Device for analyzing biological sample

Abstract

The invention relates to a device for analyzing a biological sample, comprising: a sample stage; a charged particle source configured to deliver a charged particle beam to a location on the sample stage; and a first laser source and a first focusing optic configured to direct the laser beam emitted by the first laser source toward the sample stage, where the charged particle source, the first laser source, and the first focusing optic are configured such that the charged particle beam and the laser beam are directed toward opposite sides of the sample stage.

Description

[0001] This application is a divisional application of the patent application with application number 201980053879.7 and invention name “High-resolution imaging device and method”, which entered the national stage on February 10, 2021, and has an international application date of June 18, 2019 and international application number PCT / US2019 / 037644. The entire contents of the application are incorporated herein by reference.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 686,341, filed on June 18, 2018, entitled “HIGH RESOLUTION IMAGING APPARATUS AND METHOD,” and U.S. Provisional Application No. 62 / 792,759, filed on January 15, 2019, entitled “HIGH RESOLUTION IMAGING APPARATUS AND METHOD,” the entire contents of both applications are incorporated herein by reference. Technical Field

[0004] The present invention relates to the use of mass spectrometry imaging technology (IMS) to perform high-resolution imaging of samples and mass spectrometry flow cytometry technology (IMC TM ) to image biological samples. Background Art

[0005] LA-ICP-MS (a form of IMS in which a sample is ablated by a laser and the ablated material is then ionized in an inductively coupled plasma before the ions are detected by mass spectrometry) has been used for the analysis of a variety of substances, for example, mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological substances.

[0006] Imaging of biological samples by IMC has been previously reported to image at cellular resolution, and more recently, imaging at subcellular resolution has been reported to provide details.

[0007] Despite these recent advances, equipment and techniques are still needed to further improve the resolution of IMCs to the submicron level. Two major challenges in improving the resolution of IMCs to the submicron level are:

[0008] 1) limiting the sampling spot area to a size below about 200 nm (the value below or above referred to herein means including the value itself); and

[0009] 2) Ensure that the amount of analyte in the ablated material produces an adequate signal-to-noise ratio.

[0010] It is an object of the present invention to provide further improved apparatus and techniques that overcome these two challenges in order to provide high resolution imaging of samples. Summary of the invention

[0011] In general, the analyzer apparatus disclosed herein includes two broad characterization systems for performing imaging elemental mass spectrometry.

[0012] The first is a sampling and ionization system. The system includes a sample chamber, which is a component in which the sample is placed when it is analyzed. The sample chamber includes a stage for holding the sample (usually the sample is on a sample carrier, such as a microscope slide, such as a tissue section, a monolayer of cells or a single cell, for example, where a cell suspension has been dripped onto a microscope slide and the slide is placed on the stage). The sampling and ionization system is used to remove material from the sample in the sample chamber (the removed material is referred to herein as sample material), which is converted into ions as part of a process that causes the removal of material from the sample, or via a separate ionization system downstream of the sampling system. To produce elemental ions, hard ionization techniques are used.

[0013] The ionized material is analyzed by a second system, a detector system. The detector system may take different forms depending on the specific characteristics of the ionized sample material being determined, for example, a mass detector in an analyzer device based on mass spectrometry.

[0014] The present invention provides improvements to current IMS and IMC equipment and methods by applying various equipment and techniques that take advantage of the analysis of tissue sections with a thickness of less than 100nm. When such ultrathin sections are used, the opportunity for further analysis techniques is increased. The inventors have found that a lens (e.g., an immersion lens) that maintains a particularly high refractive index around the ablation spot can be used to focus the laser used for laser ablation to a particularly small ablation spot. The inventors have determined that when analyzing ultrathin sections, such a lens can be used to focus laser radiation without causing damage to sample areas outside the ablation target area. This is because the high refractive index of the lens material ensures that the laser is focused into a tight focal spot through the lens all the way down to the ablation spot. Therefore, the tight focal spot is smaller than would be possible without the high refractive index material, and because the size of the focal spot determines the lateral resolution, the present invention provides equipment and techniques that provide improved lateral resolution. In addition, the focus of the lens is not just two-dimensional. It is a three-dimensional volume that concentrates the radiation. Therefore, the diameter of the ablation spot as well as the depth (i.e., the focus of the radiation) are both worthy of attention. If the focal length is set so that the focus of the radiation is in the substrate rather than at the surface of the substrate, this may lead to unpredictable cracking and fragmentation of the sample. This problem is caused, among other things, by the fact that the inventors have discovered that in order to use the immersion lens most efficiently, it is best to ablate the sample in such a way that the laser radiation is directed through the sample carrier.

[0015] Likewise, the inventors have discovered that, when thin sections are used, electron microscopy can be performed on a sample that is also analyzed by IMS or IMC (e.g., prior to analyzing the sample by IMC). Thus, a high-resolution structural image can be obtained by electron microscopy, e.g., transmission electron microscopy, and then the high-resolution image is used to refine the resolution of the image data obtained by IMS or IMC to a resolution that exceeds that obtainable using laser radiation ablation (due to the much shorter wavelength of electrons than photons). Alternatively or in addition, the electron microscopy image can be correlated with an IMC image of the same area of ​​the sample, e.g., by correlating the localization of a protein target imaged by IMC with subcellular (e.g., nanoscale) structures identified by electron microscopy. As described in more detail below, electron microscopy imaging can be performed "offline" in a separate device, or components of the electron microscope can be incorporated into the IMS or IMC device.

[0016] In addition, the inventors have developed further techniques for high-resolution imaging based on a combination of charged particle (e.g., ion or electron) bombardment of a sample coupled to laser ionization of the sample material after sputtering the sample material from the sample, or at the time of sampling. Since the charged particles can be more tightly focused while still removing material from the sample, this method can achieve higher resolution than conventional laser ablation-based techniques. Ion optics can be used to direct the charged particles toward the sample, and / or to direct the ionized sample to a mass spectrometer (e.g., a TOF or magnetic sector detector). The ion optics may be similar to those used in electron or ion microscopes. The ion optics may include ion scanning optics (e.g., for scanning charged particles over the sample), ion focusing optics (e.g., for determining the spot size or focusing sample ions toward a detector), and ion accelerating optics (for determining the energy of charged particles that strike the sample, or for accelerating ions toward a detector). The ion optics may include one or more charged surfaces (e.g., plates) of appropriate shape, charge, and / or orientation.

[0017] Thus, in operation of an apparatus according to the invention, a sample is placed into the apparatus, the sample is sampled using a laser system including an optical device to produce ionized material, wherein laser radiation is directed onto the sample through an immersion lens (the sample may produce a vapor / specific material which is then ionized by the ionization system), and ions of the sample material are transmitted to a detector system.

[0018] Thus, in operation of another device according to the present invention, a sample is placed in the device, the sample is imaged using electron microscopy (e.g., transmission electron microscopy), and then the sample is sampled using a sampling and ionization system to produce ionized material (sampling can produce ions, or can produce uncharged vapor / specific material (or uncharged vapor / specific material can be formed by charge neutralization of any ions formed during sampling), in which case the material is subsequently ionized by a separate ionization system), and the ions of the sample material are transmitted to a detector system.

[0019] Finally, in operation of another device of the present invention, a sample is placed in the device and the sample is sampled using a sputtering-based sampling system (a charged particle-based sampling system) to produce a material, wherein, after the sample material is sputtered from the sample or while the sample is being sampled, the sample material is ionized by a laser post-ionization system and ions of the sample material are transmitted to a detector system.

[0020] Although the detector system of the apparatus of the present invention can detect many ions, most of the ions will be ions of atoms that themselves make up the sample. In some cases, for example, when analyzing biological samples, the elemental composition of the sample itself may not have appropriate information. This is because, in general, all proteins and nucleic acids are composed of the same major constituent atoms, so although it is possible to distinguish areas containing protein / nucleic acid from areas that do not contain such protein or nucleic acid material, it is not possible to distinguish a specific protein from all other proteins. However, by labeling the sample with atoms that are not present in the material being analyzed under normal conditions, or at least not present in large quantities (e.g., certain transition metal atoms, such as rare earth metals; see the section on labeling below for further details), specific characteristics of the sample can be determined. As with IHC and FISH, the detectable label can be attached to a specific target on or in the sample (e.g., a fixed cell or tissue sample on a slide), in particular by using an SBP such as an antibody, nucleic acid or lectin to the target molecule on or in the sample. To detect the ionizing label, a detector system is used because it will detect ions from atoms naturally present in the sample. By correlating the detected signals with known sampling locations of the sample that produced those signals, an image of the atoms present at each location can be generated, including both the native elemental composition and any labeled atoms. Insofar as the native elemental composition of the sample is depleted prior to detection, the image may be of only the labeled atoms. This technique allows for the analysis of many labels in parallel (also called multiplexing), which is a great advantage in the analysis of biological samples, now made faster by the use of laser scanning systems in the apparatus and methods disclosed herein.

[0021] Therefore, the present invention provides an apparatus for analyzing a biological sample, comprising:

[0022] Sample table;

[0023] a laser source; and

[0024] focusing optics comprising an objective lens, the focusing optics being adapted to direct a radiation beam from a laser source towards a position on a sample stage; and wherein,

[0025] The apparatus also includes an immersion medium located between the objective lens and the sample stage.

[0026] The present invention also provides a method for preparing a biological sample for analysis, comprising:

[0027] staining specimens with contrast agents for electron microscopy;

[0028] Label the sample with a marker atom.

[0029] The present invention also provides a method for analyzing a biological sample, comprising:

[0030] Imaging samples by electron microscopy;

[0031] directing a radiation beam emitted by a laser source toward a location on the sample to produce an ablation plume of sample material;

[0032] an ablation plume that ionizes sample material; and

[0033] Sample ions are detected from the sample material.

[0034] The present invention also provides a device for analyzing a biological sample, comprising:

[0035] Sample table;

[0036] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0037] The first laser source and the first focusing optics are configured to direct a laser beam emitted by the first laser source toward the sample stage.

[0038] The present invention also provides a method for analyzing a biological sample, comprising: delivering a charged particle beam to a position on the sample to sputter material from the sample; irradiating the sputtered sample material with a pulse of laser radiation to produce a material plume containing sample ions; and detecting the sample ions by mass spectrometry.

[0039] The present invention also provides a method for analyzing a biological sample, comprising: delivering a charged particle beam to a location on the sample to produce a sample ionization state on the sample; irradiating the sample with a laser radiation pulse to produce a sample material plume containing sample ions from the location; and detecting the sample ions by mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the optical device assembled in the existing equipment;

[0041] Figure 2 is a schematic diagram of an optical device assembled in another existing device;

[0042] Figure 3 is a schematic diagram of an optical device arrangement of an exemplary embodiment of the present invention;

[0043] Figure 4 is a schematic diagram of an optical device arrangement according to another exemplary embodiment of the present invention;

[0044] Figure 5 a) is a schematic diagram of an optical device arrangement according to another exemplary embodiment of the present invention, showing a path of a laser beam through a hemispherical solid immersion lens;

[0045] Figure 5 b) is a schematic diagram of an optical device arrangement of another exemplary embodiment of the present invention, showing the path of a laser beam through a Weierstrass solid immersion lens;

[0046] Figure 6 is a schematic diagram of the optical arrangement of an apparatus for analyzing biological samples using laser post-ionization;

[0047] Figure 7 is a schematic diagram of an optical arrangement of an apparatus for analyzing a biological sample using laser pumping of the sample;

[0048] Figure 8 is a schematic diagram of an optical arrangement of an apparatus for analyzing a biological sample using laser pumping of the sample through a sample carrier;

[0049] Fig. 9 is a schematic diagram of an optical arrangement for an apparatus for analyzing a biological sample using laser post-ionization of the sample through a sample carrier;

[0050] Fig.10 is a schematic diagram of an optical arrangement of an apparatus for analyzing a biological sample, the apparatus comprising at least two laser sources, one for laser post-ionization and one for laser pumping of the sample;

[0051] Fig.11 shows images showing the detection of various osmium isotopes in a sample prepared according to an exemplary method of the present invention;

[0052] Fig.12 Shows the display for Fig.11An image of the detection of various osmium isotopes in a sample prepared according to an exemplary method of the present invention, Fig.12 An area of ​​2100 × 1087 μm is shown, with a step size of 250 nm;

[0053] Fig.13 is a schematic diagram of a dual-pulse sampling and ionization system;

[0054] Fig.14 is a schematic diagram of a dual-pulse sampling and ionization system, in which the first pulse and the second pulse are focused onto the sample from the same side;

[0055] Fig.15 is a schematic diagram of a dual pulse sampling and ionization system, wherein the sample is placed on a material that is at least semi-transparent to a first pulse and a second pulse;

[0056] Fig.16 is a schematic diagram of a dual-pulse sampling and ionization system, wherein the objective lens has an opening that allows the ions generated after ablation to pass through;

[0057] Fig.17 is a schematic diagram of the three-pulse sampling and ionization system. DETAILED DESCRIPTION

[0058] Thus, various types of analyzer devices may be used to practice the present disclosure, some of which are discussed in detail below, for example, devices including immersion lenses, devices including electron microscopes (or components thereof), and devices for performing secondary neutral mass spectrometry.

[0059] Analyzer equipment based on quality inspection

[0060] 1. Sampling and ionization system

[0061] a. Laser ablation sampling and ionization system

[0062] Laser ablation-based analyzers typically include three components. The first component is a laser ablation sampling system, which is used to generate a plume of gaseous and particulate matter from a sample for analysis. In order for the atoms in the plume of ablated sample material (including any detectably labeled atoms discussed below) to be detected by a detector system (a mass spectrometer component (MS component; a third component)), the sample must be ionized (and atomized). Therefore, the device includes a second component, which is an ionization system that ionizes the atoms to form elemental ions so that the MS component can detect these elemental ions based on mass / charge ratio (some ionization of the sample material may occur at the ablation point, but space charge effects cause the charge to be neutralized almost immediately). The laser ablation sampling system is connected to the ionization system by a transfer conduit.

[0063] Laser Ablation Sampling System

[0064] Briefly, the components of the laser ablation sampling system include a laser source that emits a beam of laser radiation toward a sample. The sample is placed on a stage within a chamber (sample chamber) of the laser ablation sampling system. The stage is typically a translation stage that allows the sample to be moved relative to the laser radiation beam, thereby allowing different locations on the sample to be sampled for analysis (e.g., due to the relative movement of the laser beam (the term "laser beam" can be used interchangeably with the term "laser radiation"), the laser scanning system described herein can induce locations farther away than can be ablated). As discussed in more detail below, a gas flows through the sample chamber, and the gas flow carries away a plume of atomized material generated when the laser source ablates the sample for analysis and construction of an image of the sample based on the elemental composition of the sample (including labeled atoms, for example, labeled atoms from elemental tags). As further explained below, in an alternative mode of operation, the laser system of the laser ablation sampling system can also be used to release material from the sample.

[0065] For biological samples in particular (cells, tissue sections, etc.), the samples are usually heterogeneous (although in other application areas of the present disclosure heterogeneous samples are known, i.e. samples of a non-biological nature). A heterogeneous sample is a sample that contains regions composed of different materials, so that some regions of the sample can be ablated at a lower threshold flux than other regions at a given wavelength. Factors that influence the ablation threshold are the absorption coefficient of the material and the mechanical strength of the material. For biological tissues, the absorption coefficient will have a major influence, as it can vary by several orders of magnitude with the laser radiation wavelength. For example, in a biological sample, when using nanosecond laser pulses, regions containing proteinaceous matter absorb more easily in the 200-230 nm wavelength range, while regions containing mainly DNA absorb more easily in the 260-280 nm wavelength range.

[0066] Laser ablation can be performed at a flux near the ablation threshold of the sample material. Performing ablation in this manner often improves aerosol formation, which in turn helps improve the quality of the data after analysis. Usually, in order to minimize damage and to maximize the resolution of the final image, a Gaussian beam is used. The cross-section of a Gaussian beam records the energy density distribution with a Gaussian distribution. In this case, the flux of the beam varies with the distance from the center. As a result, the diameter of the ablation spot size is a function of two parameters: (i) the Gaussian beam waist (1 / e 2 ) and (ii) the ratio between the applied flux and the threshold flux.

[0067] Therefore, to ensure that a reproducible amount of material is consistently removed with each ablation laser pulse, and thus maximize the quality of the imaging data, it is useful to maintain a consistent ablation diameter, which in turn means adjusting the ratio of the energy delivered to the target by the laser pulse to the ablation threshold energy of the ablated material. This requirement becomes a problem when ablating inhomogeneous samples where the threshold ablation energy varies across the sample (e.g., biological tissues where the ratio of DNA and protein material varies or geological samples where the threshold ablation energy varies with the specific composition of the minerals in the sample region). To address this issue, laser radiation of more than one wavelength can be focused onto the same ablation location on the sample to more effectively ablate the sample based on the composition of the sample at that location.

[0068] Laser system for laser ablation sampling system

[0069] The laser system can be arranged to generate laser radiation at a single or multiple (i.e. two or more) wavelengths. Usually, the wavelength of laser radiation in question is the wavelength with the highest intensity (the "peak" wavelength). If the system generates different wavelengths, they can be used for different purposes, for example, to target different materials in the sample (targeted here means that the selected wavelength is well absorbed by the material).

[0070] Where multiple wavelengths are used, at least two of the two or more wavelengths of the laser radiation may be discrete wavelengths. Thus, when a first laser source emits radiation of a first wavelength that is discrete from radiation of a second wavelength, this means that the first laser source produces no or very small amounts of radiation of the second wavelength in pulses of the first wavelength, e.g., less than 10% of the intensity of the first wavelength, e.g., less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Typically, when laser radiation of different wavelengths is produced by harmonic generation or other nonlinear frequency conversion processes, then when a specific wavelength is mentioned herein, it will be understood by those skilled in the art that there will be some degree of variation in the specific wavelength in the spectrum produced by the laser. For example, a reference to X nm includes lasers that produce a spectrum within the range of X ± 10 nm, e.g., X ± 5 nm, e.g., X ± 3 nm.

