less, 50 nm or less, or 30 nm or less. The distance referred to as the spot size corresponds to the longest internal dimension of the ion beam, for example, a beam with a diameter of 2 μm for a circular beam, the length of the diagonal between opposite corners for a square beam, and the length of the longest diagonal for a quadrilateral. Beam shaping and beam masking can be used to provide the shape and size of the spot.

[0307] When used for the analysis of biological samples, the spot size of the ion beam used to analyze individual cells depends on the size and spacing of the cells. For example, if cells are densely packed together (e.g., within a tissue section), and single-cell analysis is performed, the charged particle beam may have a spot size no larger than these cells. This size depends on the specific cells in the sample, but generally, ion beam spots have a diameter of less than 4 μm, e.g., in the range of 0.1–4 μm, 0.25–3 μm, or 0.4–2 μm. Thus, charged particle beam spots can have a diameter of about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.5 μm, or less than 0.5 μm, e.g., about 400 nm or less, or about 300 nm or less. In certain embodiments, the spot size is about 200 nm or less, about 100 nm, or less than 100 nm. To analyze cells at intracellular resolution, the system uses a primary ion beam spot size no larger than these cells, and more specifically, a primary ion beam spot size that can ablate the material at intracellular resolution. In some cases, single-cell analysis can be performed using a spot size larger than the size of the cells, for example, if the cells are spread out on the slide and there are gaps between the cells. Therefore, the specific spot size used can be appropriately selected depending on the size of the cells being analyzed. In biological samples, it is rare for all cells to be the same size, so if intracellular resolution imaging is desired, the ion beam spot size must be smaller than the smallest cell, provided that a constant spot size is maintained throughout the sputtering procedure.

[0308] laser In this invention, a laser can be used to ablate the material or to assist in sputtering a material by irradiating the position after a charged particle pulse has passed toward the position on the sample (see, for example, Figures 7 and 8 and the accompanying description). Furthermore, a laser can be used to ionize a sputtered material that has been previously sputtered by a charged particle beam (see, for example, Figures 6 and 9 and the accompanying description). Furthermore, the laser can be used in combination with the above; see, for example, Figure 10 and the accompanying description.

[0309] As those skilled in the art will understand, the requirements for lasers to ablate / assist in sputtering materials differ from those for lasers to ionize materials. Generally, lasers to assist in sputtering or ablation materials provide energy on the order of μJ for pulse lengths of 300–400 fs or more. In contrast, lasers for ionizing sputtered materials are more powerful, providing energy on the order of 1 mJ or more for pulse lengths shorter than 100 fs. The apparatus of the present invention may use different lasers for the first and second laser sources, or it may use one laser for the first and second laser sources, and a beam splitter may be used to split the laser and provide the first and second laser sources.

[0310] A variety of different lasers can be used in SNMS, including commercially available lasers as described above in relation to the laser of a laser ablation sampling system appropriately adapted to enable sputtering of materials. Generally, the laser operates at the maximum achievable intensity to maximize ionization. Various types of lasers that can be used with the present invention are described below. The femtosecond lasers described above are also advantageous for certain SNMS applications. For example, in the embodiments of the present invention described above, which include first and second laser sources, a single femtosecond laser can be used to provide both laser sources. The laser may have a repetition rate of 1 MHz, a pulse width of 200 fs, and an energy pulse of 1 μJ. Those skilled in the art will understand that a beam splitter can be used to split the laser beam of the laser to provide the first and second laser sources of the present invention.

[0311] Generally, lasers useful for ionization include those that supply energy on the scale of a few microjoules per pulse, but can generate those pulses at high frequencies. The laser is used to generate elemental ions from a sample material. The generated elemental ions can then be analyzed by a mass spectrometer in the apparatus. The laser can be a picosecond laser, a femtosecond laser, or a nanosecond laser. In some embodiments, the laser is a femtosecond laser.

[0312] Femtosecond lasers can be solid-state lasers. Passive mode-locked solid-state bulk lasers can typically emit high-quality ultrashort pulses with durations of 30 fs to 30 ps. Examples of such lasers include diode-pumped lasers, such as those based on neodymium-doped or ytterbium-doped crystals. Titanium-sapphire lasers can be used with pulse durations of less than 10 fs, and in extreme cases up to about 5 fs (e.g., the Octavius ​​Ti:sapphire laser available from Thorlabs). Pulse repetition rates are most commonly between 1 kHz and 500 MHz.

[0313] Femtosecond lasers can be fiber lasers. Various types of ultrafast fiber lasers, which can also be passively mode-locked, typically offer pulse durations of 50–500 fs, repetition rates of 0.10–100 MHz, and average power outputs of a few milliwatts to a few watts (femtosecond fiber lasers are commercially available from Tooptica, IMRA America, and Coherent, Inc.).

[0314] Femtosecond lasers can be semiconductor lasers. Some mode-locked diode lasers can generate pulses with femtosecond durations. Directly at the laser output, pulse durations are typically at least several hundred femtoseconds, but much shorter pulse durations can be achieved through external pulse compression.

[0315] In some embodiments, the laser is a nanosecond laser. The nanosecond laser may be an excitation laser such as the Quantel Q-smart DPSS, Solar Laser LQ929 high-power pulsed Nd:YAG laser, or Litron high-energy pulsed Nd:YAG laser. All of these lasers can generate deep ultraviolet radiation with short pulse durations in the mJ region, which is suitable for ionization.

[0316] It is also possible to passively mode-lock vertical external cavity surface-emitting lasers (VECSELs). These are particularly interesting because they can offer a combination of short pulse duration, high pulse repetition rate, and potentially high average power output, but they are not suitable for high pulse energies.

[0317] In some embodiments, the laser is adapted to generate pulses with pulse durations on the nanosecond, picosecond, or femtosecond scale. For example, the laser may have durations of 500 fs or less, e.g., 400 fs or less, 300 fs or less, 200 fs or less, 100 fs or less, 50 fs or less, 45 fs or less, 25 fs or less, 20 fs or less, or 10 fs or less. A femtosecond laser is adapted to generate pulses with durations of less than 1 ps.

[0318] In some embodiments, the laser is adapted to have a pulse repetition rate of at least 100,000 Hz, for example, at least 1 MHz, at least 2 MHz, at least 3 MHz, at least 4 MHz, at least 5 MHz, at least 10 MHz, at least 20 MHz, at least 50 MHz, at least 100 MHz, at least 200 MHz, at least 500 MHz, or 1 GHz or higher.

[0319] In some embodiments, the laser has a beam width of 50 μm or less, 20 μm or less, 10 μm or less, or 5 μm or less (1 / e 2 The laser is adapted to have the following characteristics: The laser's focal point is where the beam's energy is most concentrated, and therefore where maximum ionization is achieved.

[0320] In some embodiments, the laser is adapted to have a pulse energy ranging from 1 nanojoule to a maximum of 50 millijoules. Lasers for assisting sputtering or ablation of materials can be adapted to have a pulse energy of 1 nanojoule to 100 microjoules, e.g., 10 nanojoules to 100 microjoules, 100 nanojoules to 10 microjoules, 500 nanojoules to 5 microjoules, e.g., about 1 microjoule, about 2 microjoules, about 3 microjoules, or about 4 microjoules. Lasers for post-ionization can be adapted to have a pulse energy of 1 millijoule to 50 millijoules, e.g., 5 millijoules to 40 millijoules, 10 millijoules to 30 millijoules, 20 millijoules to 35 millijoules, or about 25 millijoules or 35 millijoules.

[0321] In some embodiments, the laser is adapted to generate pulses having a pulse energy of about 1 microjoule, a pulse repetition rate of at least 10 MHz, and a duration of less than 100 fs, for example, 50 fs or less, 45 fs or less, 25 fs or less, 20 fs or less, or 10 fs or less.

[0322] As those skilled in the art will understand, the present invention can be used with one or a combination of the preferred lasers described herein. However, for completeness, some specific combinations of lasers that can be used with the present invention are as follows:

[0323] [Table 1]

[0324] Post-ionization is performed at 5 μm 3 The following methods require high energy density from laser radiation in small volumes. Because the post-ionization volume is extremely small, there is a limit to the amount of material that can be ionized at once. When a large amount of positive and negative charges are generated in a small volume, the motion of the formed ions is governed by a local field caused by the space charge induced by the ions and electrons. If too many charged ions are present in a small volume, the external field, for example, the field from the ion optics present in the mass spectrometer used to guide the resulting ions to the detector, is not effective in separating the positive and negative charges, and such an ion cloud eventually neutralizes, reducing ionization efficiency. For example, an ion cloud on the scale of 10 μm (diameter) containing 10,000 elemental charges generates a electrostatic potential of approximately 3 V. Since a few eV is the energy required to hold electrons in atoms, this can also be considered the energy level of free electrons after ionization. Consequently, the ion density on the scale of 10,000 ions in a 10-micrometer volume is close to the limit where space charge behavior begins to dominate.