[0071] Focusing optics and objectives

[0072] As a general rule, optical components may be used to direct the laser radiation beam towards a focus. Figure 11 is a schematic diagram of the optical devices of the existing device assembly. Here, a laser source (e.g., a pulsed laser source, optionally including a pulse picker) 101 emits a laser radiation beam, which is directed through an energy control module 102 and then through an optical device 103. The beam / illumination combination optical device 104 then directs the radiation beam through a focusing optical device and an objective lens 105 to the sample. The sample is located on a sample stage 107 in a sample chamber 106. The sample stage 107 (e.g., a slide) can be mounted on a three-axis (i.e., x, y, z) translation stage 108 in the sample chamber 106. Figure 1 The assembly also includes a camera 111 for viewing the sample using the same focusing optics and objective 105. An illumination source 109 emits visible light which is directed by illumination / inspection separation optics 110 through beam / illumination combining optics 104 and focusing optics 105 to the sample.

[0073] Figure 2 Another configuration of the existing equipment assembly is shown. Here, the sample 215 is located on the side of the sample stage 207 opposite the objective lens 205, and the laser radiation ablates the sample 215 through the sample stage 207 or through the sample chamber. In some cases, the sample chamber is maintained under vacuum or partial vacuum. Ray diagrams are shown to represent possible paths of the laser beam 216 from the objective lens 205 through the slide 207.

[0074] As mentioned above, in conventional IMC and IMS, a major challenge in achieving a spatial resolution of less than 200 nm (e.g., less than 150 nm, e.g., less than 100 nm) is to limit the spot size of the laser to a size less than 200 nm (e.g., less than 150 nm or less than 100 nm). For systems with numerical aperture (NA) exceeding 0.7, the full width at half maximum of the laser spot size is given by , where λ is the wavelength of the laser (laser is used interchangeably with laser radiation in this article), and NA is the numerical aperture of the objective lens 105, 205 of the focusing optics. Therefore, in conventional IMC and IMS, a shorter wavelength laser, such as a deep UV laser with a wavelength of 213 nm, or a focusing optic with a NA greater than 0.7 (e.g., greater than 0.8, such as a high NA (greater than 0.9)) is used to reduce the laser spot size and thus improve the resolution.

[0075] However, the numerical aperture of the lens is expressed as NA = n × sinθ, where n is the refractive index of the medium between the lens and the sample stage 107, 207, and θ is half the acceptance angle of the objective lens. Figure 1 and Figure 2The maximum theoretical numerical aperture of the objective lens 105, 205) of the prior art device configuration shown in is limited to 1.0 because the refractive index of a vacuum is 1.0 and the refractive index of air is approximately 1.0.

[0076] Immersion lens

[0077] The present invention overcomes the limitations of conventional IMC and IMS by using 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 the present invention achieves a numerical aperture greater than 1.0, so that the spot size of the laser is less than 200nm, less than 150nm, or less than 100nm. Thus, the present invention provides an apparatus for imaging mass spectrometry flow cytometry with a spatial resolution of 200nm or more, 150nm or more, or 100nm or more.

[0078] Therefore, the present invention provides a laser sampling system, comprising:

[0079] Sample table;

[0080] a laser source; and

[0081] focusing optics comprising an objective lens, the focusing optics being adapted to direct radiation from the laser source toward a position on the sample stage; and wherein,

[0082] The laser sampling system also includes an immersion medium located between the objective lens and the sample stage.

[0083] Therefore, the present invention provides an apparatus for analyzing a biological sample, comprising:

[0084] Sample table;

[0085] a laser source; and

[0086] focusing optics comprising an objective lens, the focusing optics being adapted to direct radiation from the laser source toward a position on the sample stage; and wherein,

[0087] The apparatus also includes an immersion medium located between the objective lens and the sample stage.

[0088] Typically, the device also includes a mass spectrometry based detector.

[0089] Thus, in operation, the sample stage holds the sample, typically wherein the sample is placed on a sample carrier, and the same stage holds the sample carrier. Laser radiation is then directed through the optics of the apparatus, through the objective lens and the immersion medium to the sample, wherein the radiation ablates material in the sample.

[0090] 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 greater, 1.50 or greater, 2.00 or greater, or 2.50 or greater.

[0091] Furthermore, in order to reconstruct images of a single layer thickness (or less) of a biological cell or to read thicker samples layer by layer and generate 3D images, as discussed further herein, the sample preferably has a thickness of 100 micrometers or less, e.g., 10 micrometers or less, 5 micrometers or less, 2 micrometers or less, 100 nm or less, 50 nm or less, 30 nm or less. In some embodiments described in more detail herein, the combination of an objective lens and an immersion medium is referred to as an immersion lens.

[0092] In some embodiments, the apparatus or sampling system comprising an immersion medium as described above comprises a medium or high NA objective lens, for example, a NA of 0.7 or greater, 0.8 or greater, or 0.9 or greater. Such a system typically also comprises a mass detector, for example, a TOF mass spectrometer detector.

[0093] Liquid immersion media

[0094] In some embodiments of the invention, the immersion medium is a liquid immersion medium, Figure 3 is a schematic diagram of an optical device configuration of an exemplary embodiment of the present invention. Figure 1 and Figure 2 The same elements are shown in the configuration shown. Focusing optics including objective lens 305 direct a radiation beam 316 from a laser source (source not shown) toward a location on a sample stage 307, and a liquid immersion medium 312 is located between objective lens 305 and sample stage 307. Here, a biological sample 315 is located on the opposite side of sample stage 307 from the liquid immersion medium 312. The ray diagram shows how the liquid immersion medium 312 provides a relative Figure 2The conventional configuration shown provides a tighter focusing condition. Suitable liquids for the liquid immersion medium are water with a refractive index n of 1.333, glycerol (n=1.4695), oils such as paraffin oil (n=1.480), cedar 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). Commercial immersion oils are also available, and some of these commercial oils have properties that are particularly advantageous for use in the present invention. For example, low-fluorescence or non-fluorescent immersion oils (e.g., Olympus Low Autofluorescence Immersion Oil and Nikon) are particularly suitable for short-wavelength lasers because they provide a higher signal-to-noise ratio than general-purpose immersion oils. Other oils have very high viscosities, for example, Cargille Laboratory Type NVH and OVH, which are useful when the distance between the objective lens 305 and the sample stage 307 is large.

[0095] Commercial oil immersion objectives can achieve a maximum numerical aperture of approximately 1.49 (close to the refractive index of immersion oil) and are capable of focusing a 515 nm laser beam to a FWHM focal diameter of approximately 160 nm.

[0096] When using liquid immersion media, the sample needs to be placed on the side of the sample carrier opposite to the liquid medium (e.g. Figure 3 ) so that the carrier gas can collect the ablated material. Therefore, a through-sample carrier ablation technique must be applied here. This has the added benefit of a smaller working distance achievable by the ablated material collection hardware and does not require a bend in the delivery conduit between the sample chamber and the detector. This also results in a reduction in the transient time, thereby increasing the achievable spot ablation rate per second.

[0097] Liquid immersion lenses (eg, water-in-oil objectives) are commercially available from Olympus, ThorLabs, and Leica.

[0098] Solid immersion medium

[0099] In some embodiments of the invention, the invention provides an apparatus wherein the immersion medium is a solid immersion medium. Figure 4 is a schematic diagram of an optical device configuration according to another exemplary embodiment of the present invention, wherein a solid immersion member 413 is located between an objective lens 405 and a sample stage 407 . Figure 4 The other components correspond to Figure 3 Parts in.

[0100] Similar to liquid immersion media, solid immersion lenses have a refractive index n greater than air. Suitable materials for solid immersion lenses are glass, for example, S-LAH79 TMGlass type, with a refractive index of 2.0 when operating at a wavelength of ~520nm. Alternative suitable materials for the solid immersion medium of the present invention are diamond or fused silica. At 266nm, diamond becomes very suitable for optical applications because its refractive index under UV is >2.5, providing the opportunity to focus 266nm light to a spot size of 100nm or even smaller. From a cost perspective, fused silica may be more attractive. It can be used as both a sample substrate and a solid immersion lens material. However, the refractive index of fused silica under UV is only 1.5, so the spot size will become correspondingly larger.

[0101] There are two standard optical solutions for solid immersion media: hemispherical immersion lenses and Weierstrass immersion lenses.

[0102] Hemispherical solid immersion lens;

[0103] Figure 5 a) shows the geometry of a hemispherical solid immersion lens. A hemispherical solid immersion lens is capable of increasing the numerical aperture of an optical system by the refractive index n of the lens material.

[0104] Weierstrass Solid Immersion Lens:

[0105] Figure 5 b) shows the geometry of the Weierstrass solid immersion lens. The Weierstrass solid immersion lens is a truncated sphere with a height of (1+1 / n)r from the slide 507, where r is the radius of the lens sphere. The Weierstrass lens can increase the numerical aperture of the optical system by n 2 Therefore, the Weierstrass lens can further increase the numerical aperture of the optical system than the hemispherical lens.

[0106] Therefore, the present invention provides an apparatus wherein the solid immersion medium is a hemispherical solid immersion lens or a Weierstrass solid immersion lens. Figure 3 and Figure 4 As shown, when the biological sample 315, 415 is mounted on the sample stage 307, 407, the biological sample can be mounted on the side of the sample stage opposite to the solid immersion material. The stage on which the sample is mounted can be made of a material having 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 to maintain the focus position.

[0107] Combination of immersion lens and biological sample

[0108] The present invention including an immersion medium between the objective lens and the sample stage provides further advantages when used to analyze biological samples prepared according to other methods of the present invention described herein (page 112 of the PCT publication). In particular, the present invention provides further advantages when the thickness of the biological sample is less than 100 microns, for example, less than 10 microns, or less than 100 nm, or less than 50 nm, or less than 30 nm.

[0109] For example, an apparatus of the present invention including an immersion medium between the objective lens and the sample stage can be used to analyze biological samples having a thickness of less than 100 nm (e.g., less than 50 nm, or less than 30 nm). Since the apparatus of the present invention provides an apparatus for imaging mass cytometry with a spatial resolution of 200 nm or better (preferably 100 nm or less), the spot size of the laser 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). Therefore, using the present invention to analyze biological samples having a thickness of less than 200 nm will completely ablate through the sample (or when the laser spot size is less than 100 nm, preferably less than 100 nm). Therefore, the present invention provides the possibility of using the apparatus to reconstruct an image of the thickness of a biological cell monolayer by serially sectioning the biological cell.

[0110] Alternatively, the apparatus of the present invention including an immersion medium between the objective lens and the sample stage can be used to analyze biological samples below 100 microns, for example, below 10 microns, below 5 microns, below 2 microns. As described above, the sharp focusing of the laser beam by the immersion medium produces a very short depth of focus. Therefore, the present invention provides the opportunity to read thicker samples layer by layer and generate 3D images. Those skilled in the art will understand that the more homogeneous the sample, the more direct the analysis of thicker samples. In heterogeneous tissue samples, the laser light will be distorted as it propagates through the tissue, resulting in a larger spot size at the focus than expected.

[0111] Therefore, the present invention provides an imaging mass spectrometry flow cytometer or an imaging mass spectrometer comprising a biological sample, wherein the thickness of the biological sample is less than 100 nm, for example, less than 50 nm or less than 30 nm. The present invention also provides an imaging mass spectrometry flow cytometer or an imaging mass spectrometer comprising a biological sample, wherein the thickness of the biological sample is less than 100 nm, for example, less than 50 nm, or less than 30 nm, and wherein the imaging mass spectrometry flow cytometer or the imaging mass spectrometer comprises a solid immersion lens. The present invention also provides an imaging mass spectrometry flow cytometer or an imaging mass spectrometer comprising a biological sample, wherein the thickness of the biological sample is less than 100 nm, for example, less than 50 nm, or less than 30 nm, and wherein the imaging mass spectrometry flow cytometer or the imaging mass spectrometer comprises a solid immersion lens.

[0112] Alternatives to immersion lenses in conjunction with thin biological specimens:

[0113] As described above, in conventional IMC and IMS, a short wavelength laser (e.g., a deep UV laser with a wavelength of 213 nm) or a focusing optical device with a NA greater than 0.6 (e.g., greater than 0.7, e.g., greater than 0.8, e.g., high NA (greater than 0.9)) is used to reduce the size of the laser spot size, thereby improving the resolution to 100 nm.

[0114] The high NA objective lens can be a refractive optical component and / or a reflective optical component. For example, an aspherical mirror can be used to allow the reflective objective lens to have a numerical aperture of up to 0.99 (Inagawa et al., Scientific Reports 5, 12833 (2015)). A reflective refractive mirror system can also be used. In addition, a short wavelength laser (300nm or lower) can be provided by using a laser with a wavelength of 266nm or 213nm, a solid-state laser of 193nm, or the sixth harmonic (e.g., 13.5nm and 7nm wavelength) generated from ND:Yag, ArF, F2 lasers, extreme ultraviolet light (CO2 laser + Sn plasma)).

[0115] Therefore, the present invention provides an apparatus for analyzing a biological sample, comprising:

[0116] a sample stage comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a biological sample; and

[0117] a laser source; and

[0118] focusing optics comprising an objective lens, the focusing optics being adapted to direct a radiation beam from the laser source towards the second face to a position on the sample stage; and wherein,

[0119] The numerical aperture of the objective lens is at least 0.7, at least 0.8 or at least 0.9, for example at least 0.9.

[0120] Therefore, the present invention provides an apparatus for analyzing a biological sample, comprising:

[0121] a sample stage comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a biological sample; and

[0122] a laser source; and

[0123] focusing optics comprising an objective lens, the focusing optics being adapted to direct a radiation beam from the laser source towards the second face to a position on the sample stage; and wherein,

[0124] The wavelength of the laser source is equal to or less than 300 nm.

[0125] Therefore, the present invention provides an apparatus for analyzing a biological sample, comprising:

[0126] a sample stage comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a biological sample; and

[0127] a laser source, wherein the laser source has a wavelength of 300 nm or less; and

[0128] focusing optics comprising an objective lens, the focusing optics being adapted to direct a radiation beam from the laser source towards the second face to a position on the sample stage; and wherein,

[0129] The numerical aperture of the objective lens is at least 0.7, at least 0.8 or at least 0.9, for example at least 0.9.

[0130] As further described herein, the ablated material can be ionized or re-ionized near the sample surface, for example, by laser ionizing an ablation plume that has expanded beyond a significant (effective) charge neutralization point (primary ionization / re-ionization). In such systems and methods, a gas jet may not be required because the plume does not need to be delivered to the ICP for ionization. Alternatively, ion optics located near the sample can direct the ionized labeled atoms directly to a mass spectrometer (e.g., a TOF mass spectrometer or a magnetic sector mass spectrometer). As described herein, in some cases, a small ablation spot size can reduce the neutralization and space charge effects of ions produced during ablation, making it easier to ionize and direct ions to a mass spectrometer. In this case, a second pulse for re-ionization is not required. The sample stage and directly surrounding optics (e.g., high NA lenses and / or immersion media) can operate under vacuum or low pressure (e.g., to reduce charge by collision), as further described herein.

[0131] As will be appreciated by those skilled in the art, the apparatus may include focusing optics with an objective lens having a numerical aperture of at least 0.9 and / or a laser source having a wavelength of 300 nm or less. Furthermore, although high numerical aperture or short wavelength may be used as an alternative or supplement to the immersion lens described herein, the other components of the system remain the same as the immersion lens system.

[0132] Scanning system

[0133] In certain aspects, ablation of a sample can be performed by a scanning system. A radiation source (e.g., a laser beam or a charged particle beam (e.g., an ion beam or electron beam) source) can scan a portion of a sample to produce a single transient (e.g., a single instance of ablated material). The transient can be an ablation plume that is delivered to the ICP for atomization and ionization prior to detection by the MS. Alternatively, the transient can be ionized by laser radiation at or near the sample surface, as described herein. Scanning with an ion beam or electron beam (e.g., using ion optics) can allow a small region of interest (e.g., a single organelle) of a sample to be ablated with high resolution. For example, the area of ​​the region of interest can be less than 100,000 nm 2 , less than 50,000nm 2 , or less than 20,000nm 2 , or less than 10,000nm 2 Alternatively, the region of interest may be larger, for example, a single cell or even a group of cells.

[0134] In certain embodiments, a laser scanning system directs laser radiation onto a sample to be ablated. Since a laser scanner can redirect the position of the laser focus on a sample faster than moving the sample stage relative to a stationary laser beam (due to much lower or no inertia in the operating components of the scanning system), ablation of discrete points on the sample can be performed faster. This faster speed can allow significantly larger areas to be ablated and recorded as a single pixel, or the speed at which the laser spot moves can simply translate into, for example, an increase in the pixel acquisition rate, or a combination of both. In addition, the rapid change in the position of the spot to which the laser radiation pulses can be directed allows for ablation of arbitrary patterns, for example, such that a train of pulses / shots of laser radiation directed in rapid succession to locations on a sample by a laser scanning system ablates an entire cell of uneven shape, which is then ionized and detected as a single cloud of material, thereby enabling single cell analysis (see the "Sample Chamber of a Laser Ablation Sampling System" section of the PCT publication starting at page 33). Similar rapid burst pulse techniques can also be deployed in methods that use release to remove sample material from a sample carrier, i.e., cell LIFTing (laser induced forward transfer).

[0135] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0136] (i) a sampling and ionization system for removing material from a sample and ionizing said material to form elemental ions, including a laser scanning system and a sample stage;

[0137] (ii) a detector for receiving elemental ions from the sampling and ionization system and detecting the elemental ions.

[0138] Likewise, as will be appreciated by those skilled in the art, this combination of features may be combined with any other embodiment described herein. Using a scanning system to increase the acquisition rate provides many advantages over other strategies for increasing the sample imaging rate. For example, an area of ​​100 μm x 100 μm can be ablated with a single laser pulse using appropriate equipment. However, such ablation can lead to a number of problems. Ablating a large area of ​​a sample at once with a single laser pulse results in the ablated material breaking into large chunks that initially fly at speeds close to the speed of sound, and rather than small particles, rather than material that is quickly carried away from the sample in the carrier gas flow (described in more detail below), large chunks may take longer to be carried away than small chunks (extending the flushing time of the sample chamber), fail to be carried away, or simply randomly fly out of the sample or onto another part of the sample. If a large chunk of material flies out of the sample, any information in the form of detectable atoms in the large chunk of material (e.g., marker atoms) will be lost. If a chunk of material lands on another part of the sample, information about the ablated area is lost, and any detectable atoms in the chunk of material are now on top, interfering with the signal acquired from another part of the sample. Larger ablation spot sizes can also complicate sample fragmentation because differences in biological material in the ablation spot (e.g., cartilage material versus muscle) can also affect how the product is fragmented, with some types of material being carried away in the gas flow to a lesser extent than others. In addition, as described herein, in many applications, small spot sizes are preferred, on the order of μm rather than 100 times μm, and switching between laser spot sizes of multiple orders of magnitude (e.g., 100 μm versus 1 μm) also presents technical challenges. For example, a laser capable of ablating a 1 μm spot size may not have the energy to ablate a 100 μm spot size area in a single laser pulse, and complex optics are required to facilitate switching between 1 μm and 100 μm without significant loss of energy in the laser beam or loss of sharpness of the ablation spot.

[0139] Therefore, instead of ablating 100 μm, 100×100 (i.e., 10,000) 1 μm diameter spots can be used to ablate the area by raster scanning across the area. 2 A single spot of light. Smaller spot sizes for ablation are naturally not affected to any great extent by the above problems, as the particles produced by smaller ablation spots are themselves necessarily much smaller in size. In addition, with smaller spots, the smaller particles produced by ablation have shorter and more defined washout times from the sample chamber. This in turn leads to faster acquisition of data when each smaller spot needs to be resolved individually, since the transients of each ablation laser pulse do not overlap (or overlap to an acceptable degree, as described below) when detected in the detector.

[0140] However, as mentioned above, moving the stage in 1 μm increments along a row and then down is relatively slow due to inertia. Therefore, by using the laser scanning system to raster scan across the area without moving the stage, or moving the stage less frequently or at a constant speed, the relatively slow speed of the stage does not limit the rate at which the sample is ablated.

[0141] Therefore, in order to be able to scan quickly, the laser scanning system must be able to quickly switch the location where the laser radiation is directed on the sample. The time taken to switch the ablation location of the laser radiation is called the response time of the laser scanning system. Therefore, 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.

[0142] The laser scanning system can direct the laser beam in at least one direction relative to a sample stage carrying the sample during the ablation process. In some cases, the laser scanning system can direct the laser radiation in two directions relative to the sample stage. For example, the sample stage can be used to incrementally move the sample in the X-axis, and the laser can be scanned across the sample in the Y-axis (see Figure 7-Figure 9 When using a 1 μm spot size, movement in the X-axis can be in 1 μm increments. At a given position in the X-axis, the laser scanning system can be used to direct the laser to a series of positions in the Y-axis that are 1 μm apart. Because the laser scanning system can direct the laser radiation to different positions in the Y-axis much faster than the stage can be moved in increments in the X-axis, the ablation rate is significantly increased in this simple illustration of the scanner operation.

[0143] In some cases, the laser scanning system directs the laser beam in both the X-axis and the Y-axis. Thus, in such cases, more advanced ablation patterns can be produced. For example, when the laser scanning system can direct the laser radiation in both the X-axis and the Y-axis, the sample stage can move at a constant speed in the X-axis (thereby eliminating the inefficiencies associated with the inertia of the sample stage during movement across each row, rather than accelerating / decelerating at the beginning / end of the row), while the laser scanning system directs the laser radiation pulses up and down on the sample while compensating for the movement of the sample stage. To achieve such 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 will be appreciated by those skilled in the art, it may be desirable to apply a sawtooth drive signal to the scanner in the Y-direction, depending on the processing algorithm used. As another alternative, one of the scanner components may be pre-rotated slightly to pre-compensate for a tilted scanning pattern. In some embodiments, the controller of the laser scanning system will cause the laser scanning system to move the beam in a figure-8 pattern as the sample stage moves.

[0144] If the laser used in the laser sampling system has a sufficiently high repetition rate (as described below), the significantly faster (re)direction of the laser radiation to different locations on the sample correspondingly enables faster ablation of large areas of the sample. For example, if less than 5 pulses per second can be directed to different locations on the sample, the time required to study a 1mm×1mm area by ablation with a spot size of 1μm will be more than two days. At a frequency of 200Hz, this will be about 80 minutes, and the analysis time is further reduced as the pulse frequency is further increased. However, the samples are usually much larger. The average microscope slide on which a tissue section can be placed is 25×75mm. Ablation at a rate of 200Hz requires about 110 days. However, this time can be significantly shortened if a laser scanning system is used, for example, the sample stage moves at a constant speed (1mm / s) along the X-axis, while the laser scanning system moves the laser beam back and forth in the Y-axis direction. 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, the frequency is 500Hz. This will produce a 1μm spacing between adjacent lines in the raster pattern at this speed. The degree to which the laser scanning system deflects the laser radiation is then selected to match the maximum laser repetition rate. Here, to produce a peak-to-peak amplitude of 100 microns, a laser repetition rate of 100kHz is required. This enables the device to handle 0.1mm 2 / s, while current equipment can only process 0.0004mm at most 2 / s. Compared to the 110-day figure discussed above, using the laser scanning system discussed in this paragraph, it would only take about 5 hours to process the slides.

[0145] Another application is the shaping of arbitrary ablation areas. If a high repetition rate laser is used, a train of dense laser pulses can be delivered in the same time that a nanosecond laser delivers one pulse. By quickly adjusting the X and Y positions of the ablation spot during a train of laser pulses, ablation pits of arbitrary shapes and sizes (down to the diffraction limit of light) can be produced. For example, the n and n+1 positions in the pulse train can be no more than a distance equal to 10 times the diameter of the laser spot (based on the center of the ablation spot of the nth spot and the (n+1)th spot), for example, less than 8 times, less than 5 times, less than 2.5 times, less than 2 times, less than 1.5 times, about 1 times, or less than 1 times the spot diameter. Specific methods using this technology will be discussed in the method section below (page 32 of the PCT publication).

[0146] Thus, in some embodiments, the laser scanning system includes a positioner to provide a first relative motion of a laser beam emitted by the laser with respect to the sample stage (eg, with respect to the Y-axis of the sample surface).

[0147] In some embodiments, the positioner of the laser scanning system can provide a second relative motion of the laser beam relative to the sample stage, wherein the first relative motion and the second relative motion are not parallel, for example, wherein the relative motions are orthogonal (e.g., the first motion direction is on the Y-axis relative to the sample surface and the second motion direction is on the X-axis relative to the sample surface).

[0148] In some embodiments, the laser scanning system also includes a second positioner capable of providing a second relative motion of the laser beam relative to the sample stage, wherein the first relative motion and the second relative motion are not parallel, for example, wherein the relative motions are orthogonal (e.g., the first motion direction is on the Y-axis relative to the sample surface and the second motion direction is on the X-axis relative to the sample surface).

[0149] Any component capable of rapidly directing laser radiation to different locations on a sample can be used as a positioner in a laser scanning system. The various types of positioners discussed below are commercially available and can be selected by the skilled person based on the specific application in which the device will be used, as each has inherent strengths and limitations. In some embodiments of the present invention, as described below, multiple positioners discussed below can be combined in a single laser scanning system. Positioners can generally be grouped into positioners that rely on moving parts to introduce relative motion into the laser beam (examples of which include galvanometer mirrors, piezoelectric mirrors, MEMS mirrors, polygon scanners, etc.) and those that do not require moving parts (examples include such acousto-optic devices and electro-optic devices). The types of positioners listed in the previous sentence are used to controllably deflect the laser radiation beam to different angles, which results in translation of the ablation spot. The laser scanning system may include a single positioner, or may include a positioner and a second positioner. The description of two positioners in a laser scanning system, "positioner" and "second positioner", does not define the order in which the laser radiation pulses are incident on the positioners on their path from the laser source to the sample.

[0150] Galvanometer motors with mirrors mounted on the shaft can be used to deflect laser radiation to different locations on the sample. Movement can be achieved by using a fixed magnet and a moving coil, or a fixed coil and a moving magnet. The arrangement of a fixed coil and a moving magnet produces a faster response time. Typically, sensors are present in the motor to sense the position of the shaft and mirror to provide feedback to the controller of the motor. One galvanometer mirror can steer a laser beam in one axis, so using this technique pairs of galvanometer mirrors can be used to achieve the direction of the beam in both the X and Y axes.

[0151] One advantage of a galvanometer mirror is that it is able to achieve large deflection angles (e.g., much larger than a solid-state deflector), thereby allowing for less movement of the sample stage. However, since the moving parts of the motor and mirror have mass, they will be affected by inertia, so the time for the components to accelerate must be taken into account in the sampling method. Typically, non-resonant galvanometer mirrors are used. Those skilled in the art will understand that resonant galvanometer mirrors can be used, but a device using only such a resonant component as a positioner for a laser scanning system will not be able to perform arbitrary (also known as random access) scanning patterns. Because it is based on a mirror, the galvanometer mirror deflector will reduce the quality of the laser radiation beam and increase the size of the ablation spot, so those skilled in the art will again understand that it is most suitable for situations where such effects on the beam can be tolerated.

[0152] Galvanometer mirror-based devices may be susceptible to positioning errors due to sensor noise or tracking errors. Therefore, in some embodiments, each mirror is associated with a position sensor that feeds back the mirror's position 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 that is serially connected to the galvanometer mirror, which corrects for mirror positioning errors.

[0153] Galvanometer mirror systems and components are available from various manufacturers, for example, Thorlabs (New Jersey, USA), Laser2000 (United Kingdom), ScanLab (Germany), and Cambridge Technology (Massachusetts, USA).

[0154] In an embodiment that includes only a galvanometer mirror-based positioner, the rate at which ablation laser pulses can be directed to the sample can be between 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-1 MHz, 5 kHz-1 MHz, 10 kHz-1 MHz, 50 kHz-1 MHz, 100 kHz-1 MHz, 1 kHz-100 kHz, or 10 kHz-100 kHz.

[0155] Thus, in some embodiments of the present invention, the laser scanning system includes one or more positioners that are galvanometer mirrors, such as a galvanometer mirror array.

[0156] Figure 1 1 is a schematic diagram of the optical devices of the existing device assembly. Here, a laser source (e.g., a pulsed laser source, optionally including a pulse picker) 101 emits a laser radiation beam, guides the laser radiation beam through an energy control module 102, and then through an optical device 103. The optical device 103 may include a beam shaping optical device. The beam / illumination combining optical device 104 then guides the radiation beam through a focusing optical device and an objective lens 105 to the sample. The sample can be placed on a support, such as a slide 107, on a three-axis (i.e., x, y, z) translation stage 108 located in a sample chamber 106. Figure 1 The assembly also includes a camera 111 for viewing the sample using the same focusing optics and objective 105. An illumination source 109 emits visible light, which is directed by illumination / inspection separation optics 110 through beam / illumination combining optics 104 and focusing optics 105 to the sample.

[0157] In more advanced configurations, the optics (e.g., optics 103) may also include a positioner (e.g., a movable mirror or scanner as described herein) to enable scanning of the laser light across the sample. For example, a positioner (e.g., a galvanometer mirror, a piezoelectric mirror, a MEMS mirror, or a polygon scanner) deflects the laser radiation beam before it is shaped by the beam shaping optics of optics 203. For example, a single mirror in a galvanometer mirror-based device allows the beam to be scanned in one direction, e.g., relative to the Y-axis of the sample. The deflection introduced by the positioner propagates through the optical system, resulting in ablation of different locations on the sample depending on the position of the mirror. The positioner may be coordinated with a position on the sample stage (e.g., by a controller) to determine a specific location on the sample to be ablated by the laser radiation beam. The controller may also be connected to the laser source to coordinate the generation of laser pulses (such that the laser source generates pulses when the positioner is in a defined position, rather than when it is moved between positions).

[0158] However, instead of a single mirror positioner, a pair of mirror positioners may be used to introduce deflection into the laser radiation beam. As described elsewhere herein, the mirror pair may be arranged to provide scanning in two orthogonal directions (X and Y), which may compensate for movement of the sample on the sample stage.

[0159] Similarly, a piezoelectric actuator on a shaft mounted with a mirror can be used as a positioner to deflect the laser radiation to different locations on the sample. Again, as a mirror positioner based on the movement of a component with mass, there is inherent inertia and an inherent time overhead in the movement of the mirror caused by the component. Therefore, those skilled in the art will understand that this positioner can be applied in certain embodiments where the nanosecond response time of the laser scanning system is not mandatory. Similarly, because it is based on a mirror, the piezoelectric mirror positioner may reduce the quality of the laser radiation beam and increase the size of the ablation spot, so those skilled in the art will again understand that it is most suitable for use in situations where such effects on the beam can be tolerated.

[0160] In a piezoelectric mirror based tilt mirror configuration, the laser radiation is directed onto the sample in both the X-axis and the Y-axis provided in a single component.

[0161] Piezoelectric mirrors are commercially available from suppliers such as Physik Instrumente (Germany).

[0162] Therefore, in some embodiments of the present invention, the laser scanning system includes a piezoelectric mirror, for example, a piezoelectric mirror array or a tilting mirror.

[0163] In embodiments that include only piezoelectric mirror-based positioners (e.g., piezoelectric mirror arrays or tilting mirrors), the rate at which ablation laser pulses can be directed at the sample can be between 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-1 MHz, 5 kHz-1 MHz, 10 kHz-1 MHz, 50 kHz-1 MHz, 100 kHz-1 MHz, 1 kHz-100 kHz, or 10 kHz-100 kHz. A third type of positioner is a MEMS (micro-electromechanical system) mirror, which relies on physical motion to direct laser radiation toward the surface of the sample. The micro-mirrors in this component can be driven by electrostatic, electromechanical, and piezoelectric effects. Many advantages of this type of component derive from their small size, such as light weight, ease of positioning in an apparatus, and low power consumption. However, since the deflection of the laser radiation is still ultimately based on the motion of a part in the component, the part will experience inertia. Again, because it is mirror based, the MEMS mirror positioner will degrade the laser radiation beam quality and increase the ablation spot size, so again those skilled in the art will understand that this scanner component can therefore be applied in situations where such effects on the laser radiation are tolerated.

[0164] MEMS mirrors are commercially available from suppliers such as Mirrorcle Technologies (California, USA), Hamamatsu (Japan), and Preciseley Microtechnology Corporation (Canada).

[0165] Therefore, in some embodiments of the present invention, the laser scanning system includes a MEMS mirror.

[0166] In an embodiment that includes only a MEMS mirror-based positioner, the rate at which ablation laser pulses can be directed to the sample can be between 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-1 MHz, 5 kHz-1 MHz, 10 kHz-1 MHz, 50 kHz-1 MHz, 100 kHz-1 MHz, 1 kHz-100 kHz, or 10 kHz-100 kHz. Another type of positioner is a polygon scanner, which relies on physical motion to direct laser radiation toward the surface of the sample. Here, a reflective polygon or multifaceted mirror rotates on a mechanical axis and an angularly deflected scan beam is produced each time a plane of the polygon crosses the incident beam. A polygon scanner is a one-dimensional scanner that can direct the laser beam along a scan line (thus requiring an auxiliary positioner to introduce a second relative motion in the laser beam relative to the sample, or the sample needs to be moved on the sample stage). Once the end of a line of a raster scan is reached, the beam is directed back to the position where the scan line began, as opposed to the back-and-forth motion of, for example, a galvanometer-based scanner. The polygons can be regular or irregular depending on the application. The spot size depends on the facet size and flatness, 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, such positioners actually have the disadvantage of having low positioning / feedback accuracy due to facet manufacturing tolerances and axial runout and potential wavefront distortion from the mirrors. The skilled person will again understand that such scanner components can therefore be applied in situations where such influences on the laser radiation are tolerated.

[0167] Polygon scanners are commercially available, for example, from Precision Laser Scanning (Arizona, USA), II-VI (Pennsylvania, USA), Nidec Copal Electronics Corp (Japan), etc.

[0168] In an embodiment that includes only a polygon scanner based positioner, the rate at which ablation laser pulses can be directed to the sample can be between 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-10 MHz, 5 kHz-10 MHz, 10 kHz-10 MHz, 50 kHz-10 MHz, 100 kHz-10 MHz, 1 kHz-1 MHz, 10 kHz-1 MHz, or 100 kHz-1 MHz. Unlike the components of the laser scanning systems of the aforementioned type, EODs are solid state components, i.e., they contain no moving parts. Therefore, they have no mechanical inertia in deflecting the laser radiation and therefore have very fast response times of the order of about 1 nanosecond. They are also not as susceptible to wear as mechanical components. The EODs are formed of an optically transparent material (e.g., a crystal) whose refractive index varies depending on an electric field applied thereto, which in turn is controlled by a voltage applied to the medium. The refraction of the laser radiation is caused by the introduction of a phase delay in the cross section of the beam. If the refractive index varies linearly with the electric field, this effect is called the Pockels effect. If it varies quadratically with the field strength, this 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 optical prism interfaces and on refraction of a refractive index gradient perpendicular to the direction of propagation of the laser radiation. In order to apply an electric field on the EOD, electrodes are bonded to opposite sides of an optically transparent material that acts as a medium. Combining a set of opposing electrodes produces a one-dimensional scanning EOD. Combining a second set of electrodes orthogonally to the first set of electrodes produces a two-dimensional (X, Y) scanner.

[0169] For example, the deflection angle of an EOD is smaller than that of a galvanometer mirror, but the deflection angle can be increased if required for a given equipment assembly by placing several EODs in sequence. Exemplary materials for the refractive medium in an EOD include potassium tantalate niobate (KTN). x Nb 1-x O3), LiTaO3, LiNbO3, BaTiO3, SrTiO3, SBN (Sr 1-x Ba x Nb2O6) and KTiOPO4, under the same field strength, KTN shows a larger deflection angle.