[0325] Therefore, such effects can be avoided by ensuring that the amount of material being sputtered is kept at a moderately small level. For example, ablation of material on a scale from a 10 × 10 × 10 nm cube to a 30 × 30 × 30 nm cube or similar volume represents the maximum amount of material that can be transferred to a post-ionization region of a few micrometers in size without resulting in strong spatial changes and ion neutralization. Since the system can only process a 30 × 30 × 30 nm cube per single event, this creates an opportunity to perform imaging with a spatial resolution of 30 nm or 10 nm, which allows the ion beam to be focused (as described herein).

[0326] The post-ionization beam is co-aligned with the sputtering beam to within micrometer precision, as is commonly achieved in optical setups.

[0327] In some embodiments, the laser beam is guided at an angle to the sample to the ablated region ahead of the specimen (see Figure 6). This configuration minimizes interaction between the laser light and the unablated specimen. The laser beam can be focused to a narrow focal point that overlaps with the volume of the ablation plume. Focusing the laser beam at a high numerical aperture (NA) facilitates sharp focusing within the overlapping region and allows for rapid spreading of laser energy outside the overlapping region, minimizing the possibility of damage to the specimen in the region surrounding the sampled area.

[0328] Due to the constraints of the ion optics in Figure 6, which focus the primary beam and transport the secondary beam for mass spectrometry, the space for the laser beam optics may be limited in some embodiments. As a result, in some embodiments, the numerical aperture of the laser beam may be constrained to one of the planes. For example, the laser beam may have a low NA in the plane of the drawing and a high NA in a plane perpendicular to the plane of the drawing to avoid interference with the ion optics. Such an arrangement yields an elliptical focus extending in the plane of the low NA. An elliptical focus can improve the degree of overlap with the sputtered / ablated plume. Therefore, in some embodiments, the laser in the post-ionization system has an elliptical focus.

[0329] In addition to spatial adjustment, sputtering with a primary ion or electron beam must be synchronized with the delivery of laser radiation in order to ionize the sputtered material. The speed at which the sputtered material leaves the target is typically on the scale of sound, i.e., 1000 m / s. Therefore, a timing accuracy on the scale of 1 ns is required to reliably align the sputtered cloud and the laser beam with an accuracy of 1 micrometer. Bunching and timing of the ion beam down to 1 ns is usually achieved by an accelerating voltage of about 5-20 kV. The same or similar bunching techniques can be applied to the primary beam. This technique is used, for example, in current TOF MS instruments.

[0330] Therefore, in this operating mode, a primary ion beam or electron beam sputters the material from the sample, and immediately afterward, the emitted material is post-ionized by a laser radiation pulse.

[0331] To further facilitate constant sputtering / emission of material from the target, the target may, in some cases, be irradiated with a laser light pulse synchronized with a primary ion pulse (instead of or in addition to the post-ionization described above). The energy of the laser pulse should be set below the ablation threshold of the material not exposed to the primary ion or electron beam. In the region where the primary ion or electron beam interacts with the target, the state of matter is similar to a plasma and is limited to the nanoscale, hence it is referred to herein as nanoplasma. Thus, the light beam interacts with the nanoplasma, pumping additional energy into this volume and resulting in constant desorption of the material. This desorption mode can be seen as an ignition state (brought about by the primary ion or electron beam interacting with the target and generating nanoplasma) followed by an energy-pumping state in which the laser light pumps additional energy into the nanoplasma, facilitating its emission.

[0332] This operating mode can help overcome the limitations of relatively slow sputtering with primary ion or electron beams. The sputtering rate with a primary ion / electron beam is limited by the total number of primary ions / electrons colliding with a given region. The focusing ability of the primary ion / electron beam is limited by ion / electron current and space charge effects. This, coupled with the need to time the primary ion / electron beam to sputter only the material when the post-ionization laser is off, imposes limitations on the sputtering rate. Using laser radiation to enhance sputtering can overcome this limitation because fewer primary particles are needed to seed the sample material with the excitation energy required for the laser beam to affect sputtering / ablation.

[0333] If a laser is used for both post-ionization and sputtering enhancement, the same laser may also be suitable for pumping energy into a nanoplasma to facilitate material desorption. For example, a single femtosecond laser can satisfy the needs of both applications. The laser may have a repetition rate of 1 MHz, a pulse width of 200 fs, and an energy of 1 μJ per pulse (i.e., an average power of 1 W). Such laser pulses can be split (e.g., using a beam splitter), with some of them used directly for energy pumping into the nanoplasma. The remainder can be used for post-ionization. A compression step can be applied to the post-ionization beam to reduce the amount of energy per pulse required for post-ionization.

[0334] In some embodiments, laser-induced post-ionization is performed by pumping energy into a nanoplasma on a surface. Thus, the desorption pumping and post-ionization processes are combined into a single process activated by laser pulses delivered to a nanoplasma on a target.

[0335] Furthermore, energy pumping into the plasma on the target can be achieved by tuning the laser wavelength to the plasma's absorption band while avoiding the absorption band of the non-ionized target material. Thus, the collision of the primary ion beam / electron beam helps to change the state of the material within the target from a state that does not absorb the pumping light to a state that does. The duration of the pumping light can be on a picosecond scale, from a few nanoseconds to a few femtoseconds. At the same time, the picosecond pulse is short enough to avoid significant diffusion and broadening of the plasma volume within the sample.

[0336] In some embodiments, laser radiation for energy pumping is delivered via a sample carrier (see Figure 8). For example, while a primary electron beam or primary ion beam can be focused to a spot on the scale of 10–30 nm, the focusing of laser light may be limited by diffraction effects and remain on the scale of 100–1000 nm.

[0337] Delays can be introduced between laser pulses using devices known in the art. Therefore, in some embodiments, the device includes an optical delay line for introducing delays between laser pulses. An example of an optical delay line suitable for use in the present invention is one of the optical delay lines commercially available from ThorLabs.

[0338] By using a variable delay line, the arrival of the ionization pulse can be synchronized with the primary ion or electron packet, and therefore, depending on the relative timing between the primary ion or electron packet and the laser pulse, the same laser can be made to function in post-ionization or pumping mode.

[0339] Sample chamber The sample chamber of a sputtering-based sampling system shares many features with the sample chamber of the laser ablation-based sampling system described above. The sample chamber comprises a stage that supports the sample. The stage may be a translational stage movable within the xy or xyz axes. The sample chamber also includes an outlet through which the material removed from the sample by laser radiation can be guided. The outlet is connected to a detector, making it possible to analyze the sample ions.

[0340] In some cases, the sample chamber is maintained at a pressure of less than 133322 to 133.3 Pa, for example, 1333.22 to 133.322 Pa. In some cases, the sample chamber is maintained under vacuum. Therefore, in some cases, the sample chamber pressure is less than 50,000 Pa, less than 10,000 Pa, less than 5,000 Pa, less than 1,000 Pa, less than 500 Pa, less than 100 Pa, less than 10 Pa, less than 1 Pa, about 0.1 Pa, or less than 0.1 Pa, for example, 0.01 Pa or less. For example, the partial vacuum pressure may be about 50 to 2000 Pa, 100 to 1000 Pa or 200 to 700 Pa, and the vacuum pressure may be less than 10 Pa, 1 Pa or 0.2 Pa. Suitable gases include argon, helium, nitrogen and mixtures thereof.

[0341] As those skilled in the art will understand, the choice of whether the sample pressure is at atmospheric pressure, partial vacuum, or vacuum depends on the specific analysis being performed. For example, at atmospheric pressure, the sample is easier to handle and relatively gentle ionization can be applied. Furthermore, the presence of gas molecules may be desirable to allow the phenomenon of impact cooling to occur, which can be interesting when the label is a large molecule and its fragmentation, e.g., molecular fragments containing the labeled atom or combinations thereof, is undesirable. Alternatively, when post-ionizing a material using laser radiation, the presence of gas molecules (e.g., at partial vacuum) may be advantageous. For example, impact cooling can allow for the cooling of nanoplasma generated on the surface of the sample (e.g., after charged particle impact or laser irradiation of the sample to bring about an energy pumping state), the expansion of the plume of the ablated material, and pre-re-ionization within the post-ionization system. Impacts can also allow for at least partial charge reduction, reduction of space charge effects, and / or improvement of the ability of the ion optics to guide the ions. Reducing the charge state to one charge per ion makes it easier to read signals from various mass tags.

[0342] By maintaining the sample chamber under vacuum, collisions between the generated sample ions and other particles within the chamber can be prevented.

[0343] The main difference between the sample chamber of an SNMS system and a sample chamber of a laser ablation-based system is that the chamber is kept under vacuum to prevent collisions between sample ions and other particles in the chamber. This, conversely to laser ablation and desorption-based sample chambers, can result in charge loss from ions, on a similar basis. Loss of secondary ions reduces the sensitivity of the instrument.

[0344] Ion microscope Sample ions are captured from the sample via an electrostatic lens placed near the sample, known in this art as an immersion lens (or extraction lens). The immersion lens immediately removes secondary ions from the sample's locality. This is typically achieved by a sample and lens with a large potential difference. Depending on the polarity of the sample relative to the immersion lens, positive or negative secondary ions are captured by the immersion lens. The polarity of the sample ions captured by the immersion lens is independent of the polarity of the ions in the charged particle beam.

[0345] Next, the sample ions are transferred to the detector via one or more additional electrostatic lenses (known in the art as transfer lenses). The transfer lenses focus the beam of secondary ions onto the detector. Typically, in a system with multiple transfer lenses, only one transfer lens is involved in a given analysis. Each lens may provide a different magnification of the sample surface. Generally, additional ion manipulation components, such as one or more apertures, mass filters, or sets of deflector plates, are present between the immersion lens and the detector. The immersion lens, transfer lenses, and any additional components together form an ion microscope. Components for manufacturing ion microscopes are available from commercial suppliers such as Agilent.

[0346] camera The system may also be equipped with a camera. Camera systems have been described above in relation to laser ablation sampling systems, and the features of the camera described above may also be present in secondary ion generation systems, except in unsuitable cases (for example, it can be connected to an optical microscope such as a confocal microscope, but since one beam is ions and the other beam is photons, the primary ion beam cannot be focused through the same optical system as the light guided to the camera).

[0347] c.2 Sampling and ionization system based on pulsed laser Similar to the specific apparatus, systems, and techniques described in Section 1.b above, electron seeding to bring about a “sample ignition state” can be achieved at a certain location by a laser radiation pulse. Subsequently, since the energy required to ablate the pre-seeded location is lower than that of the surrounding area, a second laser pulse with a larger laser spot diameter than that location but with a fluence lower than the sample ablation threshold can be directed to the sample to induce ablation only at the location targeted by the first laser pulse. Thus, electrons can be seeded using a densely focused, low-energy first pulse, and ablation can be induced at the electron-seed location using a higher-energy, less focused pulse. Therefore, the present invention provides further additional means for high-resolution imaging based on this technique.

[0348] The optical behavior of optical systems is well known: the minimum spot size generated by an optical system is directly proportional to the wavelength of light and inversely proportional to the numerical aperture of the objective lens. Since the wavelength of visible light is approximately 500 nm and the numerical aperture of a typical objective lens rarely exceeds 1.0, the size of a focused spot using such light ultimately becomes less than 1 micrometer. Therefore, by operating with a relatively short-wavelength laser, light can be focused to a relatively small spot on the sample. For example, an excimer laser can generate VUV light with a wavelength of approximately 200 nm. Furthermore, modern lithography tools use EUV light with a wavelength of 13.5 nm. Unfortunately, generating VUV and EUV light pulses with sufficient energy for ablation requires large, complex, and expensive equipment.

[0349] This invention solves the problem of reduced ablation spots by pre-seeding spots to be ablated with a small amount of UV, EUV, or XUV light. This pre-seeding generates free electrons in the other dielectric material. This change in material properties lowers the ablation threshold by subsequent pulses. The amount of energy required for pre-seeding is orders of magnitude less than the amount of energy required for direct ablation. This makes the equipment for generating such pulses relatively inexpensive. Because the pre-seeding pulses are short wavelength, they can be focused to a diameter of 100 or 30 nm. Immediately after pre-seeding, a pulse with a more general wavelength, such as IR or visible light, is applied to the same sample. This allows energy to accumulate much more efficiently in the pre-seeded region. As a result, the pre-seeded region can be ablated, while the remaining region to which the infrared light has spread is not ablated. Thus, the size of the ablation spots is controlled by the diffraction limit imposed by the UV, VUV, or EUV wavelengths. This limit is substantially lower than the ablation resolution of the visible or infrared light applied to it itself.

[0350] Furthermore, the present invention can result in the ionization of the ablated material. The ionic composition of the ablated material can be analyzed to provide imaging mass spectrometry or imaging mass cytometry workflows. The material to be ablated is ionized in a plasma, generating a significant portion of its elemental ions. The efficiency of ionization and ion sampling from the plasma can be high enough to facilitate single-copy detection of antibodies when using MaxPar reagents or similar reagents. Here, ions can be directly extracted for analysis via ion microscopy, as described below.

[0351] When operating at such high spatial resolution (e.g., 30 nm), several analytical problems arise. One problem is the total acquisition rate versus the field of view (or region of interest) size. Existing Hyperion systems operate at 200 pixels / s with a pixel size of 1 micrometer. As a result, it takes approximately 5000 seconds to record an image of a biologically relevant ROI on a 1 × 1 mm scale. If the pixel size is reduced to 1 / 30th (to approximately 30 nm), the amount of time required to record an image from a similar area increases to 500,000 s. This is clearly an impractically long time. This invention solves this problem by operating ionization sampling in at least a partial vacuum (see the description of the sample chamber below for details), in which case the pixel rate can be as high as 1 M pixels / s, meaning that a 1 × 1 mm area can be acquired in just 100 seconds or less. Multiple serial sections can be analyzed to provide a 3D image on a biological scale of approximately 0.03 mm. This requires 1000 sections (each 30 nm) and approximately one day of experimentation. This is extremely fast considering the multi-parametric, high spatial resolution nature of the acquired data. Increasing the acquisition speed to 10 M pixels / s makes it possible to collect such images in under 2.5 hours. Sample analysis is also approximately 30 times faster at a spatial resolution of 100 nm.

[0352] As mentioned above, using MaxPar reagents (mass-tagged antibody reagents as described below) allows for the compensation of sensitivity loss due to smaller ablation by having approximately 100 atoms per copy of the antibody. Ion sampling and transmission of more than 5% enable the detection of a single copy of the antibody. When imaging objects with a pixel size of 30 nm, only a very small number of antibodies may be found in such pixels. Therefore, in imaging at such a small scale, enabling single-copy detection becomes essential.

[0353] The plasma generated by the two-pulse technology of the present invention can solve the problems of efficient ionization and sampling. A smaller ablation scale results in a smaller plasma scale, which in turn reduces the possibility of plasma neutralization during ion sampling. Furthermore, the density of the solid material being ablated leads to an initial plasma pressure on the scale of 10,000 atm, which then leads to a local thermal equilibrium model of the plasma. Local thermal equilibrium enables the generation of an optimal plasma temperature, promoting nearly 100% efficient ionization of labeled atoms from mass tags, and forming elemental ions that can be controlled via the parameters of the second pulse.

[0354] An apparatus based on a two-pulse laser ablation system typically comprises three components. The first component is a first laser source for seeding electrons into the sample (which is housed on the sample stage in the apparatus and system disclosed herein). The second component is a second laser source for ablating the sample seeded by the first laser source. (Together these two components form a two-pulse laser sampling and ionization system.) The third component is a detector component for detecting the ionized material, e.g., a mass detector. The laser sources are typically pulsed. In some cases, the first (pre-seeding) pulse can be derived from the same laser that delivers the second (ablation) pulse, for example, by harmonic generation. The reference in this section to directing the laser beam toward a position on the sample stage refers to the arrangement of components in the apparatus and system, but as will be understood by those skilled in the art, when the apparatus / system is used to analyze a sample, the laser radiation from the first and second laser sources strikes the sample being analyzed.

[0355] Therefore, the present invention is Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a sample stage.

[0356] The apparatus typically includes a mass detector, such as a TOF detector.

[0357] Therefore, the present invention is Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, The present invention provides a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, comprising a second laser source configured to ablate a sample material from which electrons have been pre-seed by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a sample stage.

[0358] In some embodiments, a first focusing optical system configured to guide a laser beam emitted by a first laser source toward a sample stage is configured to guide the laser beam emitted by the first laser source toward a position on the sample stage, and a second focusing optical system configured to guide a laser beam emitted by a second laser source toward a sample stage is configured to guide the laser beam emitted by the second laser source toward a position on the sample stage.