[0170] The angular accuracy of the EOD is very high, depending mainly on the accuracy of the drivers connected to the electrodes. In addition, as mentioned above, the response time of the EOD is very fast, even faster than that of the AOD discussed below (due to the fact that the (changing) electric field in the crystal is established at the speed of light in the material rather than at the speed of sound in the material; see and Bechtold, 2014, Physics Procedia 56: 29–39).

[0171] Therefore, in some embodiments of the present invention, the laser scanning system includes an EOD. In some embodiments, the EOD is an EOD with two sets of electrodes orthogonally connected to a refractive medium.

[0172] In embodiments including EOD-based positioners, the rate at which ablative laser pulses can be directed to the sample can be between 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz, 10 kHz-100 MHz, 50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10 MHz-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. Such positioners are also solid-state elements. The deflection of the element is based on the propagation of sound waves in an optically transparent material to cause a periodic change in the refractive index. The change in the refractive index occurs due to compression and rarefaction (i.e., density changes) of the material caused by the propagation of sound waves in the material. The periodically changing refractive index diffracts the laser beam propagating through the material like a grating.

[0173] An AOD is produced by bonding a transducer (usually a piezoelectric element) to an acousto-optic crystal (e.g., TeO2). The transducer, driven by an electronic amplifier, introduces sound waves into a refractive medium. At the opposite end, the crystal is usually beveled and fitted with sound-absorbing material to avoid reflection of the sound waves back into the crystal. When the wave propagates through the crystal in one direction, a one-dimensional scanner is created. A two-dimensional scanner can be created by placing two AODs orthogonally in series, or by bonding two transducers on orthogonal crystal planes.

[0174] For EOD, the AOD has lower deflection angles than galvanometer mirrors, but the angle accuracy is high compared to such mirror-based scanners, and the frequency of the drive crystal is digitally controlled and can typically be resolved to 1 Hz. and Bechtold (2014) pointed out that the drift common to galvanometer-based scanners and the temperature dependence compared to analog controllers is not usually a problem encountered with AODs.

[0175] Exemplary materials for use as refractive media for AODs include tellurium dioxide, fused quartz, crystalline quartz, sapphire, AMTIR, GaP, GaAs, InP, SF6, lithium niobate, PbMoO4, arsenic trisulfide, tellurite glass, lead silicate, Ge 55 As 12 S 33 , mercury(i) chloride and lead(II) bromide.

[0176] To change the deflection angle, the frequency of the sound introduced into the crystal must be changed, and it takes a finite time for the sound wave to fill the crystal (depending on the speed of the sound wave in the crystal and the size of the crystal), which means there is a certain degree of delay. However, compared to laser system positioners based on moving parts, the response time is relatively fast.

[0177] Another property of an AOD that can be exploited in certain circumstances is that the acoustic power applied to the crystal determines the amount of diffraction of the laser radiation relative to the zero-order (i.e., undiffracted) beam. The undiffracted beam is typically directed toward a beam dump. Thus, an AOD can be used to efficiently control (or modulate) the intensity and power of a deflected beam at high speed.

[0178] The diffraction efficiency of an AOD is generally nonlinear, and therefore, the diffraction efficiency versus power curve can be mapped for different input frequencies. The mapped efficiency curve for each frequency can then be recorded as an equation or in a lookup table for subsequent use in the devices and methods disclosed herein.

[0179] Therefore, in some embodiments of the present invention, the laser scanning system includes an AOD.

[0180] A solid-state positioner (e.g., AOD or EOD) can replace the rotating mirror and be used to introduce deflection into the laser radiation beam instead of a mirror-based positioner. As described elsewhere herein, a solid-state scanner can scan in two orthogonal directions (X and Y) by attaching orthogonal electrodes to the EOD medium, or by arranging two AODs in orthogonal series.

[0181] In embodiments including an AOD-based positioner, the rate at which ablation laser pulses can be directed to the sample can be between 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz, 10 kHz-100 MHz, 50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10 MHz-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. In the preceding paragraphs, two types of laser scanning system positioners were discussed: mirror-based, including moving parts; and solid-state positioners. The former are characterized by large deflection angles, but relatively slow response times due to inertia. In contrast, solid-state positioners have a smaller range of deflection angles, but faster response times. Therefore, in some embodiments of the present invention, the laser scanning system includes a mirror-based component and a solid-state component connected in series. This arrangement takes advantage of the advantages of both, e.g., the large range provided by mirror-based components, but takes into account the inertia of mirror-based components. See, e.g., Matsumoto et al., 2013 (Journal of Laser Micro / Nanoengineering 8:315:320).

[0182] Thus, a solid-state positioner (i.e., AOD or EOD) may be used, for example, to correct for errors in a mirror-based scanner component. In this case, a position sensor associated with the mirror position provides feedback to the solid-state component, and the deflection angle introduced into the laser radiation beam by the solid-state component may be appropriately altered to correct for position errors in the mirror-based scanner component.

[0183] An example of a combined system includes a galvanometer mirror and an AOD (wherein the AOD can achieve deflection in one or two directions (by using two AODs in series, or by coupling two drivers to orthogonal planes of the crystal of a single AOD)). The system may include two galvanometer mirrors to combine with an AOD (wherein the AOD can achieve deflection in one or two directions (by using two AODs in series, or by coupling two drivers to orthogonal planes of the crystal of a single AOD)) to produce a two-dimensional scanning system. In such a system, the rate at which ablation laser pulses can be directed to the sample can be between 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz, 10 kHz-100 MHz, 50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10 MHz-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. Alternative examples of combined systems include galvanometer mirrors and EODs (where the EODs can deflect in one or two directions (by coupling two orthogonally arranged electrodes to the crystal)). The system can include two galvanometer mirrors to combine with the EODs (where the EODs can achieve deflection in one or two directions (by coupling two orthogonally arranged electrodes to the crystal)) to produce a two-dimensional scanning system. In such a system, the rate at which ablation laser pulses can be directed to the sample can be between 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 200 Hz-50 kHz, 200 Hz-10 kHz, 1 kHz-100 MHz, 5 kHz-100 MHz, 10 kHz-100 MHz, 50 kHz-100 MHz, 100 kHz-100 MHz, 1 MHz-100 MHz, 10 MHz-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. To control the positioner of the laser scanning system, the laser scanning system can include a scanner control module (e.g., a computer or programming chip) that coordinates the movement of the positioner in the Y-axis and / or X-axis and the movement of the sample stage. In some cases, such as rastering back and forth, the appropriate pattern will be pre-programmed into the chip. However, in other cases, the control module can apply a reverse motion to determine the appropriate ablation pattern to follow. Reverse motion may be particularly useful, for example, when generating arbitrary ablation patterns to map optimal ablation processes 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, also by coordinating the operation of a pulse picker.

[0184] Sometimes, a positioner causes the laser radiation beam it directs to disperse. Therefore, in some embodiments of the apparatus described herein, the laser scanning system includes at least one dispersion compensator between the positioner and / or the second positioner and the sample, the dispersion compensator being suitable for compensating for any dispersion caused by the positioner. When 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 (i.e., appropriate material, thickness, and prism angle) suitable for compensating for the dispersion caused by the positioner and / or the second positioner; (iii) a combination of a diffraction grating (i) and a prism (ii); and / or (iv) another acousto-optic device. In the case where the first positioner causes dispersion and the second positioner causes dispersion, the laser scanning system may include a first dispersion compensator that compensates for any dispersion caused by the first positioner and a second dispersion compensator that compensates for any dispersion caused by the second positioner. WO03 / 028940 describes how the dispersion caused by an AOD positioner can be compensated using another appropriately adjusted AOD.

[0185] Sometimes, the focal length of the radiation beam can vary relative to the position of the sample due to movement of a positioner that directs the laser radiation to different locations. This can be compensated for in a number of ways. For example, a movable focusing lens can be moved so as to maintain a constant or near constant diameter spot size on the sample, regardless of the specific location on the sample to which the laser radiation is directed. Alternatively, an adjustable focusing lens (available from Optotune) can be used. It is also possible to compensate for changes in spot size by changing the height of the sample stage in the z-axis. However, both of these techniques rely on moving parts, introducing timing overhead into the operation of the system. If the AOD is used with a Gaussian beam, the ablation spot size can be controlled by the power applied to the crystal in the AOD in order to rapidly modulate the first and zero order beam intensities.

[0186] exist Figures 2 to 4 In the alternative arrangement shown, the components may be similar to Figure 1 Such an arrangement may be preferred, for example, when it is desired to impart additional kinetic energy to the sample material being ablated, to assist in removing material from the area near the ablation spot. Alternatively or additionally, Figure 2 , Figure 3 and / or Figure 4 The configuration presented in may allow for smaller spot sizes as described herein. In some embodiments, ablation through a sample carrier may be combined with laser scanning optics.

[0187] Laser

[0188] In general, the choice of wavelength and power of the laser used for sample ablation can follow normal use in cell analysis. The laser must have sufficient fluence to ablate to the desired depth without significantly ablating the sample carrier. 0.1-5J / cm 2 Laser fluences between 3 and 4 J / cm are usually suitable, for example, 2 or about 3.5 J / cm 2 , and the laser would ideally be able to produce pulses with this flux at a rate of 200 Hz or more. In some cases, a single laser pulse from such a laser should be sufficient to ablate cellular material for analysis so that the laser pulse frequency matches the frequency at which the ablation plume is produced. In general, to be a useful laser for imaging biological specimens, the laser should produce pulses with a duration of less than 100 ns (preferably less than 1 ns) that can be focused to a specific spot size such as discussed herein.

[0189] For example, the ablation frequency of the laser system is in the range of 200 Hz-100 MHz, 200 Hz-10 MHz, 200 Hz-1 MHz, 200 Hz-100 kHz, 500 Hz-50 kHz, or in the range of 1 kHz-10 kHz.

[0190] At these frequencies, the instrument must be able to analyze the ablated material quickly enough to avoid substantial signal overlap between successive ablations if it is desired to resolve each ablation plume individually (which may not necessarily be necessary when bursts of pulses are fired at the sample, as described below). Preferably, the overlap in intensity between signals originating from successive plumes is less than 10%, more preferably less than 5%, and ideally less than 2%. The time required for plume analysis depends on the flushing time of the sample chamber (see Sample Chamber section below), the transit time of the plume aerosol to and through the laser ionization system, and the time required to analyze the ionized material. Each laser pulse can be associated with a pixel on a subsequently created sample image, as discussed in more detail below.

[0191] In some embodiments, the laser source comprises a laser with a nanosecond pulse duration or an ultrafast laser (pulse duration of 1 ps (10 -12s) or faster), for example, femtosecond lasers. Ultrafast pulse durations offer many advantages because they limit heat diffusion from the ablated area, thereby providing more precise and reliable ablation pits, and minimize debris scattering for each ablation event. Femtosecond lasers are particularly useful in the systems and devices described herein. In particular, femtosecond lasers are highly compatible with systems that include laser scanning components, and because of the short pulse duration, ablation techniques based on multiphoton events and electron implantation processes are implemented. Three characteristics of femtosecond lasers make them particularly suitable for this application. The first characteristic is the high repetition rate of a typical configuration on a femtosecond laser, typically between 1 MHz and 100 MHz. In contrast, the pulse frequency of nanosecond lasers is typically limited to less than 100 kHz. The second characteristic is the nonlinear ablation mechanism. Nonlinear ablation sharpens the definition of the ablation edge and allows ablation to be performed with higher spatial resolution. Third, the nonlinear ablation threshold can also be less material-specific, which makes it easier to obtain a consistent ablation spot size when studying inhomogeneous materials (e.g., tissue).

[0192] 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. Such short pulses are usually generated using active mode-locking techniques. Femtosecond lasers can be generated using a variety of laser types. Typical durations between 30 fs and 30 ps can be achieved using passively mode-locked solid-state bulk lasers. Similarly, various diode-pumped lasers (e.g., based on neodymium- or ytterbium-doped gain media) operate in this regime. Titanium-sapphire lasers with advanced dispersion compensation are even suitable for pulse durations of less than 10 fs, down to about 5 fs in extreme cases. Most often, pulse repetition rates are between 10 MHz and 500 MHz, but there are also low-repetition-rate versions with repetition rates of several megahertz for higher pulse energies (available from Lumentum (CA, USA), Radiantis (Spain), Coherent (CA, USA)). This type of laser can be equipped with an amplifier system for increasing the pulse energy.

[0193] There are also various types of ultrafast fiber lasers, which are also passively mode-locked in most cases, typically providing pulse durations between 50 and 500 fs and repetition rates between 10 and 100 MHz. Such lasers are available from, for example, NKTPhotonics (Denmark; formerly Fianium), Amplitude Systems (France), Laser-Femto (California, USA). The pulse energy of such lasers can also be increased by an amplifier, usually in the form of an integrated fiber amplifier.

[0194] Some mode-locked diode lasers can generate pulses of femtosecond duration. Directly at the laser output, the pulse duration is typically of the order of a few hundred femtoseconds (available from, for example, Coherent (CA, USA)).

[0195] Femtosecond lasers are particularly well suited for use with immersion lenses, such as oil immersion lenses (described above). Oil immersion lenses can achieve laser spot diameters of about 160 nm, but because ablation using femtosecond lasers is a multiphoton process, the effective spot diameter is reduced when used with femtosecond lasers. times, where m is the order of magnitude of the nonlinear process. In this way, an effective spot diameter of less than 100 nm can be obtained. When used with a femtosecond laser, all possible configurations of the device including the immersion lens as described above can achieve an ablation spot size of 100 nm, so the device and method provided by the present invention provide a ten-fold improvement in spatial resolution compared to traditional IMC and IMS. The wavelength of femtosecond lasers is typically in the near-infrared region (700-1300 nm), which means that frequency conversion techniques must be used to shorten their wavelength in order to achieve an ablation spot diameter of 100 nm. The most direct and well-known of these is second harmonic generation (SHG), which can effectively convert near-infrared laser beams into visible wavelengths, where commercial off-the-shelf microscope optics can be used.

[0196] In some cases, picosecond lasers are used. Many of the types of lasers already discussed in the previous paragraphs can also be adapted to produce pulses with durations in the picosecond range. The most common sources are actively or passively mode-locked solid-state bulk lasers, e.g., actively mode-locked Nd-doped YAG, glass, or vanadate lasers. Likewise, picosecond mode-locked lasers and laser diodes are commercially available (e.g., NKT Photonics (Denmark), EKSPLA (Lithuania)).

[0197] Nanosecond pulse duration lasers (gain switched and Q switched) are also available in specific equipment packages (Coherent (CA, USA), Thorlabs (NJ, USA)). Nanosecond UV lasers are particularly suitable for use with the above-mentioned solid immersion lenses. In particular, nanosecond UV lasers with a wavelength of 266nm can be used with diamond and fused silica solid immersion lenses to focus light to a spot size of less than 100nm. As those skilled in the art will understand, laser ablation using UV lasers is generally a thermal process, which means that the area around the laser spot size will be affected by the heat from the ablation process. This sets a limit on the achievable ablation spot size, and therefore, those skilled in the art will accordingly understand that UV lasers will be suitable for some situations, but other lasers may be more useful in other situations.

[0198] Alternatively, continuous wave lasers can be used, externally modulated to produce pulses of sub-nanosecond duration.

[0199] Typically, the laser beam used for ablation in the laser systems discussed herein has a spot size of less than 100 μm, e.g., less than 50 μm, less than 25 μm, less than 20 μm, less than 15 μm, or less than 10 μm, e.g., less than about 3 μm, less than about 2 μm, less than about 1 μm, less than about 500 nm, less than about 250 nm, at the sampling location. In the case of using an immersion medium between the objective and the sample, (as described in part on page 8 of the PCT publication), smaller spot sizes can be obtained, e.g., less than 200 nm, less than 150 nm, or less than 100 nm. The distance referred to as the spot size corresponds to the longest internal dimension of the beam, e.g., for a circular beam it is the beam diameter, for a square beam it corresponds to the length of the diagonals between opposite corners, for a quadrilateral it is the length of the longest diagonal, and so on (as described above, the diameter of a circular beam with a Gaussian distribution is defined as the distance from the peak flux to the beam diameter). 2times the distance between points). As an alternative to a Gaussian beam, beam shaping and beam shielding can be employed to provide the desired ablation spot. For example, in some applications, a square ablation spot with a top-hat energy distribution may be useful (i.e., the beam has a near-uniform flux, as opposed to a Gaussian energy distribution). This configuration reduces the dependence of the ablation spot size on the ratio between the flux at the peak of the Gaussian energy distribution and the threshold flux. Ablation close to the threshold flux provides more reliable ablation crater generation and controls the generation of debris. Therefore, the laser system may include a beam shield and / or a beam shaping component, such as a diffractive optical element, which is disposed in a Gaussian beam to re-shame the beam and produce a laser focus of uniform or near-uniform flux, for example, with a flux variation across the beam of less than ±25%, for example, less than ±20%, ±15%, ±10%, or less than ±5%. Sometimes, the laser beam has a square cross-sectional shape. Sometimes, 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 location, i.e., the spot size is the lateral dimension of the beam, so, for example, the spot size of a circular beam is the diameter. However, those skilled in the art will appreciate that the focal spot of an objective is a three-dimensional volume, and the axial dimension of the focused spot size is typically longer than the lateral dimension, so in some cases the axial dimension of the focal spot may be longer than the distance referred to as the "spot size" in the context of the present invention. Beam shaping and masking can also be used to enable the device to switch between high-resolution spot sizes achievable with immersion lenses and larger spot sizes if desired by the user. In addition to high-resolution imaging under certain conditions, large spots can be used when project specifications require (e.g., where lower resolution is appropriate, or where a larger area of ​​tissue needs to be sampled and analyzed in a given time).