[0359] For example, the present invention is Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a position on the sample stage, The present invention provides an apparatus for analyzing samples such as biological samples, comprising a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a position on the sample stage.

[0360] The apparatus typically includes a mass detector, such as a TOF detector.

[0361] For example, the present invention is Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a position on the sample stage, The present invention provides a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, comprising a second laser source configured to ablate a sample material from which electrons have been pre-seed by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a position on the sample stage.

[0362] Figure 13 is a schematic diagram of an exemplary embodiment of the present invention showing a reduction in ablation spot size by two-pulse ablation. A support target 20 holds a thin section of specimen 30. In some embodiments (e.g., nanomachining applications), the support target and specimen may be a single body. A pulse of UV, or VUV, EUV, or XUV light 40, is focused onto the specimen 30 by an objective lens 60 (50). Special objective lenses for UV, VUV, and EUV optics are known in the art and are often based on reflective optics configurations. For example, a pulse of EUV light generates a seed of free electrons at the focal point. Since specimens are generally nonconductive, pre-seeding free electrons alters the properties of the material at the laser focal point. A second pulse of light 70 is delivered to the same region including the location where the electrons were pre-seeded, providing energy to generate plasma at the pre-seeded location. The second pulse of light 70 is delivered by another objective lens 80. Because the wavelength of the second pulse is significantly longer, the focus of the second pulse (90) is much larger than that of the first pulse. The process by which electrons multiply in the sample during the second pulse can be called avalanche ionization. In practice, many phenomena can contribute to the preferential excitation of the pre-seeded region. As long as the energy required for preferential excitation is substantially lower than the threshold for direct ablation, a scheme that defines the ablation zone by pre-seeding using, for example, EUV pulses is effective.

[0363] Accordingly, the present invention provides an apparatus and system for analyzing a sample such as a biological sample, wherein the second focusing optical system is configured to synchronize a second laser radiation pulse from a second laser source with a second laser radiation pulse from a second laser source so that it reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source.

[0364] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a sample stage. The present invention provides a device configured to synchronize a second laser radiation pulse from a second laser source with a second laser radiation pulse from a second laser source so that the second laser radiation pulse from the second laser source reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source. The apparatus typically includes a mass detector, such as a TOF detector.

[0365] Therefore, the present invention relates to a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a sample stage. The present invention provides a two-pulse laser sampling and ionization system configured such that a second focusing optical system synchronizes a second laser radiation pulse from a second laser source with the laser radiation pulse from the second laser source so that it reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source. In some embodiments, a first focusing optical system configured to guide a laser beam emitted by a first laser source toward a sample stage is configured to guide the laser beam emitted by the first laser source toward a position on the sample stage, and a second focusing optical system configured to guide a laser beam emitted by a second laser source toward a sample stage is configured to guide the laser beam emitted by the second laser source toward a position on the sample stage.

[0366] Accordingly, the present invention provides an apparatus and system for analyzing a sample such as a biological sample, wherein a first focusing optical system and a second focusing optical system are configured to guide laser radiation to the opposite side of the sample stage.

[0367] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a first surface of a sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a second surface of the sample stage. The present invention provides a device configured to synchronize a second laser radiation pulse from a second laser source with a second laser radiation pulse from a second laser source such that the second focusing optical system reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source.

[0368] The apparatus typically includes a mass detector, such as a TOF detector.

[0369] Therefore, the present invention relates to a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a first surface of a sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a second surface of the sample stage. The present invention provides a two-pulse laser sampling and ionization system configured to synchronize a second laser radiation pulse from a second laser source with a second laser radiation pulse from a second laser source so that the second focusing optical system reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source.

[0370] The present invention also relates to an apparatus for analyzing samples such as biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A first laser source configured to seed electrons in the sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a second surface of the sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of the sample stage. The present invention provides a device configured to synchronize a second laser radiation pulse from a second laser source with a second laser radiation pulse from a second laser source such that the second focusing optical system reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source.

[0371] The apparatus typically includes a mass detector, such as a TOF detector.

[0372] Therefore, the present invention relates to a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A first laser source configured to seed electrons in the sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a second surface of the sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of the sample stage. The present invention provides a two-pulse laser sampling and ionization system configured to synchronize a second laser radiation pulse from a second laser source with a second laser radiation pulse from a second laser source so that the second focusing optical system reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source.

[0373] Figure 14 shows another preferred embodiment of the present invention, where the first pulse 40 and the second pulse 70 are focused onto the sample from the same side. A single objective lens 60 is used to combine and focus the two light pulses. Thus, the present invention provides an apparatus and system for analyzing a sample such as a biological sample, wherein the first and second focusing optics are configured to guide the laser radiation to the opposite side of the sample stage.

[0374] Therefore, the present invention relates to an apparatus for analyzing samples such as biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a first surface of a sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of the sample stage. The second focusing optical system is configured to synchronize the second laser radiation pulse from the second laser source with the second laser radiation pulse from the second laser source so that it reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source. The first and second focusing optical systems provide a device that focuses radiation from first and second laser sources using the same objective lens.

[0375] The apparatus typically includes a mass detector, such as a TOF detector.

[0376] Therefore, the present invention relates to a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a sample, A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward a first surface of a sample stage, The system comprises a second laser source configured to ablate a sample material that has been pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a first surface of the sample stage. The second focusing optical system is configured to synchronize the second laser radiation pulse from the second laser source with the second laser radiation pulse from the second laser source so that it reaches a position on the sample stage immediately after the laser radiation pulse from the first laser source. A first focusing optical system and a second focusing optical system provide a two-pulse laser sampling and ionization system that focuses radiation from first and second laser sources using the same objective lens.

[0377] Figure 15 shows yet another embodiment in which the sample (30) is placed on a material that is at least semi-transparent to a first pulse and even more transparent to a second pulse. This material may be in the form of a metal mesh used in electron microscopy. This shape causes the ablated plasma to expand into a cloud 100 of ions, electrons, and neutral material, where the ions can be sampled within a mass spectrometer for imaging mass spectrometry and imaging mass cytometry applications.

[0378] Accordingly, in some embodiments of the two-pulse laser-based apparatus and sampling and ionization systems disclosed herein, the sample stage is at least partially composed of a material that is at least semi-transparent to laser radiation from a first laser source and even more transparent to radiation from a second laser source. Alternatively, if laser radiation is directed to reach a sample on a first surface via the sample stage, as described elsewhere herein and repeated here for completeness, the sample stage should include a gap through which the laser radiation can pass (through the sample carrier) to reach the sample. In situations where the sample stage has a gap, the sample is placed on a sample carrier that is at least partially composed of a material that is at least semi-transparent to radiation from a first laser source and even more transparent to radiation from a second laser source.

[0379] Figure 16 shows another embodiment that facilitates efficient sampling of ablated ions. Here, a first laser pulse 40 is delivered from the first side. The first laser pulse is formed by an objective lens 60 having a central aperture to allow the passage of ions 100 generated after ablation.

[0380] Accordingly, in some embodiments of the two-pulse laser-based apparatus and sampling and ionization systems disclosed herein, the objective lens of the first focusing optical system has a central aperture to allow ionized material from the sample to pass through the aperture.

[0381] Components of the 2-Laser Sampling and Ionization System First laser source - laser for pre-seeding electrons As described above, the requirement for achieving high-resolution imaging is the wavelength of the laser radiation that can be focused to a focal point with a diameter of 100 nm or less. Therefore, the wavelength of the laser radiation emitted by the first laser source must be UV light. In some embodiments, the laser radiation emitted by the first laser source is UV, VUV, EUV, or XUV. In some embodiments, the laser radiation emitted by the first laser source is 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, 50 nm or less, 25 nm or less, 20 nm or less, or 15 nm. For example, in some embodiments, the laser radiation emitted by the first laser source is 10-200 nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, 10-50 nm, 10-25 nm, 10-20 nm, or 10-15 nm.

[0382] As described above, the pulse energy of the first laser source is selected so that it seeds electrons into the material but does not ablate the material. In some embodiments, the pulse energy of the first laser source is in the range of picoJ to femtoJ. For example, 1 picoJ to 100 femtoJ, 10 picoJ to 100 femtoJ, or 100 picoJ to 1 femtoJ.