[0200] When used to analyze biological samples, in order to analyze individual cells, the spot size of the laser beam used will depend on the size and spacing of the cells. For example, in the case where cells are closely packed with each other (e.g., in a tissue slice), one or more laser sources in the laser system may have a spot size no larger than these cells. The size depends on the specific cells in the sample, but typically the diameter of the laser spot is less than 4 μm, for example, about 3 μm or less, about 2 μm or less, about 1 μm or less, about 500 nm or less, about 250 nm or less. In order to analyze a given cell at subcellular resolution, the system uses a laser spot size no larger than these cells, more specifically, a laser spot size that can ablate the material at subcellular resolution. The smaller the spot size, the greater the image resolution obtained. Therefore, in the case of high-resolution subcellular imaging, the techniques described herein can be used. Sometimes, single cell analysis can be performed using a spot larger than the size of the cell, for example, when the cells are spread on a slide with space between the cells (e.g., after analysis by electron microscopy, it has been determined that the internal structure of the cell is separated from the preliminary analysis of the cell). Here, larger spot sizes can be used and single cell characterization is achieved because the additional ablation area around the cell of interest does not include additional cells. Therefore, the specific spot size used can be appropriately selected based on the size of the cell being analyzed. In biological samples, cells are rarely all the same size, so if subcellular resolution imaging is required, the ablation spot size should be smaller than the smallest cell if a constant spot size is maintained throughout the ablation process. Small spot sizes can be achieved using laser beam focusing. A laser spot diameter of 1 μm corresponds to a laser focus of 1 μm (i.e., the diameter of the laser beam at the beam focus), but the laser focus can vary by +20% or more due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and changes in the total laser energy relative to the ablation threshold energy. Suitable objectives for focusing the laser beam include reflective objectives, such as those of the Schwarzschild Cassegrain design (reverse Cassegrain). Refractive objectives can also be used, as can combinations of reflective-refractive objectives. A single aspheric lens can also be used to achieve the desired focus. Solid immersion lenses or diffractive optics can also be used to focus the laser beam. Another method of controlling the laser spot size is to pass the beam through an aperture before focusing, which can be used alone or in combination with the objectives described above. Different beam diameters can be achieved by passing the beam through apertures of different diameters from a range of diameters. In some cases, there is a single aperture of variable size, for example, when the aperture is an aperture. Sometimes, the aperture is an iris. Variation of the spot size can also be achieved by dithering the optics. One or more lenses and one or more apertures are located between the laser and the sample stage.

[0201] For the sake of completeness, the standard lasers for LA with subcellular resolution known in the art are excimer or exciplex lasers. Suitable results can be obtained using argon fluoride lasers (λ=193 nm). The pulse duration of these lasers is 10-15 ns, which can achieve sufficient ablation for some applications.

[0202] In general, the laser pulse frequency and intensity are chosen in conjunction with the response characteristics of the MS detector to allow for distinct detection of individual laser ablation plumes. Combined with the use of a small laser spot and a sample chamber with short washout times, fast and high-resolution imaging is now feasible.

[0203] Laser ablation focus

[0204] To maximize the efficiency of the laser in ablating material from the sample, the sample should be in an appropriate position relative to the focal point of the laser, for example, at the focus, since the focus is where the laser beam has its smallest diameter and where the energy is most concentrated. This can be achieved in a number of ways. A first approach is that the sample can be moved on the axis of the laser pointed at it (i.e., up and down along the path of the laser / toward and away from the laser source) to a desired point where the light has sufficient intensity to achieve the desired ablation. Alternatively or in addition, a lens can be used to move the focus of the laser, thereby increasing its effective ability to ablate material at the sample location, for example, by microscaling. One or more lenses are located between the laser and the sample stage. A third approach is to change the position of the laser, which can be used alone or in combination with one or both of the above two approaches.

[0205] To assist the user of the system in placing the sample in the most appropriate position for ablating material from the sample, a camera can be directed toward a stage holding the sample (discussed in more detail below). Accordingly, the present disclosure provides a laser ablation sampling system that includes a camera directed toward a sample stage. The image detected by the camera can be focused to the same point at which the laser is focused. This can be achieved by using the same objective lens for both laser ablation and optical imaging. By aligning the focus of both, the user can be sure that laser ablation will be most effective when the optical image is focused. Precise movement of the stage, and thus focusing the sample, can be achieved using piezoelectric actuators, which are available from Physik Instrumente, Cedrat-technologies, Thorlabs, and other suppliers.

[0206] In another mode of operation, laser ablation is directed toward the sample through the sample carrier. In this case, the sample support should be selected to be transparent (at least partially transparent) to the frequency of the laser radiation used to ablate the sample. Ablation through the sample can be advantageous in certain circumstances because this mode of ablation can provide additional kinetic energy to the plume of material ablated from the sample, driving the ablated material further away from the sample surface, thereby facilitating the transport of the ablated material out of the sample for analysis in a detector.

[0207] In order to achieve 3D imaging of the sample, the sample or its limited area can be ablated to a first depth, which does not completely pass through the sample. After this, the same area can be ablated again to a second depth, and so on to a third, fourth, etc. depth. In this way, a 3D image of the sample can be established. In some cases, it may be preferred to ablate all areas to be ablated to a first depth before ablation at a second depth. Alternatively, ablation can be repeated at the same point to ablate through different depths before advancing to the next position of the ablation area. In both cases, the signal obtained on MS can be deconvoluted into the position and depth of the sample by imaging software. Thick tissue staining can be used, and the tissue is stabilized in a wet state, similar to the workflow used in confocal imaging (Clendenon et al., 2011, Microsc Microanal, 17: 614–617).

[0208] Sample chamber for laser ablation sampling system

[0209] When a sample is subjected to laser ablation, the sample is placed in a sample chamber. The sample chamber includes a stage that holds the sample (usually the sample is placed on a sample carrier). As ablation occurs, the material in the sample forms a plume, and the gas flow from the gas inlet through the sample chamber to the gas outlet carries away the plume of atomized material, including any labeled atoms at the ablation site. The gas carries the material to the ionization system, which ionizes the material so that it can be detected by the detector. The atoms in the sample, including the labeled atoms, can be distinguished by the detector, so their detection reveals the presence or absence of multiple targets in the plume, thereby determining which targets are present at the ablation site on the sample. The sample chamber therefore serves the dual purpose of containing the solid sample being analyzed and as the starting point for delivering the atomized material to the ionization and detection system. This means that the gas flow through the chamber affects how diffuse the ablated material plume is as it passes through the system. A measure of how diffuse the ablation plume is is the sample chamber flushing time. This number is a measure of the time required for the material ablated from the sample to be carried out of the sample chamber by the gas flowing through the sample chamber.

[0210] The spatial resolution of the signal generated by laser ablation in this manner (i.e., when ablation is used for imaging rather than specifically for clearing, as described below) depends on factors including: (i) the spot size of the laser, since the signal is integrated over the total area ablated; and the speed at which the plume is generated relative to the motion of the sample relative to the laser, and (ii) the speed at which plumes can be analyzed relative to the speed at which the plumes are generated, in order to avoid overlap of signals from successive plumes as described above. Thus, if individual plumes need to be analyzed, being able to analyze plumes in the shortest possible time can minimize the likelihood of plume overlap (and thus allow plumes to be generated more frequently).

[0211] Therefore, sample chambers with short washout times (e.g., under 100 ms) are advantageous for use with the apparatus and methods disclosed herein. Sample chambers with long washout times can limit the speed of image generation or can result in overlap between signals originating from consecutive sample spots (e.g., Kindness et al. (2003; Clin Chem 49: 1916-23), where signal duration exceeds 10 seconds). Therefore, aerosol washout time is a key limiting factor in achieving high resolution without increasing the total scan time. Sample chambers with washout times < 100 ms are known in the art. For example, Gurevich & (2007; J. Anal. At. Spectrom., 22: 1043-1050) discloses a sample chamber with a washout time of less than 100 ms. Wang et al. (2013; Anal. Chem. 85: 10107-16) (see also WO 2014 / 146724) discloses a sample chamber with a washout time of 30 ms or less, thereby allowing high ablation frequencies (e.g., greater than 20 Hz) and thus fast analysis. Another such sample chamber is disclosed in WO 2014 / 127034. The sample chamber in WO2014 / 127034 includes a sample capture unit configured to be operably arranged near a target, the sample capture unit including: a capture chamber having an opening formed in a surface of the capture unit, wherein the capture chamber is configured to receive a target material ejected or generated from a laser ablation site through the opening; and a guide wall exposed in the capture chamber, configured to guide a carrier gas flow in the capture chamber from an inlet to an outlet, so that at least a portion of the target material received in the capture chamber can be transferred to the outlet as a sample. The volume of the capture chamber in the sample chamber of WO2014 / 127034 is less than 1 cm 3 , and can be less than 0.005cm 3Sometimes, the sample chamber has a flushing 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 flushing time of the sample chamber may be 10 μs or more. Typically, the flushing time of the sample chamber is 5 ms or less.

[0212] For the sake of completeness, sometimes the plume from the sample may be generated more frequently than the flushing time of the sample chamber, and the resulting image will be blurred accordingly (eg, if the highest possible resolution is deemed unnecessary for the particular analysis being performed).

[0213] The 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 using an operating mode that requires the laser radiation to pass through the sample carrier to the direction of the sample, the stage holding the sample carrier should also be transparent to the laser radiation used.

[0214] Thus, when laser radiation is directed onto the sample, the sample can be positioned on a side of a sample carrier (e.g., a glass slide) facing the laser radiation, such that the ablation plume is released on and captured from the same side as the laser radiation is directed onto the sample. Alternatively, when laser radiation is directed onto the sample (i.e., the laser radiation passes through the sample carrier before reaching the sample), the sample can be positioned on a side of the sample carrier opposite to the laser radiation, and the ablation plume is released and captured on the opposite side of the laser radiation. Directing the laser radiation onto the sample through the sample carrier is particularly useful when an immersion medium (e.g., a solid immersion lens or a liquid immersion lens) is used.

[0215] One feature of the sample chamber is a wide range of movement, wherein the sample can be moved relative to the laser in the x-axis and y-axis (i.e. horizontally) (where the laser beam is directed onto the sample in the z-axis), the x-axis and y-axis being perpendicular to each other, a feature that is particularly useful for ablating specific portions of the sample in discrete regions. By moving the stage within the sample chamber and keeping the position of the laser fixed in the laser ablation sampling system of the apparatus, a more reliable and more accurate relative position can be obtained. The greater the range of movement, the greater the distance between discrete ablation regions. The sample is moved relative to the laser by moving the stage on which the sample is placed. Thus, the range of movement of the sample stage within the sample chamber is at least 10 mm in the x-axis and y-axis, for example, 20 mm in the x-axis and y-axis, 30 mm in the x-axis and y-axis, 40 mm in the x-axis and y-axis, 50 mm in the x-axis and y-axis, for example, 75 mm in the x-axis and y-axis. Sometimes, the range of movement is such as to allow the entire surface of a standard 25 mm x 75 mm microscope slide to be analyzed within the chamber. Of course, in order to be able to achieve subcellular ablation, in addition to a wide range of movement, the movement should be precise. Therefore, the stage can be configured to move the sample in increments of less than 10 μm on the x-axis and y-axis, for example, 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, 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 can be performed in increments of about 1 μm, for example, 1 μm ± 0.1 μm. Commercially available microscope stages can be used, for example, available from Thorlabs, Prior Scientific and Applied Scientific Instrumentation. Alternatively, the motorized stage can be constructed from components, providing a desired range of movement and appropriate precise movement based on a positioner, for example, Smaract's SLC-24 positioner. The movement speed of the sample stage will 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.

[0216] Of course, when there is a large range of movement of the sample stage in the sample chamber, the size of the sample chamber must be appropriate to allow for the movement of the sample stage. Therefore, the size of the sample chamber depends on the size of the sample involved, which in turn determines the size of the moving sample stage. Exemplary dimensions of the sample chamber have an inner chamber of 10×10 cm, 15×15 cm, or 20×20 cm. The depth of the chamber can be 3 cm, 4 cm, or 5 cm. The technician will be able to select the appropriate size based on the teachings of this article. The internal dimensions of the sample chamber for analyzing biological samples using a laser ablation sampler must be larger than the range of movement of the sample stage, for example, larger than at least 5 mm, for example, at least 10 mm. This is because if the wall of the chamber is too close to the edge of the stage, the carrier gas flow through the chamber will become turbulent, where the carrier gas flow carries the ablation plume of material away from the sample and into the ionization system. Turbulence disrupts the ablation plume, so that after the material plume is ablated and brought to the ionization system of the device, the material plume begins to spread instead of remaining as a compact ablation material cloud. The broader peak of the ablated material has a negative impact on the data produced by the ionization and detection systems because it causes interference due to peak overlap, ultimately resulting in data with lower spatial resolution unless the ablation rate is reduced to a rate that is no longer of experimental interest.

[0217] As described above, the sample chamber includes a gas inlet and a gas outlet to bring the material to the ionization system. However, it may also contain other ports used as inlets or outlets to direct the gas flow in the chamber and / or provide a gas mixture to the chamber, which is determined by the technician according to the specific ablation process being performed.

[0218] camera

[0219] In addition to identifying the most effective positioning of the sample for laser ablation, including a camera (e.g., a charge coupled device (CCD) based image sensor camera or an active pixel sensor based camera) or any other light detection device in the laser ablation sampling system enables a variety of further analysis and techniques. A CCD is a means of detecting light and converting it into digital information that can be used to generate an image. In a CCD image sensor, an array of capacitors that detect light are included, with each capacitor representing a pixel on a determined image. These capacitors can convert incident photons into electrical charges. These charges are then read out 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 containing an array of pixel sensors, each pixel containing a photodetector and an active amplifier, for example, a CMOS sensor.

[0220] A camera can be included in any laser ablation sampling system discussed herein. The camera can be used to scan the sample to identify cells of particular interest or areas of particular interest (e.g., cells with a particular morphology), or fluorescent probes specific to antigens or intracellular or structures. In certain embodiments, the fluorescent probe is a histochemical stain or antibody that also contains a detectable metal tag. Once such cells have been identified, laser pulses can be directed to these specific cells to ablate material for analysis, for example, in an automated (wherein the system identifies and ablates features / areas of interest, e.g., cells) or semi-automated process (wherein the user of the system (e.g., a clinical pathologist) identifies features / areas of interest, and then the system ablates in an automated manner). This enables the speed of analysis to be significantly increased because the entire sample does not need to be ablated to analyze a particular cell, but rather the cell of interest can be specifically ablated. This results in improved efficiency in the method of analyzing biological samples in terms of the time spent performing ablation, particularly in terms of the time spent interpreting the data from the ablation, and in terms of building images from it. Building images from data is one of the more time-consuming parts of the imaging process, so by minimizing the collected data to data from the relevant portion of the sample, the overall analysis speed is increased.

[0221] The camera can record images from a confocal microscope. Confocal microscopy is a form of optical microscopy that has 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. Confocal microscopy can be used to assess the cellular morphology of unstained samples, or to assess whether a cell is a discrete cell or part of a cluster of cells. Typically, the sample is specifically labeled with a fluorescent marker (e.g., a labeled antibody or a labeled nucleic acid). These fluorescent markers can be used to stain specific cell populations (e.g., expressing certain genes and / or proteins) or specific morphological features on cells (e.g., nuclei or mitochondria), and these areas of the sample are specifically identifiable when illuminated with light of the appropriate wavelength. Therefore, some systems described herein may include a laser for exciting fluorophores in a label used to label the sample. Alternatively, an LED light source can be used to excite the fluorophore. Non-confocal (e.g., wide-field) fluorescence microscopy can also be used to identify certain areas of a biological sample, but the resolution is lower than confocal microscopy.

[0222] An alternative imaging technique is two-photon excitation microscopy (also called nonlinear or multiphoton microscopy). This technique typically uses near-infrared light to excite fluorophores. Each excitation event absorbs two infrared light photons. Scattering in tissue is minimized by the IR. In addition, due to the multiphoton absorption, the background signal is strongly suppressed. The excitation spectra of the most commonly used fluorophores are in the 400-500nm range, and the laser used to excite two-photon fluorescence is in the near-infrared range. If a fluorophore absorbs two infrared photons simultaneously, it will absorb enough energy to rise to an excited state. The fluorophore will then emit a single photon, the wavelength of which depends on the type of fluorophore used, which can be detected.

[0223] When a laser is used to excite fluorophores for fluorescence microscopy, sometimes the laser is the same laser that produces the laser used to ablate material from a biological specimen, but the power used is insufficient to ablate material from the specimen. Sometimes the fluorophores are excited by the wavelength of light used by the laser to ablate the specimen. In other cases, different wavelengths can be used, for example, by generating different harmonics of the laser to obtain light of different wavelengths, or by utilizing different harmonics generated in a harmonic generation system, as described above, in addition to the harmonic used to ablate the specimen. For example, if the fourth and / or fifth harmonics of an Nd:YAG laser are used, the fundamental or the second to third harmonics can be used for fluorescence microscopy.

[0224] As an example technique combining fluorescence and laser ablation, the nucleus in a biological sample can be marked with an antibody or nucleic acid that is bound to a fluorescent moiety. Therefore, by stimulating the fluorescent marker and then using a camera to observe and record the position of the fluorescence, the ablation laser can be specifically directed to the nucleus or an area that does not include nuclear material. The sample is divided into the nucleus and cytoplasm area, which will be particularly used in the field of cell chemistry. By using an image sensor (e.g., a CCD detector or an active pixel sensor, such as a CMOS sensor), the process of identifying features / areas of interest and then ablating them can be fully automated by using a control module (e.g., a computer or a programming chip), which associates the position of the fluorescence with the x, y coordinates of the sample and then directs the ablation laser to the position. As part of the process, the first image taken by the image sensor can have a low objective lens magnification (low numerical aperture), which allows the study of samples over a large area. After this, it is possible to switch to an objective lens with a higher magnification to focus on specific features of interest, which have been determined to be fluorescent by optical imaging with a higher magnification. These features recorded as fluorescent can then be laser ablated. Using a lower numerical aperture lens first has the further advantage of increasing the depth of field, thus meaning that features buried in the sample can be detected with greater sensitivity than if screening was performed from the outset with a higher numerical aperture lens.

[0225] In methods and systems using fluorescence imaging, one or more lenses and / or one or more filters may be included in the fluorescence emission path from the sample to the camera. By including filters adapted to pass selected spectral bandwidths from one or more fluorescent labels, the system is adapted to handle chromatic aberration associated with emissions from the fluorescent labels. Chromatic aberration is the result of the inability of a lens to focus light of different wavelengths to the same focal point. Therefore, by including filters, the background in the optical system is reduced and the resulting optical image has a higher resolution. Another way to minimize the amount of emission light of undesirable wavelengths that reaches the camera is to intentionally attenuate the chromatic aberration of the lenses by using a series of lenses that are designed to transmit and focus light of the wavelengths transmitted by the filters, similar to the system explained in WO 2005 / 121864.

[0226] Higher resolution optical images are advantageous in this coupling of optical techniques and laser ablation sampling because the accuracy of the optical image determines the precision with which the ablation laser can be directed to ablate the sample.

[0227] Thus, in some embodiments disclosed herein, the apparatus of the present invention includes a camera. The camera can be used to identify features / regions of a sample online, e.g., specific cells, which can then be sampled, e.g., by firing a train of pulses at the feature / region of interest to ablate sample material from the feature / region of interest. When the train of pulses is directed at the sample, the material detected in the final plume can be a continuous event (the plumes produced by each individual ablation actually form a single plume, which is then transmitted for detection). Although each cloud of sample material formed by the aggregated plume from a location within the feature / region of interest can be analyzed together, the sample material in the plume from each different feature / region of interest remains discrete. That is, sufficient time is left between ablations of different features / regions of interest to allow ablation of sample material from the nth feature / region of interest before ablation of the (n+1)th feature / region of interest begins.

[0228] In another mode of operation that combines fluorescence analysis and laser ablation sampling, rather than analyzing the fluorescence of the entire slide before targeting laser ablation to those locations, a spot on the sample can be pulsed from the laser (at low energy so as to excite only the fluorescent portion of the sample, rather than ablating the sample), and if fluorescence emission of the expected wavelength is detected, the sample at that spot can be ablated by firing the laser at full energy at that spot, and the resulting plume analyzed by the detector, as described below. This has the advantage of maintaining the raster mode of analysis, but at a faster pace, because the fluorescence can be pulsed and tested and the results obtained immediately from the fluorescence (rather than taking the time to analyze and interpret the ion data from the detector to determine if the area is of interest), again enabling only the sites of interest to be targeted for analysis. Therefore, when applying this strategy to imaging a biological sample containing multiple cells, the following steps can be performed: (i) labeling multiple different target molecules in the sample with one or more different labeling atoms and one or more fluorescent labels to provide a labeled sample; (ii) illuminating a known location of the sample with light to excite one or more fluorescent labels; (iii) observing and recording whether there is fluorescence at the location; (iv) if there is fluorescence, directing laser ablation to the location to form a plume; (v) performing inductively coupled plasma mass spectrometry on the plume, and (vi) repeating steps (ii)-(v) for one or more other known locations on the sample, thereby detecting the labeled atoms in the plume, thereby allowing the construction of a sample image of the ablated area.

[0229] In some cases, a sample or sample carrier may be modified to include optically detectable (e.g., by optical or fluorescent microscopy) portions at specific locations. The fluorescent locations can then be used to locate the sample in the device. The use of such marked locations is useful, for example, in situations where the sample may have been visually examined "offline," i.e., in a piece of equipment other than the device of the present invention. Before an optical image with features / regions of interest is highlighted and the sample is transferred to a device according to the present invention, such an optical image may be marked, for example, by a physician, with features / regions of interest corresponding to specific cells. Here, by reference to the marked locations in the annotated optical image, the device of the present invention can identify the corresponding fluorescent locations by using a camera and calculate an ablation plan for the laser pulse locations accordingly. Therefore, in some embodiments, the present invention includes a directional controller module capable of performing the above steps.

[0230] In some cases, the selection of features / regions of interest may be performed using the apparatus of the present invention based on an image of the sample taken by a camera of the apparatus of the present invention.

[0231] Electron microscopy

[0232] In some embodiments of the invention, the apparatus further comprises means for performing electron microscopy.

[0233] In general, an electron microscope includes an electron gun (e.g., with a tungsten cathode), an electrostatic / electromagnetic lens, and an aperture that controls the light beam to direct it toward the sample in a sample chamber. The sample is held under a vacuum so that gas molecules cannot obstruct or diffract the electrons on their way from the electron gun to the sample. In transmission electron microscopy (TEM), electrons pass through the sample and are then deflected. The deflected electrons are then detected by a detector, such as a fluorescent screen or, in some cases, 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.

[0234] TEM requires ultrathin sections to allow enough electrons to pass through the sample so that an image can be reconstructed from the deflected electrons that strike the detector. Typically, TEM specimens are 100nm or thinner and are prepared by using an ultramicrotome. Biological tissue specimens are chemically fixed, dehydrated, and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers, and similar materials may need to be stained with heavy atom markers to obtain the desired image contrast, since unstained biological specimens in their natural unstained state rarely interact strongly with electrons to deflect them to allow recording of electron microscopy images.

[0235] As mentioned above, when thin sections are used, electron microscopy can be performed on samples that are also analyzed by IMS or IMC. Thus, a high-resolution structural image can be obtained by electron microscopy, for example, transmission electron microscopy, and this high-resolution image is then used to increase the resolution of the image data obtained by IMS or IMC beyond the resolution obtainable by ablation using laser radiation (due to the much shorter wavelength of electrons than photons). In some cases, electron microscopy and elemental analysis performed by IMS or IMC are performed on samples in a single device (since IMC / IMS are destructive processes, electron microscopy is performed before IMC / IMS)

[0236] Thus, the present invention provides an imaging mass cytometer or imaging mass spectrometer as described herein, which also includes an electron microscope, for example, including components as described above, for example, an electron source, such as an electron gun. As will be appreciated by those skilled in the art, the specific settings of the components will vary (for example, the direction in which electrons are directed onto the sample and the direction in which laser radiation is directed onto the sample), and a conventional setting of the components can be implemented without undue burden. In some cases, the sample is not moved within the device between the analysis performed by electron microscopy and the subsequent ablation. As will be appreciated 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 carries away particulate material in the plume produced by the ablation of the sample. Therefore, after completion of the electron microscopy analysis phase, the sample chamber will be allowed to return to a pressure close to atmospheric pressure before elemental analysis is performed.

[0237] In ICP and IMS devices including electron microscopes, the components may be arranged so that the laser radiation used for ablation is directed to the sample through the sample carrier, e.g. Figure 3 or Figure 4 As shown, the sample carrier may be part of the sample chamber wall, allowing the sample chamber to be maintained under vacuum for electron microscopy purposes. Thus, in some embodiments herein, the apparatus comprises an electron microscope and an immersion medium, such as a liquid immersion lens or a solid immersion lens.

[0238] Laser ablation

[0239] According to variations related to this document (e.g., not mandating the use of an ICP to ionize sample material, nor mandating the use of a TOFMS detector), laser ablation can be performed in a manner previously described, for example, by Giesen et al. in WO2014169394 of 2014. For example, as described herein, methods and systems for ionization at or near a sample surface can use ion optics to transfer labeled atoms directly from the sample to a mass spectrometer detector (e.g., a TOF detector or a magnetic sector detector) without requiring a gas jet to transport the sample to the ICP. In some cases, the methods and systems can use non-laser forms of radiation (e.g., electron beams or ion beams) instead of or in addition to lasers.

[0240] In embodiments where laser ablation is performed without continuous ionization of the ablated sample, the ablation plume may be delivered to an ICP-MS as described below.

[0241] Delivery catheter

[0242] The delivery conduit forms a connection between the laser ablation sampling system and the ionization system and allows the sample material plume generated by the laser ablation sample to be transferred from the laser ablation sampling system to the ionization system. A portion (or all) of the delivery conduit can be formed, for example, by drilling through a suitable material, to create a lumen (e.g., a lumen having a circular, rectangular, or other cross-section) for generating the delivery plume. The inner diameter of the delivery conduit is sometimes in the range of 0.2 mm to 3 mm. Sometimes, the inner diameter of the delivery conduit can vary along its length. For example, the delivery conduit can be tapered (thinned) at the end. The length of the delivery conduit is sometimes in the range of 1 cm to 100 cm. Sometimes, the length is no more than 10 cm (e.g., 1-10 cm), no more than 5 cm (e.g., 1-5 cm), or no more than 3 cm (e.g., 0.1-3 cm). Sometimes, the delivery conduit lumen is straight over the entire distance or nearly the entire distance from the ablation system to the ionization system. Other times, the delivery conduit lumen is not straight over the entire distance, but changes orientation. For example, the delivery conduit can be made to have a gentle 90° turn. This configuration allows the plume generated by sample ablation in a laser ablation sampling system to initially move in a vertical plane, while the axis at the entrance of the transfer conduit will point upward and move horizontally as it approaches the ionization system (e.g., an ICP torch that is typically oriented horizontally to utilize convection cooling). The transfer conduit can be straight, at least 0.1 cm, at least 0.5 cm, or at least 1 cm from the entrance orifice through which the plume enters or is formed. In general, the transfer conduit is adapted to minimize the time it takes to transfer material from the laser ablation sampling system to the ionization system.

[0243] Delivery catheter inlet, including sample cone

[0244] The delivery conduit includes an inlet in the laser ablation sampling system (particularly 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 delivery conduit receives sample material ablated from the sample in the laser ablation sampling system and delivers it to the ionization system. In some cases, the laser ablation sampling system inlet is the source of all gas flows along the delivery conduit to the ionization system. In some cases, the laser ablation sampling system inlet that receives material from the laser ablation sampling system is a hole in the conduit wall, and a second "delivery" gas flows along the conduit from a separate delivery flow inlet (for example, as disclosed in WO2014146724 and WO2014147260). In this case, the delivery gas accounts for a large proportion, and in many cases, most of the gas flows to the ionization system. The sample chamber of the laser ablation sampling system includes a gas inlet. Gas flows into the chamber through the inlet, generating a gas flow that flows out of the chamber through the inlet of the delivery conduit. The gas flow captures the plume of ablated material and entrains it into a transfer conduit (typically the laser ablation sampling system inlet of the transfer conduit is conical in shape, referred to herein as the sample cone), and out of the sample chamber into a conduit passing over the chamber. The conduit also has gas flowing into it from a separate transfer stream inlet, (on the left hand side of the figure, indicated by the transfer stream arrow). The assembly comprising the transfer stream inlet, the laser ablation sampling system inlet and beginning with the transfer conduit that transfers the ablated sample material to the ionization system may also be referred to as a flow cell (as in WO2014146724 and WO2014147260).

[0245] The transport flow performs at least three functions: flushes the plume entering the transport conduit in the direction of the ionization system and prevents the plume material from contacting the side walls of the transport conduit; forms a "protection zone" above the sample surface to ensure ablation is performed under a controlled atmosphere; and increases the flow rate in the transport conduit. Typically, the viscosity of the capture gas is lower than the viscosity of the transport gas. This helps to confine the sample material plume to the capture gas in the center of the transport conduit and minimizes the diffusion of the sample material plume downstream of the laser ablation sampling system (because the transport rate is more constant and almost flat in the center of the flow). The gas can be, for example, but not limited to, argon, xenon, helium, nitrogen, or mixtures thereof. A common transport gas is argon. Argon is particularly suitable for preventing the diffusion of the plume before it reaches the walls of the transport conduit (and also helps to increase instrument sensitivity in devices where the ionization system is an argon-based ICP). The capture gas is preferably helium. However, the capture gas can be replaced by or contain other gases, such as hydrogen, nitrogen, or water vapor. At 25°C, the viscosity of argon is 22.6 μPas, while the viscosity of helium is 19.8 μPas. Sometimes, the capture gas is helium and the transport gas is argon.

[0246] As described in WO2014169394, the use of a sample cone minimizes the distance between the target and the laser ablation sampling system inlet of the delivery conduit. This results in improved capture of sample material, reduced turbulence, and therefore reduced spread of the plume of ablated sample material, due to the reduced distance between the sample and the point of the cone where the capture gas can flow through the cone. Thus, the inlet of the delivery conduit is a hole at the end of the sample cone. The cone extends into the sample cavity.

[0247] An optional modification to the sample cone is to make it asymmetric. When the cone is symmetric, then at the exact center, the airflow from all directions is neutral, so at the axis of the sample cone, the total airflow along the sample surface is zero. By making the cone asymmetric, a non-zero velocity is created along the sample surface, which helps flush plume material from the sample chamber of the laser ablation sampling system.

[0248] In fact, any modification to the sample cone can be adopted that enables a non-zero vector airflow along the sample surface on the axis of the cone. For example, an asymmetric cone may include a notch or a series of notches suitable for generating a non-zero vector airflow along the sample surface on the axis of the cone. An asymmetric cone may include a hole on the side of the cone suitable for generating a non-zero vector airflow along the sample surface on the axis of the cone. The hole will unbalance the airflow around the cone, thereby generating a non-zero vector airflow along the sample surface at the cone axis again at the target. The side of the cone may include more than one hole, and may include one or more notches and one or more holes. The edges of the notches and / or holes are typically smooth, rounded or chamfered to prevent or minimize turbulence.

[0249] Depending on the choice of capture and delivery gases and their flow rates, different orientations of the cone asymmetry will be appropriate for different situations, and the skilled person will be able to appropriately identify the combination of gas and flow rate for each orientation.

[0250] As used in the present invention, all of the above adjustments can be present in a single asymmetric sample cone. For example, the cone can be asymmetrically truncated and formed from two different elliptical cone halves, the cone can be asymmetrically truncated and include one or more holes, etc.

[0251] Thus, the sample cone is adapted to capture a plume of material ablated from a sample in a laser ablation sampling system. In use, the sample cone is operably positioned adjacent to the sample, for example, by manipulating the sample within the laser ablation sampling system on a removable sample carrier tray, as described above. As described above, the plume of ablated sample material enters the delivery conduit through the aperture at the narrow end of the sample cone. The diameter of the aperture may be a) adjustable; b) sized to prevent disturbance of the ablation plume as it enters the delivery conduit; and / or c) approximately equal to the cross-sectional diameter of the ablation plume.

[0252] Conical catheter

[0253] In a tube with a smaller inner diameter, the same flow rate of gas flows at a higher velocity. Therefore, by using a tube with a smaller inner diameter, a plume of ablated sample material carried in a gas stream can be transported faster through a defined distance (e.g., from a laser ablation sampling system to an ionization system in a delivery conduit) at a given flow rate. A key factor in analyzing the velocity of a single plume is how much the plume diffuses from the time the plume is generated by ablation to the time its component ions are detected at the mass spectrometer portion of the device (the detector's transient time). Therefore, by using a narrow delivery conduit, the time between ablation and detection is reduced, which means that diffusion is reduced because there is less time for diffusion to occur, with the end result being a reduced transient time for each ablation plume at the detector. Less transient time means that more plumes can be generated and analyzed per unit time, resulting in higher quality and / or faster images.

[0254] The cone can include a gradual change in the inner diameter of the delivery conduit along the portion of the length of the delivery conduit (i.e., the inner diameter of the tube is a cross-section taken through it that decreases along the portion from the end of the portion toward the inlet (at the laser ablation sampling system end) to the outlet (at the ionization system end)). Typically, the conduit has a relatively wide inner diameter near the area where ablation occurs. The larger volume of the conduit before the cone helps to confine the material produced by ablation. When the ablated particles fly out of the ablation spot, they move at a high speed. Friction in the gas slows the speed of these particles, but the plume can still be diffuse in the sub-millimeter to millimeter range. Allowing sufficient distance from the wall helps to contain the plume near the center of the gas flow.

[0255] Because the wide ID section is only short (approximately 1-2 mm), it does not contribute significantly to the total transient time if the plume spends more time in the longer section of the narrower ID delivery conduit. Therefore, the larger ID section is used to capture the ablation products and the smaller ID conduit is used to quickly deliver these particles to the ionization system.

[0256] The diameter of the narrow inner diameter portion is limited by the diameter corresponding to the onset of turbulence. The Reynolds number can be calculated for a round tube and a known flow rate. In general, Reynolds numbers above 4000 will indicate turbulent flow and should be avoided. Reynolds numbers above 2000 will indicate transitional flow (between non-turbulent and turbulent flow) and may also need to be avoided. For a given gas mass flow rate, the Reynolds number is inversely proportional to the conduit diameter. The inner diameter of the narrow inner diameter portion of the delivery conduit is typically narrower than 2 mm, for example, narrower than 1.5 mm, narrower than 1.25 mm, narrower than 1 mm, but larger than the diameter when the Reynolds number is greater than 4000 for a helium flow of 4 liters per minute in the conduit.

[0257] Rough or even angled edges in the transition between the constant diameter portion and the taper of the delivery conduit may cause turbulence in the gas flow and are generally avoided.

[0258] Sacrifice Flow

[0259] At higher flow rates, the risk of turbulence in the conduit increases. This is especially true if the delivery conduit has a small inner diameter (e.g., 1 mm). However, if a light gas such as helium or hydrogen is used instead of argon, which is traditionally used as the gas delivery flow, high-speed delivery (up to and exceeding 300 m / sec) can be achieved in a delivery conduit with a small inner diameter.

[0260] High-speed delivery is problematic because it can cause a plume of ablated sample material to pass through the ionization system without an acceptable level of ionization occurring. The level of ionization will decrease because the increase in cooling gas flow will reduce the temperature of the plasma at the end of the torch. If the plume of sample material is not ionized to an appropriate level, information about the ablated sample material is lost because its composition (including any marker atom / element labels) cannot be detected by the mass spectrometer. For example, in an ICP ionization system, the sample may pass through the plasma at the end of the torch so quickly that the plasma ions do not have sufficient time to act on the sample material to ionize it. This problem caused by high-flow, high-speed delivery in a narrow bore delivery conduit can be solved by introducing a flow sacrificial system at the outlet of the delivery conduit. The flow sacrificial system is adapted to accommodate the gas flow from the delivery conduit and forward only a portion of the gas flow (the central portion of the gas flow including any plume of ablated sample material) into the injector leading to the ionization system. To facilitate diffusion of gas from the delivery conduit in a flow sacrificial system, the delivery conduit outlet can be flared outward.