[0383] The pulse duration must be short enough to minimize the diffusion of electrons seeded before the second laser pulse that causes ablation of the sample. The total time between the start of the first pulse (from the first laser source) and the end of the second pulse (from the second laser source) should be kept to less than 10 ps. Therefore, in some embodiments, the pulse duration from the first laser source is 5 ps or less, for example, 2 ps or less, 1 ps or less, 500 fs or less, 400 fs or less, 300 fs or less, 200 fs or less, or 100 fs or less, 50 fs or less, 40 fs or less, 30 fs or less, 20 fs or less, or 10 fs or less.

[0384] First focusing optical system The first focusing optical system guides the radiation from the first laser source onto the sample and focuses it onto the sample. To achieve high-resolution imaging, the first focusing optical system must focus the laser radiation to a spot size of approximately 100 nm or less on the sample, for example, 75 nm or less, 50 nm or less, or approximately 30 nm.

[0385] In some embodiments, the objective lens of the first focusing optical system is a reflective objective lens.

[0386] Second laser source - laser for ablation of pre-seeded sample locations The requirements for the second laser source in the system differ due to its different function. Laser radiation pulses from the second laser source provide energy to generate plasma in the sample and control its temperature.

[0387] Therefore, the wavelength of the laser radiation emitted by the second laser source can be IR or visible light. When visible light is emitted from the second laser source, the visible light can be focused more densely, thus reducing the energy per pulse required for ablation. In some embodiments, the laser radiation emitted by the second laser source is 400 nm or greater, 500 nm or greater, 600 nm or greater, 700 nm or greater, 800 nm or greater, or 1 μm or greater. For example, in some embodiments, the laser radiation emitted by the second laser source is 400 nm to 100 μm, e.g., 200 nm to 100 μm, 200 nm to 10 μm, 200 nm to 1 μm, 400 nm to 10 μm, 400 nm to 1 μm, 400 nm to 900 nm, 400 nm to 800 nm, 400 nm to 700 nm, 400 nm to 600 nm, or 500 nm to 600 nm.

[0388] In accordance with the above description, the pulse energy of the second laser source is selected so that only the sample material with pre-seed electrons is ablated within the laser spot, i.e., the pulse fluence is below the material ablation threshold. In some embodiments, the pulse energy of the second laser source is in the nanojoule range. For example, 1 nanoJ to 1 μJ, 10 nanoJ to 500 nanoJ, 50 nanoJ to 250 nanoJ, or about 100 nanoJ.

[0389] The pulse duration must be short enough to minimize the diffusion of electrons seeded before the second laser pulse that causes ablation of the sample. The total time between the start of the first pulse (from the first laser source) and the end of the second pulse (from the second laser source) should be kept to less than 10 ps. Therefore, in some embodiments, the pulse duration from the second laser source is 5 ps or less, for example, 2 ps or less, 1 ps or less, 500 fs or less, 400 fs or less, 300 fs or less, 200 fs or less, or 100 fs or less, 50 fs or less, 40 fs or less, 30 fs or less, 20 fs or less, or 10 fs or less.

[0390] Second focusing optical system The second focusing optical system directs radiation from the second laser source onto the sample and focuses it onto the sample. High-resolution imaging is achieved by the density of the focusing of the first short-wavelength pulse, and therefore the spot size may be larger than that of the first pulse. Nevertheless, a relatively small spot size minimizes irradiation of the sample around the seeded position. Therefore, in some embodiments, the second focusing optical system focuses the laser radiation to a spot size of about 2 μm or less on the sample, for example, 1 μm or less, 750 nm or less, 500 nm, 250 nm or less, 200 nm or less, 150 nm or less, or about 100 nm.

[0391] In some embodiments, the objective lens of the second focusing optical system is a refractive or reflective objective lens, such as a lens having an NA greater than 0.7, greater than 0.8, or greater than 0.9. In some embodiments, the objective lens of the second focusing optical system is also the objective lens of the first focusing optical system, as described above.

[0392] Synchronization of the first and second laser sources The apparatus of the present invention may use different lasers for the first and second laser sources. Here, the delivery of a first pulse from the first laser source and a second pulse from the second laser source to the sample can be coordinated using techniques commonly used in the art, such as a programmed module with instructions, resulting in pre-seeding and ablation of the pre-seeded region as described above. In some embodiments, the first and second pulses are synchronized such that the elapsed time from the start of the pulse from the first laser source to the end of the pulse from the second laser source is less than 50 ps, ​​for example less than 25 ps. In some embodiments, the time is less than 10 ps, ​​for example less than 5 ps, less than 2 ps or less than 1 ps. In some embodiments, the apparatus and system use one laser for the first and second laser sources, and a beam splitter is used to split the laser and provide the first and second laser sources.

[0393] For example, a single laser engine can be used to generate IR pulses. These pulses can be converted to visible light by a second or third harmonic generator. To generate short-wavelength radiation for the first pulse, the laser can be coupled to a higher harmonic generation stage. Optical delay lines can be used to control the time separation between the first and second pulses, and between the second and third pulses.

[0394] Accordingly, in some embodiments, an apparatus or sampling system comprising a first laser source and a second laser source comprises a single laser, a beam splitter, and two harmonic generators, one of which is adapted to generate UV, VUV, EUV, or XUV laser radiation (as described above for the first laser source) from the single laser as the first laser source, and the other harmonic generator is adapted to generate IR or visible wavelength laser radiation (as described above for the second laser source) from the single laser as the second laser source.

[0395] Accordingly, in some embodiments, an apparatus or sampling system comprising a first laser source and a second laser source comprises a single laser, a beam splitter, two harmonic generators, and an optical delay line, wherein one of the harmonic generators is adapted to generate UV, VUV, EUV, or XUV laser radiation (as described above for the first laser source) from the single laser as the first laser source, and the other harmonic generator is adapted to generate IR or visible wavelength laser radiation (as described above for the second laser source) from the single laser as the second laser source, and the optical delay line is adapted to deliver a pulse from the second laser source, derived from a pulse of the single laser, after a pulse from the first laser source, derived from the same pulse of the single laser, so that the end of the second pulse is less than 50 ps after the start of the first pulse, e.g., less than 25 ps, less than 10 ps, ​​e.g., less than 5 ps, less than 2 ps, or less than 1 ps.

[0396] In some cases, the output of a harmonic generator adapted to produce UV, VUV, EUV, or XUV laser radiation can be filtered to include a range of desired wavelengths in order to pre-seed the region to be ablated.

[0397] Magnetic sector instruments appear to be relatively suitable for high-speed recording of 1 megapixel or more. For recording speeds of 100,000 kpixels / s, time-of-flight mass spectrometers are suitable and can be easily designed and constructed.

[0398] Because this type of ion source generates a very dense ion spot, the characteristics of the ion beam in this technology make the ion beam suitable for many types of mass spectrometers (currently known and planned for invention).

[0399] General considerations regarding lasers Femtosecond lasers can be solid-state lasers. Passive mode-locked solid-state bulk lasers can typically emit high-quality ultrashort pulses with durations of 30 fs to 30 ps. Examples of such lasers include diode-pumped lasers, such as those based on neodymium-doped or ytterbium-doped crystals. Titanium-sapphire lasers can be used with pulse durations of less than 10 fs, and in extreme cases up to about 5 fs (e.g., the Octavius ​​Ti:sapphire laser available from Thorlabs). Pulse repetition rates are most commonly between 1 kHz and 500 MHz.

[0400] Femtosecond lasers can be fiber lasers. Various types of ultrafast fiber lasers, which can also be passively mode-locked, typically offer pulse durations of 50–500 fs, repetition rates of 0.10–100 MHz, and average power outputs of a few milliwatts to a few watts (femtosecond fiber lasers are commercially available from Topica, IMRA America, and Coherent, Inc.). Femtosecond fiber lasers are particularly well-suited for this application. When the energy output is <1 uJ, the laser price is affordable. The laser pulse repetition rate is >1 MHz, which can result in acquisition speeds of 1 M pixels / s. The pulse duration of approximately 200 fs for such lasers is well within the 10 ps upper limit determined by the diffusive spreading of the plasma region.

[0401] Femtosecond lasers can be semiconductor lasers. Some mode-locked diode lasers can generate pulses with femtosecond durations. Directly at the laser output, pulse durations are typically at least several hundred femtoseconds, but much shorter pulse durations can be achieved through external pulse compression.

[0402] In some embodiments, the laser is a nanosecond laser. The nanosecond laser may be an excitation laser such as the Quantel Q-smart DPSS, Solar Laser LQ929 high-power pulsed Nd:YAG laser, or Litron high-energy pulsed Nd:YAG laser. All of these lasers can generate deep ultraviolet radiation with short pulse durations in the mJ region, which is suitable for ionization.