[0261] The flow sacrificial system is positioned close to the ionization system, so the length of the tube (e.g., syringe) leading from the flow sacrificial system to the ionization system is relatively short (e.g., about 1 cm long; compared to the length of the delivery conduit, which is typically on the order of tens of cm, e.g., about 50 cm). Therefore, the lower gas velocity in the tube leading from the flow sacrificial system to the ionization system does not significantly affect the overall delivery time because the relatively slow portion of the entire delivery system is much shorter.

[0262] In most configurations, it is not desirable or in some cases possible to significantly increase the diameter of the tube (e.g., syringe) leading from the flow sacrificial system to the ionization system as a way to reduce the gas velocity at the volumetric flow rate. For example, where the ionization system is an ICP, the conduit from the flow sacrificial system forms an injection tube in the center of the ICP torch. When a wider inner diameter syringe is used, the signal quality is reduced because the plume of ablated sample material cannot be injected so accurately into the center of the plasma (which is the hottest and most efficient ionization part of the plasma). Most preferred are syringes with an inner diameter of 1 mm or narrower (e.g., an inner diameter of 800 μm or less, such as 600 μm or less, 500 μm or less, or 400 μm or less). Other ionization techniques rely on the material being ionized in a relatively small volume in three-dimensional space (because the energy density required for ionization can only be achieved in a smaller volume), so a conduit with a wider inner diameter means that much of the sample material passing through the conduit is outside the region where the energy density is sufficient to ionize the sample material. Therefore, narrow diameter tubes entering the ionization system from the flow sacrificial system are also used in devices with non-ICP ionization systems. As discussed above, if the plume of sample material is not ionized to an appropriate level, information about the ablated sample material is lost because its composition (including any marker atom / element labels) cannot be detected by the mass spectrometer.

[0263] Pumping can be used to help ensure the desired split ratio between the sacrificial flow and the flow entering the ionization system inlet. Therefore, sometimes, the flow sacrificial system includes a pump connected to the sacrificial flow outlet. A controllable restrictor can be added to the pump to control the sacrificial flow. Sometimes, the flow sacrificial system also includes a mass flow controller suitable for controlling the restrictor.

[0264] In the case of using expensive gases, the gas pumped out of the sacrificial flow outlet can be purified using known gas purification methods and recycled back into the same system. As mentioned above, helium is particularly suitable as a transport gas, but it is expensive; therefore, it is advantageous to reduce the loss of helium in the system (i.e., when the helium enters the ionization system and is ionized). Therefore, sometimes, a gas purification system is connected to the sacrificial flow outlet of the flow sacrificial system.

[0265] Ionization system

[0266] To generate elemental ions, strong ionization techniques are required that can evaporate, atomize, and ionize the atomized sample.

[0267] Inductively coupled plasma torch

[0268] Typically, an inductively coupled plasma is used to ionize the material to be analyzed, which is then transferred to a mass detector for analysis. This is a plasma source in which the energy is provided by an electric current generated by electromagnetic induction. The inductively coupled plasma is maintained in a torch consisting of three concentric tubes, the innermost tube being called an injector.

[0269] An induction coil that provides the electromagnetic energy to sustain the plasma is located around the output end of the torch. The alternating electromagnetic field reverses polarity millions of times per second. Argon gas is supplied between the two outermost concentric tubes. Free electrons are introduced by the discharge and then accelerated in the alternating electromagnetic field, whereupon they collide with and ionize the argon atoms. In steady state, the plasma consists mainly of argon atoms, with a small fraction of free electrons and argon ions.

[0270] The ICP can be retained in the torch because the gas flow between the two outermost tubes keeps the plasma away from the torch walls. A second argon gas flow introduced between the injector (center tube) and the middle tube keeps the plasma away from the injector. A third gas flow is introduced into the injector at the center of the torch. The sample to be analyzed is introduced into the plasma through the injector.

[0271] The ICP may include a syringe having an inner diameter less than 2 mm and greater than 250 μm for introducing material from the sample into the plasma. The diameter of the syringe refers to the inner diameter of the syringe at the end proximal to the plasma. The syringe may have a different diameter as it extends away from the plasma, e.g., a wider diameter, wherein the difference in diameter is achieved by a step-wise increase in diameter, or because the syringe tapers along its length. For example, the inner diameter of the syringe may be between 1.75 mm and 250 μm, e.g., between 1.5 mm and 300 μm, between 1.25 mm and 300 μm, between 1 mm and 300 μm, between 900 μm and 300 μm, between 900 μm and 400 μm, e.g., around 850 μm. Using a syringe having an inner diameter less than 2 mm has significant advantages over using syringes of larger diameters. One advantage of this feature is that, with a narrower syringe, the transient of the signal detected in the mass detector is reduced when a plume of sample material is introduced into the plasma (the plume of sample material is a cloud of a particular gaseous material removed from the sample by the laser ablation sampling system). Thus, the time required from the introduction of sample material into the ICP for ionization until the generated ions are detected in the mass detector to analyze the sample material plume is reduced. The reduction in the time required to analyze the sample material plume allows more of the sample material plume to be detected in any given period of time. In addition, a syringe with a smaller inner diameter results in more precise introduction of sample material into the center of the inductively coupled plasma, where more efficient ionization occurs (as opposed to a larger diameter syringe, which may introduce more sample material into the edge of the plasma, where ionization is less efficient).

[0272] ICP torches (Agilent, Varian, Nu Instruments, Spectro, Leeman Labs, PerkinElmer, Thermo Fisher, etc.) and syringes (e.g., from Elemental Scientific and Meinhard) are commercially available.

[0273] Other ionization techniques

[0274] Electron ionization

[0275] Electron ionization involves bombarding a gas phase sample with an electron beam. An electron ionization chamber consists of an electron source and an electron trap. Typical sources for the electron beam are rhenium or tungsten filaments, typically operating at an energy of 70 electron volts. Electron beam sources for electron ionization are available from Markes International. The electron beam is directed toward the electron trap, and a magnetic field applied parallel to the direction of electron travel causes the electrons to travel in a spiral path. A gas phase sample is directed through the electron ionization chamber and interacts with the electron beam to form ions. Electron ionization is considered a hard ionization method because the process typically results in fragmentation of sample molecules. Examples of commercial electron ionization systems include the modified Markus electron ionization chamber.

[0276] Optional additional components for laser ablation based sampling and ionization systems

[0277] Ion Deflector

[0278] A mass spectrometer detects ions when they strike a detector surface. The collision of ions with the detector results in the release of electrons from the detector surface. These electrons are multiplied as they pass through the detector (first released electrons knock more electrons out of the detector, and then these electrons strike the secondary plate, further amplifying the number of electrons). The number of electrons striking the positive electrode of the detector generates an electric current. The number of electrons striking the positive electrode can be controlled by varying the voltage applied to the secondary plate. The current is an analog signal that can then be converted by an analog-to-digital converter into the number of ions striking the detector. When the detector is operating in its linear range, the current can be directly related to the number of ions. There is a limit to the number of ions that can be detected at one time (which can be expressed in terms of the number of ions that can be detected per second). Above this point, the number of electrons released by ions striking the detector no longer correlates to the number of ions. Therefore, this places an upper limit on the quantitative capability of the detector.

[0279] When ions strike the detector, its surface becomes damaged by contamination. Over time, this irreversible contamination damage results in a reduction in the electrons released from the detector surface when ions strike the detector, eventually causing the detector to need replacement. This is known as "detector aging" and is a well-known phenomenon in MS.

[0280] Therefore, the life of the detector can be extended by avoiding the introduction of excess ions into the MS. As mentioned above, when the total number of ions striking the MS detector exceeds the upper detection limit, the signal is not as informative as when the number of ions is below the limit because it is no longer quantitative. Therefore, it is desirable to avoid exceeding the upper detection limit because this will cause the detector to age faster without producing useful data.

[0281] Mass spectrometers analyze large groups of ions, involving a special set of challenges not found in conventional mass spectrometers. Specifically, typical MS techniques involve introducing a low and constant level of material into the detector that does not approach the upper detection limit or cause accelerated detector aging. Laser ablation-based techniques, on the other hand, analyze relatively large amounts of material in a very short time window in MS: for example, ions from a cell-sized block of a tissue sample are much larger than the small ion groups typically analyzed in MS. In effect, this is done intentionally to nearly overload the detector with the analytical ion groups produced by ablation or lift. Between analytical events, the signal is at baseline (the signal is close to zero because no ions from the labeled atoms are intentionally entering the MS from the sampling and ionization system; some ions will inevitably be detected because the MS is not a perfect vacuum).

[0282] Therefore, in the devices described herein, there is a high risk of accelerated detector aging, as ions from ionized sample material groups labeled with a large number of detectable atoms may exceed the upper detection limit and damage the detector without providing useful data.

[0283] To address these issues, the device may include an ion deflector located between the sampling and ionization system and the detector system (mass spectrometer), which is operable to control the ions entering the mass spectrometer. In one configuration, when the ion deflector is turned on, the ions received from the sampling and ionization system are deflected (i.e., the path of the ions is changed so that they do not reach the detector), but when the deflector is turned off, the ions are not deflected and reach the detector. How the ion deflector is deployed will depend on the configuration of the sampling and ionization system and the MS of the device. For example, if the entrance through which the ions enter the MS is not directly in a straight line with the path of the ions leaving the sampling and ionization system, then by default, the appropriately set ion deflector will be turned on so as to guide the ions from the sampling and ionization system into the MS. When an event is detected in which ions from the ionized sample material grouping (see below) are considered to be likely to overload the MS, the ion deflector is turned off so that the remaining ionized material in the event is not deflected into the MS, but can only impact the inner surface of the system, thereby preserving the life of the MS detector. After blocking ions from the destruction event from entering the MS, the ion deflector returns to its initial state, thereby allowing ions from subsequently ionized sample material packets to enter the MS and be detected.

[0284] Alternatively, in the case where (under normal operating conditions) the ions coming out of the sampling and ionization system do not change direction before entering the MS, the ion deflector will be closed and the ions from the sampling and ionization system will pass through it for analysis in the MS. To prevent damage to the detector in the event of a potential overload of the detector, in this configuration the ion deflector is stepped, thereby diverting the ions so that they do not enter the detector to prevent damage to the detector.

[0285] Ions entering the MS from ionized sample material (e.g., a plume of material from laser ablation) do not enter the MS all at once, but rather in a peak whose frequency follows a probability distribution curve about the maximum frequency: starting from the baseline, first a small number of ions enter the MS and are detected, then the frequency of the ions increases to a maximum, then the number decreases again and shrinks to the baseline. Events that could damage the detector can be identified because of the rapid increase in the count of ions hitting the detector, rather than the slow increase in frequency of the ions at the leading edge of the peak.

[0286] During the analysis of ions in the ionized sample material grouping, the ion flow that strikes the TOF MS detector (a specific type of detector as described below) is not continuous. The TOF includes a pulse generator that periodically releases ions into the flight chamber of the TOF MS in the form of pulse groups. Time-of-flight mass determination is achieved by releasing all known ions at the same time. The time between the release of ion pulses for time-of-flight mass determination is called the extraction or push of the TOF MS. The push is on the order of microseconds. Therefore, the signal from one or more ion groups of the sampling and ionization system covers many pushes.

[0287] Thus, when the ion count reading jumps from baseline to very high counts (i.e., the first portion of ions from a particular ionized sample material grouping) within one push, it can be predicted that the ion body produced by the ionized sample material grouping will be larger and therefore exceed the upper detection limit. At this point, the ion deflector can be operated to ensure that the damaging bulk ions are directed away from the detector (by being activated or deactivated, depending on the configuration of the system, as described above).

[0288] Suitable quadrupole-based ion deflectors are available in the art (eg, from Colutron Research Corporation and Dreebit GmbH).

[0289] The present invention is based on the sampling system of laser ablation

[0290] The components of the laser ablation-based sampling system can be appropriately combined depending on the analytical task being performed. An exemplary embodiment is set forth below.

[0291] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0292] Sample table;

[0293] Laser source;

[0294] Laser scanning systems; and

[0295] Focusing optics comprising an objective lens, wherein the objective lens has a numerical aperture of at least 0.7, at least 0.8 or at least 0.9, and optionally an immersion medium between the objective lens and the sample stage.

[0296] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0297] Sample table;

[0298] a laser source comprising a femtosecond laser;

[0299] Laser scanning systems; and

[0300] Focusing optics comprising an objective lens, wherein the objective lens has a numerical aperture of at least 0.7, at least 0.8 or at least 0.9, and optionally an immersion medium between the objective lens and the sample stage.

[0301] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0302] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0303] Laser source;

[0304] Laser scanning systems; and

[0305] A focusing optical device comprising an objective lens, the focusing optical device being adapted to direct a radiation beam from a laser source towards the second face to a position on a sample stage; and wherein the objective lens has a numerical aperture of at least 0.7, at least 0.8 or at least 0.9, and optionally also comprising an immersion medium between the objective lens and the sample stage.

[0306] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0307] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0308] a laser source comprising a femtosecond laser;

[0309] Laser scanning systems; and

[0310] A focusing optical device comprising an objective lens, the focusing optical device being adapted to direct a radiation beam from a laser source towards the second face to a position on a sample stage; and wherein the objective lens has a numerical aperture of at least 0.7, at least 0.8 or at least 0.9, and optionally also comprising an immersion medium between the objective lens and the sample stage.

[0311] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0312] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0313] a laser source; and

[0314] A focusing optical device comprising an objective lens, the focusing optical device being adapted to direct a radiation beam from a laser source towards the second face to a position on a sample stage; and wherein the objective lens has a numerical aperture of at least 0.7, at least 0.8 or at least 0.9, and optionally also comprising an immersion medium between the objective lens and the sample stage.

[0315] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0316] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0317] a laser source comprising a femtosecond laser; and

[0318] A focusing optical device comprising an objective lens, the focusing optical device being adapted to direct a radiation beam from a laser source towards the second face to a position on a sample stage; and wherein the objective lens has a numerical aperture of at least 0.7, at least 0.8 or at least 0.9, and optionally also comprising an immersion medium between the objective lens and the sample stage.

[0319] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0320] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0321] Laser source;

[0322] Laser scanning systems; and

[0323] Focusing optics comprising an objective lens and optionally an immersion medium between the objective lens and the sample stage.

[0324] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0325] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0326] a laser source comprising a femtosecond laser;

[0327] Laser scanning systems; and

[0328] Focusing optics comprising an objective lens and optionally an immersion medium between the objective lens and the sample stage.

[0329] In some embodiments, a laser ablation-based sampling system for analyzing a sample (e.g., a biological sample) includes:

[0330] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0331] a laser source comprising a femtosecond laser; and

[0332] Focusing optics comprising an objective lens and optionally an immersion medium between the objective lens and the sample stage.

[0333] Each of the above-described laser ablation-based sampling systems is suitable for inclusion in an apparatus for analyzing a sample according to the present invention, which apparatus comprises a mass detector (eg, TOF) mass detector, in particular comprising an ICP ionization system.

[0334] b. Sputtering-based sampling and ionization systems

[0335] Sputtering-based sampling systems and techniques provide an alternative surface analysis technique to the laser ablation-based systems and techniques described above. One such sputtering technique is secondary ion mass spectrometry (SIMS). SIMS involves bombarding a sample with a focused ion beam, sputtering material from the sample. The sputtered material includes ions and neutral atoms. In SIMS, the ions are captured by an immersion lens and then transferred to a mass detector in a vacuum. The mass detector can be any of the mass detector systems described below. Similar sputtering can be achieved by directing other charged particles, such as electrons, at the sample.

[0336] SIMS is a useful surface analysis technique for several reasons. First, the technique is very sensitive to low concentrations of the analyte. Second, because the diffraction effects of primary ions are negligible under most practical conditions, there is virtually no diffraction limit in SIMS. Therefore, SIMS has the potential to analyze materials on a scale of 10 to 30 nm.

[0337] However, the ionization efficiency of sputtered materials is very low, and ionization is also very dependent on the surface chemistry and specific elements to be ionized, so the number of ions produced by SIMS is not always enough to provide enough signal-to-noise ratio. For example, the ionization efficiency is not enough to detect a single copy of an antibody labeled with a MaxPar reagent. Since a single antibody labeled with a MaxPar mass tag carries about 100 atoms, in a traditional SIMS workflow, about 100 copies of the antibody may be required to generate a signal greater than a few ions on the detector. Another defect of SIMS is due to spectral interference from molecules in the same mass channel and due to the formation of compound ions, for example, oxides and other species involving a large number of neutral atoms present in the main marker element and biological samples. Compound ions weaken the signal of elemental ions and cause overlap with the mass channel of higher quality elements. Therefore, the resolution of imaging using SIMS may be limited by low detection sensitivity, partly due to low ionization efficiency.

[0338] The present invention overcomes the limitations of SIMS by providing improved methods and apparatus for analyzing biological samples using laser-based secondary neutral mass spectrometry (SNMS).

[0339] The laser-SNMS method and apparatus of the present invention include bombarding a sample with a focused beam of charged particles to sputter material from the sample. Neutral sputtered material is post-ionized using a laser. These ejected ions (including any detectable ions from labeled atoms as described below) can be detected by a detector system, for example, a mass spectrometer (the detector will be discussed in more detail below). Since most sputtered materials are in a neutral state, and SNMS ionizes the sputtered materials so that they can be analyzed using a mass detector, SNMS provides better surface quantitative estimates than SIMS. In addition, as described above, a major challenge to improving the spatial resolution of traditional IMS and IMC is to ensure that the amount of analyte in the analyzed material provides sufficient signal-to-noise ratio. Therefore, since SNMS uses both neutral and ionized sputtered materials, SNMS-based IMS and IMC provide higher resolution than SIMS-based IMS and IMC. For example, SNMS can perform single copy detection with antibodies labeled with MaxPar reagents. For example, if the efficiency of post-ionization reaches 10%, 100 atoms of each antibody will result in the generation of 10 ions per antibody, and if these ions are transmitted to the detector with good efficiency, this will ensure reliable detection of each antibody copy.