[0403] It is also possible to passively mode-lock vertical external cavity surface-emitting lasers (VECSELs). These are particularly interesting because they can offer a combination of short pulse duration, high pulse repetition rate, and potentially high average power output, but they are not suitable for high pulse energies.

[0404] Post-ionization in sampling systems and apparatus based on two-pulse laser ablation Figure 17 shows an embodiment in which three laser pulses are used to provide ablation and post-ionization. Here, the first two pulses 40 and 70 are used to ablate the material on a small scale, similar to the embodiment in Figure 13. A third pulse 110 is delivered to overlap with the plume of ablated material. This is used to provide additional ionization to this material. This step may be necessary to suppress the effects of neutralization that may occur in the ablated plume. The ions are then extracted through the aperture (not shown) of the objective lens 60.

[0405] Post-ionization is performed at 5 μm 3The following methods require high energy density from laser radiation in small volumes. Because the post-ionization volume is extremely small, there is a limit to the amount of material that can be ionized at once. When a large amount of positive and negative charges are generated in a small volume, the motion of the formed ions is governed by a local field caused by the space charge induced by the ions and electrons. If too many charged ions are present in a small volume, the external field, for example, the field from the ion optics present in the mass spectrometer used to guide the resulting ions to the detector, is not effective in separating the positive and negative charges, and such an ion cloud eventually neutralizes, reducing ionization efficiency. For example, an ion cloud on the scale of 10 μm (diameter) containing 10,000 elemental charges generates a electrostatic potential of approximately 3 V. Since a few eV is the energy required to hold electrons in atoms, this can also be considered the energy level of free electrons after ionization. Consequently, the ion density on the scale of 10,000 ions in a 10-micrometer volume is close to the limit where space charge behavior begins to dominate.

[0406] Therefore, such effects can be avoided by ensuring that the amount of material being ablated is kept at a moderately small level. For example, ablating material from a 10×10×10 nm cube to a 30×30×30 nm cube or a similar volume scale represents the maximum amount of material that can be transferred to a post-ionization region of a few micrometers in size without resulting in strong spatial changes and ion neutralization. Since the system can only process 30×30×30 nm cubes per single event, this creates an opportunity to perform imaging with a spatial resolution of 30 nm or 10 nm, which allows the ion beam to be focused (as described herein).

[0407] The post-ionization laser beam / radiation is co-aligned with the beam of the second laser source to within micrometer precision, as is commonly achieved in optical settings.

[0408] In some embodiments, the post-ionization laser beam / radiation is guided at an angle to the sample to an ablated region ahead of the specimen (arrangement of the charged particle-based sampling and ionization system in Figure 6). This configuration minimizes interaction between the laser radiation for post-ionization and the unablated specimen. The laser beam can be focused to a narrow focal point that overlaps with the volume of the ablation plume. Focusing the laser beam at a high numerical aperture (NA) facilitates sharp focusing within the overlapping region and allows for rapid spreading of laser energy outside the overlapping region, minimizing the possibility of damage to the specimen in the region surrounding the sampled area.

[0409] In some embodiments, the numerical aperture of the post-ionization laser beam / radiation may be constrained to one of the planes. Such an arrangement yields an elliptical focus extending in a plane of low NA. An elliptical focus can improve the degree of overlap with the sputtered / ablated plume. Therefore, in some embodiments, the laser of the post-ionization system has an elliptical focus.

[0410] In addition to spatial adjustment, the plume generation resulting from the combined action of the first and second laser sources described above must be synchronized with the delivery of the laser radiation in order to ionize the ablated material. The velocity at which the ablated material can leave the target is on the scale of the speed of sound, i.e., 1000 m / s. Therefore, a timing accuracy on the scale of 1 ns is required to reliably align the ablated plume and the post-ionization laser radiation / beam with an accuracy of 1 micrometer. In certain embodiments, the velocity can be 1 km / s to 10 km / s, for example, 2 km / s to 5 km / s, depending on the temperature and composition of the ablated material. Ablation can be at atmospheric pressure, partial vacuum, or vacuum, as described herein.

[0411] Therefore, in this operating mode, the first and second laser sources work together to ablate the material from the sample, and immediately thereafter, the emitted material is post-ionized by laser radiation pulses from the third laser source.

[0412] The nanoplasma generated by the action of the first and second lasers contains elemental ions, but these ions are not directly extracted when post-ionization is used. Rather, the emitted plume can expand, during which at least some charge neutralization occurs (because the plasma at that point is under high pressure and very high density, which means that impact cooling and charge reduction occur very rapidly). As mentioned above, the expansion rate of the nanoplasma plume is approximately 1000-5000 m / s. Therefore, after a few picoseconds, the plasma generated from the ablation of, for example, a 10 nm diameter spot on the sample will expand by only an order of magnitude, for example, 100 nm. 3 It expands to a volume of 10 to 100 times the original nanoplasma volume. Adding or re-ionizing the plume after this dimensional expansion means that the components of the original plume are now significantly more spread out, and as a result, when ionizing and restoring the micrometer-scale plasma, the likelihood of collisions is much lower (and therefore the likelihood of charge neutralization is lower), and consequently, a relatively high proportion of elemental ions can be extracted from the plume. Further improved sensitivity can be achieved by even more efficient extraction of ions, including elemental ions derived from labeled atoms.

[0413] Therefore, in some embodiments, the sampling and ablation system and apparatus based on a two-pulse laser comprises a third laser source configured to ionize the plume of sample material ablated from the sample.

[0414] Therefore, the present invention relates to a two-pulse laser sampling and ionization system for analyzing samples such as biological samples, Sample stage, A first laser source configured to seed electrons in the sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward the sample stage. A second laser source configured to ablate a sample material pre-seed with electrons by a first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward a sample stage. The system comprises a third laser source configured to ionize a plume of sample material ablated from the sample, and a third focusing optical system configured to guide the laser beam emitted by the third laser source to a volume in which the ablated sample material forms a plume. The second focusing optical system is configured to synchronize the second laser radiation pulse from the second laser source so that it reaches a position on the sample stage immediately after the first laser radiation pulse from the first laser source. The present invention provides a two-pulse laser sampling and ionization system configured such that a third focusing optical...

Claims

1. A device for analyzing biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a biological sample, Laser source and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from the laser source toward the second surface to a position on the sample stage, The apparatus further comprises an immersion medium disposed between the objective lens and the second surface of the sample stage.

2. The apparatus according to claim 1, wherein the immersion medium has a refractive index of 1.00 or higher, for example, 1.33 or higher, 1.50 or higher, 2.00 or higher, or 2.50 or higher.

3. The apparatus according to claim 1 or claim 2, wherein the immersion medium is a liquid immersion medium, and optionally the liquid immersion medium is oil or water.

4. The apparatus according to claim 1 or claim 2, wherein the immersion medium is a solid immersion medium, and optionally the solid immersion medium is S-LAH79, diamond, or fused silica.

5. The apparatus according to claim 4, wherein the solid immersion medium is a hemispherical solid immersion lens or a Weierstrass solid immersion lens.

6. The apparatus according to any one of claims 1 to 5, wherein the laser source is a femtosecond laser, a picosecond laser, or a nanosecond laser.

7. The apparatus according to any one of claims 1 to 6, wherein the laser source is a UV laser, and optionally the laser emits a radiation beam with a wavelength of 193 nm, 213 nm, or 266 nm.

8. The apparatus according to any one of claims 1 to 7, wherein the laser is a near-infrared laser and emits a radiation beam with a wavelength of 700 to 1300 nm.

9. The apparatus according to any one of claims 1 to 8, wherein the laser generates an ablation spot size of 100 nm or less at the position on the sample stage.

10. An apparatus according to any one of claims 1 to 9, comprising a biological sample, wherein during operation, the biological sample is placed on the first surface of the sample stage, the biological sample is placed on the side of the sample stage opposite to the immersion medium, and optionally the biological sample is placed on a sample carrier.

11. The apparatus according to claim 10, wherein the sample has a thickness of 100 micrometers or less, for example, 10 micrometers or less, 5 micrometers or less, 2 micrometers or less, or 1 micrometer or less, or 100 nm or less, or 50 nm or less, or 30 nm or less.

12. The apparatus according to any one of claims 1 to 11, comprising a detector for detecting sample ions.

13. The apparatus according to any one of claims 1 to 12, comprising an ionization system.

14. The apparatus according to any one of claims 1 to 13, further comprising an electron beam source, wherein the electron beam source is for electron microscopy examination of the biological sample.

15. The apparatus according to claim 14, further comprising an electron microscope if it is dependent on claim 13.

16. a) Staining the sample with a contrast agent for electron microscopy, b) Labeling the sample with labeled atoms, A method for preparing a biological sample for analysis, including [a specific component].

17. A method according to claim 16, comprising first sectioning the sample into thin sections, wherein the sample is optionally sectioned into sections with a thickness of less than 10 micrometers, for example, 1 micrometer or less, or 100 nm or less, or 50 nm or less, or 30 nm or less.

18. The method according to claim 16 or claim 17, wherein the contrast agent comprises at least one of osmium tetroxide, gold, silver, and iridium.

19. The method according to any one of claims 16 to 18, wherein the labeled atom comprises at least one of a gold-labeled antibody and a lanthanide-labeled antibody.

20. The process involves guiding a radiation beam emitted from a laser source towards a specific location on the sample to generate an ablated plume of the sample material. Ionizing the ablated plume of the sample material, and To detect sample ions from the aforementioned sample material, A method for analyzing biological samples, including [specific components / synthesis].

21. The method according to claim 20, comprising first performing an electron microscope examination.

22. The method according to claim 20 or claim 21, wherein the biological sample is prepared according to the method described in any one of claims 16 to 19.

23. The method according to any one of claims 20 to 22, using the apparatus according to any one of claims 1 to 15.

24. Sample stage and A charged particle source and a charged particle column for passing a charged particle beam to a position on the sample stage, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward the sample stage, A device equipped with the necessary components for analyzing biological samples.

25. The apparatus according to claim 24, wherein the first focusing optical system is configured to synchronize laser beam pulses so that they reach the position on the sample stage immediately after the charged particle pulse.

26. The apparatus according to claim 24, wherein the first focusing optical system is configured to synchronize laser beam pulses to ionize a plume of sputtered material by charged particle pulses.

27. The apparatus according to any one of claims 24 to 26, wherein the charged particle source and charged particle column, and the first laser source and first focusing optical system are configured such that the charged particle beam and the laser beam are directed toward the same side of the sample stage.

28. The apparatus according to any one of claims 24 to 26, wherein the charged particle source and charged particle column, and the first laser source and first focusing optical system are configured such that the charged particle beam and the laser beam are directed toward the opposite side of the sample stage.

29. The apparatus according to any one of claims 24 to 28, further comprising a second laser source and a second focusing optical system, wherein the second focusing optical system is configured to synchronize laser beam pulses from the second laser source to ionize a plume of sputtered material with charged particle pulses.

30. The apparatus according to claim 29, wherein the first laser source and the first focusing optical system, and the second laser source and the second focusing optical system are configured such that the laser beam from the first laser source and the laser beam from the second laser source are directed toward the same side of the sample stage.

31. The apparatus according to claim 29, wherein the first laser source and the first focusing optical system, and the second laser source and the second focusing optical system are configured such that the laser beam from the first laser source and the laser beam from the second laser source are directed toward the opposite side of the sample stage.

32. The apparatus according to any one of claims 24 to 31, wherein the charged particle source is one of a primary ion beam and / or an electron beam.

33. The apparatus according to any one of claims 24 to 32, wherein the laser source is a pulsed laser source, and optionally the laser source is a femtosecond laser, a picosecond laser, or a nanosecond laser.

34. The laser source has a range of 1 nanojoule to 100 microjoules, for example, 10 nanojoules to 100 microjoules, 100 nanojoules to 10 microjoules, 500 nanojoules to 5 microjoules, for example, about 1 microjoule, about 2 microjoules, about 3 microjoules or about 4 microjoules and / or The apparatus according to claim 33, which is adapted to have a pulse energy of 1 millijoule to 50 millijoules, for example, 5 millijoules to 40 millijoules, 10 millijoules to 30 millijoules, 20 millijoules to 35 millijoules, or about 25 millijoules or 35 millijoules.

35. The apparatus according to any one of claims 24 to 34, wherein the charged particle beam is pulsed.

36. The apparatus according to any one of claims 24 to 35, comprising a charged particle beam scanning system adapted to scan the charged particle beam across a plurality of positions on the sample stage.

37. The apparatus according to any one of claims 24 to 36, further comprising an electron microscope.

38. The apparatus according to claim 37, wherein the charged particle source is an electron beam, and the electron beam is an electron source in the electron microscope.

39. The apparatus according to any one of claims 34 to 38, wherein the sample stage is transparent and / or the sample stage has a notched portion.

40. The apparatus according to any one of claims 24 to 39, comprising a variable delay line.

41. The apparatus according to any one of claims 24 to 40, wherein the first and / or second laser source is configured to ionize the material by avalanche ionization.

42. Passing a charged particle beam towards a specific location on the sample, The sample is irradiated with a first laser beam pulse to generate a plume of material containing sample ions, and To detect the sample ions by mass spectrometry, A method for analyzing biological samples, including [specific components / synthesis].

43. The method according to claim 42, The method comprises passing the charged particle beam toward a position on the sample, thereby sputtering material from the sample, and irradiating the sputtered sample with the first laser beam pulse, and ionizing the sputtered material to generate sample ions.

44. The method according to claim 42, A method wherein passing the charged particle beam toward a position on the sample brings about a sample ignition state, and irradiating the sample brings about a sample energy pumping state at the position on the sample.

45. The method according to any one of claims 42 to 44, wherein the charged particle beam passes from one side of the sample toward a position on the sample, and the first laser beam pulse irradiates the sample from the same side.

46. The method according to any one of claims 42 to 44, wherein the charged particle beam passes from one side of the sample toward a position on the sample, and the first laser beam pulse irradiates the sample from the opposite side.

47. The method according to claim 44, or, if dependent on claim 44, the method according to claims 45 and 46, The method further comprises passing the charged particle beam toward a position on the sample to sputter a material from the sample, wherein the method includes irradiating the sputtered sample material with a second laser beam pulse and ionizing the sputtered material.

48. The method according to claim 47, wherein the first laser beam pulse and the second laser beam pulse irradiate the sample from the same side of the sample.

49. The method according to claim 47, wherein the first laser beam pulse and the second laser beam pulse irradiate the sample from the opposite side of the sample.

50. To guide the electron beam toward the aforementioned sample, and Imaging the sample using an electron microscope, The method according to any one of claims 42 to 49, comprising first the following:

51. The method according to any one of claims 20 to 23 and 42 to 50, comprising generating a multiplexed image of the biological sample.

52. The method according to any one of claims 42 to 50, wherein the process includes an avalanche ionization step of irradiating the sample with a first laser beam pulse to generate a plume of material containing sample ions.

53. A device for analyzing biological samples, A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a biological sample, Laser source and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from the laser source toward the second surface to a position on the sample stage, The device wherein the objective lens has an numerical aperture of at least 0.

6.

54. A sample stage comprising a first surface and a second surface, wherein the first surface and the second surface face each other, and the first surface is adapted to receive a biological sample, Laser source and A focusing optical system comprising an objective lens, wherein the focusing optical system is adapted to guide a radiation beam from the laser source toward the second surface to a position on the sample stage, A device equipped with the necessary components for analyzing biological samples.

55. The apparatus according to claim 53 or 54, further comprising a biological sample, wherein the biological sample is prepared according to the method described in any one of claims 16 to 19.

56. The apparatus according to claim 53 or 54, wherein the laser source provides a wavelength of 500 to 600 nm.

57. The apparatus according to claim 53 or 54, wherein the laser source is a UV laser source.

58. The apparatus according to claim 53 or 54, wherein the laser source is an IR laser source.

59. The apparatus according to claim 1, wherein the objective lens has an numerical aperture of at least 0.

6.

60. The apparatus according to claim 1, 53, or 54, further comprising a laser for ionizing the sample emitted from the sample at the position on the sample stage.

61. The apparatus according to claim 60, configured to hold the sample stage under partial vacuum or vacuum.

62. Sample stage and An energy source for passing a beam to a position on the sample stage, the energy source being a laser, an ion beam, or an electron beam, A first laser source, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward the sample stage, A device equipped with the necessary components for analyzing biological samples.

63. Sample stage and A first laser source configured to seed electrons in a sample, and a first focusing optical system configured to guide the laser beam emitted by the first laser source toward the sample stage, A second laser source configured to ablate a sample material from which electrons have been pre-seed by the first laser source, and a second focusing optical system configured to guide the laser beam emitted by the second laser source toward the sample stage, A two-pulse laser sampling and ionization system for analyzing samples such as biological samples.

64. The sampling and ionization system according to claim 63, wherein the second focusing optical system is configured to synchronize the second laser radiation pulse so that it reaches a position on the sample stage immediately after the first laser radiation pulse.

65. A sampling and ionization system according to claim 63 or claim 64, wherein the sample stage comprises a first surface and a second surface, the first and second surfaces facing each other, and the first surface being adapted to receive a biological sample. The first focusing optical system is configured to guide the laser beam emitted by the first laser source toward the first surface of the sample stage. A sampling and ionization system in which the second focusing optical system is configured to guide the laser beam emitted by the second laser source toward the first surface of the sample stage.

66. A sampling and ionization system according to claim 63 or claim 64, wherein the sample stage comprises a first surface and a second surface, the first and second surfaces facing each other, and the first surface being adapted to receive a biological sample. The first focusing optical system is configured to guide the laser beam emitted by the first laser source toward the first surface of the sample stage. A sampling and ionization system in which the second focusing optical system is configured to guide the laser beam emitted by the second laser source toward the second surface of the sample stage.

67. A sampling and ionization system according to claim 63 or claim 64, wherein the sample stage comprises a first surface and a second surface, the first and second surfaces facing each other, and the first surface being adapted to receive a biological sample. The first focusing optical system is configured to guide the laser beam emitted by the first laser source toward the second surface of the sample stage. A sampling and ionization system in which the second focusing optical system is configured to guide the laser beam emitted by the second laser source toward the second surface of the sample stage.

68. The sampling and ionization system according to any one of claims 63 to 67, wherein the first focusing optical system and the second focusing optical system focus radiation from the first and second laser sources using the same objective lens.

69. The sampling and ionization system according to any one of claims 63 to 68, wherein the sample stage is at least partially composed of a material that is at least semi-transparent to the first pulse and even more transparent to the second pulse.

70. The sampling and ionization system according to any one of claims 63 to 69, wherein the objective lens of the first focusing optical system has a central opening to allow ionized material from a sample to pass through the opening.

71. The sampling and ionization system according to any one of claims 63 to 70, wherein the light emitted by the first laser source is 200 nm or less, 175 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, 50 nm or less, 25 nm or less, 20 nm or less, or 15 nm.

72. A sampling and ionization system according to any one of claims 63 to 71, wherein the light emitted by the first laser source is 10-200 nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, 10-50 nm, 10-25 nm, 10-20 nm, or 10-15 nm.

73. The sampling and ionization system according to any one of claims 63 to 72, wherein the pulse energy of the first laser source is selected such that the pulse energy seeds electrons into the material but does not ablate the material.

74. The sampling and ionization system according to any one of claims 63 to 73, wherein the pulse energy of the first laser source is in the range of picoJ to femtoJ, such as 1 picoJ to 100 femtoJ, 10 picoJ to 100 femtoJ, or 100 picoJ to 1 femtoJ.

75. A sampling and ionization system according to any one of claims 63 to 74, wherein the duration of the pulse from the first laser source is 5 ps or less, for example, 2 ps or less, 1 ps or less, 500 fs or less, 400 fs or less, 300 fs or less, 200 fs or less, or 100 fs or less, 50 fs or less, 40 fs or less, 30 fs or less, 20 fs or less, or 10 fs or less.

76. The sampling and ionization system according to any one of claims 63 to 75, wherein the first focusing optical system must focus the laser radiation from the first laser source to a spot size in the sample of about 100 nm or less, for example, 75 nm or less, 50 nm or less, or about 30 nm.

77. The sampling and ionization system according to any one of claims 63 to 76, wherein the objective lens of the first focusing optical system is a reflective objective lens.

78. The sampling and ionization system according to any one of claims 63 to 77, wherein the wavelength of the laser radiation emitted by the second laser source is IR or visible light.

79. The sampling and ionization system according to any one of claims 63 to 78, wherein the wavelength of the laser radiation emitted by the second laser source is 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, or 1 μm or more.

80. A sampling and ionization system according to any one of claims 63 to 79, wherein the wavelength of the laser radiation emitted by the second laser source is 200 nm to 100 μm, for example, 200 nm to 10 μm, 200 nm to 1 μm, 200 nm to 900 nm, 200 nm to 800 nm, 200 nm to 700 nm, 200 nm to 600 nm, or 500 nm to 600 nm.

81. The sampling and ionization system according to any one of claims 63 to 80, wherein the pulse energy of the second laser source is in the range of nanojoules, such as 1 nanoJ to 1 μJ, 10 nanoJ to 500 nanoJ, 50 nanoJ to 250 nanoJ, or about 100 nanoJ.

82. A sampling and ionization system according to any one of claims 63 to 81, wherein the duration of the first pulse is 5 ps or less, for example, 2 ps or less, 1 ps or less, 500 fs or less, 400 fs or less, 300 fs or less, 200 fs or less, or 100 fs or less, 50 fs or less, 40 fs or less, 30 fs or less, 20 fs or less, or 10 fs or less.

83. The sampling and ionization system according to any one of claims 63 to 82, wherein the second focusing optical system focuses the laser radiation to a spot size in the sample of about 2 μm or less, for example, 1 μm or less, 750 nm or less, 500 nm, 250 nm or less, 200 nm or less, 150 nm or less, or about 100 nm.

84. The sampling and ionization system according to any one of claims 63 to 83, wherein the system comprises a single laser for the first and second laser sources, and a beam splitter for splitting the laser and providing the first and second laser sources.

85. A sampling and ionization system according to any one of claims 63 to 84, comprising a single laser, a beam splitter, and two harmonic generators, wherein one of the harmonic generators is adapted to generate UV, VUV, EUV, or XUV laser radiation from the single laser as a first laser source, and the other harmonic generator is adapted to generate IR or visible wavelength laser radiation from the single laser as a second laser source.

86. A sampling and ionization system according to claim 85, further comprising an optical delay line, wherein the optical delay line is adapted to deliver a pulse from the second laser source, derived from the pulse of the single laser, after a pulse from the second laser source, derived from the same pulse of the single laser, such that the end of the second pulse is less than 50 ps after the start of the first pulse, e.g., less than 25 ps, less than 10 ps, ​​e.g., less than 5 ps, less than 2 ps, or less than 1 ps.

87. The sampling and ionization system according to any one of claims 84 to 86, wherein the laser is a femtosecond laser, in particular a femtosecond fiber laser.

88. The sampling and ionization system according to any one of claims 63 to 87, further comprising a third laser source configured to ionize a plume of sample material ablated from the sample.

89. The sampling and ionization system according to claim 88, further comprising a third focusing optical system configured to guide a laser beam emitted by the third laser source into a volume in which the ablated sample material forms a plume.

90. The sampling and ionization system according to claim 88, wherein a third focusing optical system synchronizes a third laser radiation pulse so that it reaches the volume above the position on the sample stage less than 100 ps, ​​for example less than 50 ps, ​​less than 30 ps, ​​or less than 10 ps, ​​from the end of the pulse from the second laser source.

91. A sampling and ionization system according to any one of claims 63 to 90, further comprising a sample chamber, wherein the sample chamber contains argon, helium, nitrogen, or a mixture thereof.

92. The sampling and ionization system according to claim 91, wherein the sample chamber is held under partial vacuum or vacuum.

93. An apparatus comprising a sampling and ionization system according to any one of claims 63 to 92.

94. The apparatus according to claim 93, comprising a mass detector such as a TOF or magnetic sector detector.

95. a) Labeling the sample with labeled atoms, b) Irradiating a sample at a spot with radiation from an energy source, wherein the spot includes spot sizes of 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 50 nm or less, or 30 nm or less. c) Ionizing the sample from the spot, d) Detecting labeled atoms by mass spectrometry. A method that includes this.

96. The method according to claim 95, wherein the energy source is a laser, an electron beam source, or an ion beam source.

97. The method according to claim 95 or claim 96, wherein step c) ionization is performed by a laser, and the laser is not the energy source for step b).

98. The method according to claim 95, wherein the ionization step c) is performed by ICP.

99. The method according to claim 95, wherein step c) is performed at the spot.

100. The method according to any one of claims 95 to 99, wherein the spot has a depth of 50 nm or less, 30 nm or less, or 10 nm or less.

101. The method according to any one of claims 95 to 100, wherein the labeled atom is a mass tag.

102. The method according to claim 101, wherein at least some of the mass tags are subject to targeted barcoding.

103. The method according to claim 102, wherein a single target barcoded mass tag is detected at a first spot.

104. The method according to any one of claims 95 to 103, further comprising repeating steps a) to d) to generate an image of the distribution of labeled atoms in the sample.

Images