[0340] A laser SNMS system typically includes three components. The first component is a charged particle source for sputtering material from a sample for analysis (such a charged particle source will be discussed in 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 laser SNMS, the laser and the charged particle source are typically pulsed. In a related system, the first component is a charged particle source directed to a location on the sample (such a charged particle source will be discussed in more detail below). The second component is a laser for ablating and optionally ionizing tracks that have been pre-seeded with electrons by the charged particles. The third component is a detector component, such as a mass detector, for detecting the ionized material. In some aspects, the spot size of the charged particles that strike the sample is smaller than the spot size of the laser. The charged particles can ablate the sample at this location, after which the laser can ionize the sample near the sample surface (e.g., as Figure 6 Charged particles can seed electrons at the location of the sample, and then the laser can ablate and ionize the sample seeded with electrons (e.g., as shown in Figure 7 ). The laser can ionize the sample within picoseconds (e.g., 10-100ps) of the charged particle impacting the sample (e.g., within the charge ignition state). In some aspects, the spot size of the charged particles 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 can have a spot size (irradiating the sample) of less than 10 microns and / or greater than 500 nanometers, for example, between 500 nanometers and 5 microns, between 800 nm and 2 microns, or about 1 micron. The charged particles can provide a small spot size (e.g., within the ranges described herein) to allow for neutralization of minimal ionization under laser irradiation, for example, 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.

[0341] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0342] Sample table;

[0343] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0344] A first laser source and first focusing optics are configured to direct a laser beam emitted by the first laser source toward the sample stage.

[0345] The apparatus typically comprises a mass detector, for example a TOF detector.

[0346] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0347] Sample table;

[0348] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0349] A first laser source and first focusing optics are configured to direct a laser beam emitted by the first laser source toward the sample stage.

[0350] The charged particle column of the present invention includes suitable ion optics, which are configured to focus the charged particles so as to deliver the beam to a position on the sample stage. Such suitable ion optics may include mass filters, lenses and apertures and deflection plates to shape the primary ion beam, as described in more detail below.

[0351] Figure 6 is a schematic diagram of a configuration of an exemplary embodiment of the present invention. An energy source 40 emits radiation (e.g., a laser beam, a primary ion beam, or an electron beam, as discussed further below), which is transmitted to a position 55 on a sample stage 20 by an optical device 80 (e.g., an optical device or an ion optical device, such as a charged particle column). The sample 30 is positioned on the sample stage 20 so that the charged particles are transmitted to the position 55 to sputter the material 50 from the sample 30. A first laser source 60 emits a laser beam, and a focusing optical device (not shown) directs the laser beam toward the sample stage to ionize the sputtered material 50, forming a material plume containing the sample ions. The ions can then be transmitted to a mass detector, such as a time-of-flight detector or a magnetic sector detector or any other mass detector discussed in more detail below.

[0352] Thus, the present invention provides an apparatus or sampling and ionization system for analyzing a sample (e.g., a biological sample) wherein a first focusing optical device is configured to synchronize laser beam pulses to ionize a plume of material sputtered by a charged particle pulse (referred to herein as post-ionization).

[0353] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0354] Sample table;

[0355] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0356] A first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optics are configured to synchronize laser beam pulses to ionize a plume of material sputtered by a charged particle pulse from the charged particle source.

[0357] Specifically, the first focusing optics directs and focuses the laser beam to a volume above the surface of the sample stage so that when material is sputtered from the sample on the sample stage, a plume of material ejected from the sample enters the volume into which the radiation of the first laser source is focused so that the material can be ionized. This explanation applies to all devices discussed 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 optics for ionizing the plume. For the sake of brevity, it is not repeated below.

[0358] The apparatus typically comprises a mass detector, for example a TOF detector.

[0359] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0360] Sample table;

[0361] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0362] A first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optics are configured to synchronize laser beam pulses to ionize a plume of material sputtered by a charged particle pulse from the charged particle source.

[0363] As described above, the first focusing optics directs and focuses the laser beam to a volume above a location on the sample stage (more specifically, above a location on the sample on the sample stage) such that when material is sputtered from the sample on the sample stage, a plume of material ejected from the sample enters the volume into which the radiation of the first laser source is focused, such that the material can be ionized. This description applies to all systems discussed 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 optics for ionizing the plume. For the sake of brevity, this is not repeated below.

[0364] Figure 7 is a schematic diagram of the configuration of another exemplary embodiment of the present invention. Figure 7 Included with Figure 6 common elements, and these elements share the same reference numerals. Figure 7In the apparatus of the illustrated embodiment, a laser beam from a first laser source 60 is directed toward a position 55 on the sample stage 20, and an optical device 80 (e.g., a charged particle column) directs the charged particles toward the position. For example, a charged particle pulse transmitted toward the position 55 on the sample forms an excited state with free electrons at the position 55 on the sample; such an excited state is referred to herein as a "sample ignition state". After the charged particle pulse reaches the position, the laser beam from the first laser source directly irradiates the position 55 on the sample, so that the first laser source irradiates the sample in the ignition state. Since the sample is in the sample ignition state, the sample easily converts the laser pulse from the laser beam into ablation and ionization energy; such a state is referred to herein as a "sample energy pumping state". This process forms a material plume containing sample ions. Those skilled in the art will appreciate that in this embodiment, the energy of the laser pulse is less than the ablation threshold of the sample (so that the same area adjacent to the position to which the charged particle is directed will not be ablated in response to the laser pulse that causes the position to enter the "sample energy pumping state", although the laser spot on the sample can be larger than the diameter of the position to which the charged particle is directed).

[0365] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample) wherein a first focusing optical device is configured to synchronize a laser beam pulse to arrive at a position on a sample stage immediately after a charged particle pulse. This configuration of the present invention can overcome the relatively slow particle sputtering by a charged particle beam alone, thus providing a faster method of analyzing a biological sample.

[0366] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0367] Sample table;

[0368] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0369] A first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optic is configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles.

[0370] Specifically, the first focusing optics directs 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 particle. This explanation applies to all devices discussed below in this section that use a combination of features of the sample stage, the charged particle source and the first laser source and the first focusing optics so that the radiation pulse from the first laser source irradiates the same location as the location that was previously targeted by the charged particle. For the sake of brevity, it will not be repeated below.

[0371] The apparatus typically comprises a mass detector, for example a TOF detector.

[0372] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0373] Sample table;

[0374] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0375] A first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a sample stage, wherein the first focusing optic is configured to synchronize a laser beam pulse to arrive at a position on the sample stage immediately after a pulse of charged particles.

[0376] Specifically, the first focusing optics directs 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 particle. This explanation applies to all systems discussed below in this section that use a combination of features of the sample stage, the charged particle source and the first laser source and the first focusing optics that cause the radiation pulse from the first laser source to irradiate the same location as the location that was previously targeted by the charged particle. For the sake of brevity, it will not be repeated below. Figure 8 is a schematic diagram of the configuration of another exemplary embodiment of the present invention. Figure 8 Included with Figure 6 and 7 Common elements are shown in the drawings and these elements share the same reference numerals. In this embodiment, the sample stage 20 is transparent (as is any sample carrier on which the sample is placed). Those skilled in the art will appreciate that a transparent sample stage or a sample stage including a cut-away portion can be used with any embodiment of the sputtering-based sampling and ionization system described herein. The transparent sample stage is described in more detail herein.

[0377] exist Figure 8 In the illustrated embodiment, the charged particle source and charged particle column, the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to opposite sides of the sample stage. Figure 8 In the embodiment shown, the charged particles are transmitted toward a location 55 on the sample and the laser beam is also directed toward the location 55 on the sample. Figure 7 In the same manner as the embodiment shown, Figure 8 In an embodiment of the present invention, a pulsed beam of charged particles provides a sample ignition state and a pulsed laser beam provides an energy pumping state to generate a material plume including sample ions.

[0378] Fig. 9is a schematic diagram of the configuration of another exemplary embodiment of the present invention. Fig. 9 Included with Figures 6 to 8 Common elements, and these elements share the same reference numerals. Figure 8 In the embodiment shown, in this embodiment of the invention, the charged particle source and charged particle column, the first laser source and the first focusing optics are configured so that the charged particle beam and the laser beam are directed to opposite sides of the sample stage. The sample 30 is positioned on the sample stage 20 so that the charged particles are transmitted toward the position 55 to sputter the material 50 from the sample 30. The first laser source 60 emits a laser beam, and the focusing optics (not shown) direct the laser beam toward the sample stage to ionize the sputtered material 50 to form a material plume containing the sample ions. The ions can then be transmitted to a mass detector, such as a time-of-flight detector or any other mass detector discussed in more detail below.

[0379] It is noted that in any of the above embodiments, the charged particle beam can be scanned over the sample to analyze any region of interest, such as a cell organelle, as described above.

[0380] Thus, the present invention provides an apparatus wherein the charged particle source and the charged particle column and the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to the same side of the sample stage. Examples of apparatus of this type are Figure 6 and Figure 7 shown.

[0381] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0382] Sample table;

[0383] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0384] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0385] Therein, the charged particle source and the charged particle column as well as the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to the same side of the sample stage.

[0386] The apparatus typically comprises a mass detector, for example a TOF detector.

[0387] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0388] Sample table;

[0389] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0390] a first laser source and first focusing optics, the first focusing optics being configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics is configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0391] Therein, the charged particle source and the charged particle column as well as the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to the same side of the sample stage.

[0392] For example, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0393] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0394] a charged particle source and a charged particle column for delivering a charged particle beam to a position on the first surface of the sample stage; and

[0395] A first laser source and a first focusing optics, the first focusing optics being configured to direct a laser beam emitted by the first laser source toward a first side of a sample stage, wherein the first focusing optics is configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles.

[0396] The apparatus typically comprises a mass detector, for example a TOF detector.

[0397] For example, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0398] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0399] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first surface of the sample stage; and

[0400] A first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first side of a sample stage, wherein the first focusing optic is configured to synchronize a laser beam pulse to arrive at a position on the sample stage immediately after a pulse of charged particles.

[0401] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0402] Sample table;

[0403] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0404] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize the laser beam pulses to ionize a plume of material sputtered by the charged particle pulses from the charged particle source; and

[0405] Therein, the charged particle source and the charged particle column as well as the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to the same side of the sample stage.

[0406] The apparatus typically comprises a mass detector, for example a TOF detector.

[0407] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0408] Sample table;

[0409] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0410] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize the laser beam pulses to ionize a plume of material sputtered by the charged particle pulses from the charged particle source; and

[0411] Therein, the charged particle source and the charged particle column as well as the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to the same side of the sample stage.

[0412] For example, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0413] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0414] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first surface of the sample stage; and

[0415] a first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first side of the sample stage, wherein the first focusing optic is configured to synchronize the laser beam pulses to ionize a plume of material sputtered from the sample by the charged particle pulses from the charged particle source.

[0416] The apparatus typically comprises a mass detector, for example a TOF detector.

[0417] For example, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0418] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0419] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first surface of the sample stage; and

[0420] a first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first side of the sample stage, wherein the first focusing optic is configured to synchronize the laser beam pulses to ionize a plume of material sputtered from the sample by the charged particle pulses from the charged particle source.

[0421] Furthermore, the invention provides an apparatus wherein the charged particle source and the charged particle column and the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to opposite sides of the sample stage. Examples of apparatus of this type are Figure 8 and Fig. 9 As shown. One advantage of such devices is that they minimize the mechanical complexity of combining the charged particle column and focusing optics. In some aspects, the charged particles can be directed toward the sample through a support or substrate that is at least partially transparent to the charged particles and ablate the sample or plant electrons in the sample. In these aspects, the sample can be thin, for example, less than 200 nm, less than 100 nm, less than 50 nm, or less than 30 nm.

[0422] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0423] Sample table;

[0424] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0425] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0426] Therein, the charged particle source and the charged particle column as well as the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to opposite sides of the sample stage.

[0427] The apparatus typically comprises a mass detector, for example a TOF detector.

[0428] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0429] Sample table;

[0430] a charged particle source and a charged particle column for delivering a charged particle beam to a location on a sample stage; and

[0431] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0432] Therein, the charged particle source and the charged particle column as well as the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed to opposite sides of the sample stage.

[0433] For example, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0434] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0435] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first surface of the sample stage; and

[0436] A first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the second side of the sample stage, wherein the first focusing optic is configured to synchronize a laser beam pulse to arrive at a position on the sample stage immediately after a pulse of charged particles.

[0437] The apparatus typically comprises a mass detector, for example a TOF detector.

[0438] For example, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0439] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0440] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first surface of the sample stage; and

[0441] A first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the second side of the sample stage, wherein the first focusing optic is configured to synchronize a laser beam pulse to arrive at a position on the sample stage immediately after a pulse of charged particles.

[0442] As explained elsewhere herein, and reiterated here for the sake of completeness, when the laser beam passes through the sample stage to reach the sample on the first side, the sample stage should be transparent to the laser radiation (as should the sample carrier of the sample), or the sample stage should include a gap through which the laser radiation can pass to reach the sample (through the sample carrier).

[0443] The skilled person will also understand that Figures 6 to 9 The configurations can be combined in various ways. Fig.10 Another exemplary embodiment of the present invention is shown. Figures 6 to 9 The combination of configurations, similarly, with Figures 6 to 9 The same elements are marked with the same reference numerals.

[0444] Fig.10 The embodiment shown includes a second laser source 61 and second focusing optics (not shown). Figure 8 The first laser source 60 and the second focusing optical device of this embodiment are configured to direct the second laser beam to the position 55 on the sample stage. Figure 7 and Figure 8 In the same manner as the embodiment shown, the pulsed beam of charged particles provides the sample ignition state and the pulsed laser beam from the second laser source 61 provides the energy pumping state to produce a material plume containing sample ions. In this embodiment of the invention, the first laser source 60 and the first focusing optics are configured to ionize the material plume sputtered by the pulse of charged particles. Therefore, the first laser source 60 will ionize any neutral material sputtered from the surface that is not ionized by the energy pumping state. Therefore, since both the first and second laser sources are configured to ionize the material plume to produce sample ions, the present invention provides a device that further improves the ionization probability and therefore improves the signal-to-noise ratio. Therefore, the present invention provides a device for analyzing a sample with improved resolution.

[0445] Thus, in this section, the present invention provides an apparatus as described above, comprising a second laser source and a second focusing optics, wherein the second focusing optics is configured to synchronize laser pulses from the second laser source to ionize a plume of material sputtered by the charged particle pulses. In certain aspects, the first and second laser sources may comprise the same laser, wherein control of the lasers and / or optics allows for the two-step laser irradiation described herein.

[0446] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0447] Sample table;

[0448] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0449] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0450] a second laser source and second focusing optics, wherein the second focusing optics are configured to synchronize laser beam pulses from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source.

[0451] The apparatus typically comprises a mass detector, for example a TOF detector.

[0452] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0453] Sample table;

[0454] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0455] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize the laser beam pulses to ionize a plume of material sputtered by the charged particle pulses from the charged particle source; and

[0456] a second laser source and second focusing optics, wherein the second focusing optics are configured to synchronize laser beam pulses from the second laser source to ionize a plume of sample material from the location, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source.

[0457] Therefore, the invention provides an apparatus wherein a first laser source and first focusing optics and a second laser source and second focusing optics are configured such that the laser beam from the first laser source and the laser beam from the second laser source are directed towards the same side of the sample stage.

[0458] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0459] Sample table;

[0460] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0461] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0462] a second laser source and second focusing optics, wherein the second focusing optics are configured to synchronize laser beam pulses from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source,

[0463] The charged particle source and the charged particle column, the first laser source and the first focusing optical device, and the second laser source and the second focusing optical device are configured so that the charged particle beam, the laser beam from the first laser source and the laser beam from the second laser source are directed to the same side of the sample stage.

[0464] The apparatus typically comprises a mass detector, for example a TOF detector.

[0465] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0466] Sample table;

[0467] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0468] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0469] a second laser source and second focusing optics, wherein the second focusing optics are configured to synchronize laser beam pulses from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source,

[0470] The charged particle source and the charged particle column, the first laser source and the first focusing optical device, and the second laser source and the second focusing optical device are configured so that the charged particle beam, the laser beam from the first laser source and the laser beam from the second laser source are directed to the same side of the sample stage.

[0471] For example, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0472] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0473] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first side of the sample stage;

[0474] a first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first side of the sample stage, wherein the first focusing optic is configured to synchronize a pulse of the laser beam to arrive at a position on the first side of the sample stage immediately after a pulse of charged particles; and

[0475] a second laser source and a second focusing optic configured to direct a laser beam emitted by the second laser source toward the first side of the sample stage, wherein the second focusing optic is configured to synchronize the laser beam pulses from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source.

[0476] The apparatus typically comprises a mass detector, for example a TOF detector.

[0477] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0478] a sample stage, comprising a first side and a second side, the first side and the second side being opposite to each other, and wherein the first side is adapted to receive a sample;

[0479] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first side of the sample stage;

[0480] a first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first side of the sample stage, wherein the first focusing optic is configured to synchronize a pulse of the laser beam to arrive at a position on the first side of the sample stage immediately after a pulse of charged particles; and

[0481] a second laser source and a second focusing optic configured to direct a laser beam emitted by the second laser source toward the first side of the sample stage, wherein the second focusing optic is configured to synchronize the laser beam pulses from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source.

[0482] Therefore, the present invention provides an apparatus wherein a first laser source and first focusing optics and a second laser source and second focusing optics are configured such that the laser beam from the first laser source and the laser beam from the second laser source are directed to opposite sides of the sample stage.

[0483] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0484] Sample table;

[0485] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0486] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize the laser beam pulses to arrive at a position on the sample stage immediately after the pulse of charged particles; and

[0487] a second laser source and second focusing optics, wherein the second focusing optics are configured to synchronize a laser beam pulse from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulse and the laser beam pulse from the first laser source,

[0488] wherein the charged particle source and the charged particle column and the second laser source and the second focusing optical device are configured so that the charged particle beam and the laser beam from the second laser source are directed to the same side of the sample stage; and the first laser source and the first focusing optical device are configured so that the laser beam from the first laser source is directed to the opposite side of the sample stage.

[0489] The apparatus typically comprises a mass detector, for example a TOF detector.

[0490] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0491] Sample table;

[0492] a charged particle source and a charged particle column for delivering a charged particle beam to a position on a sample stage;

[0493] a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, wherein the first focusing optics are configured to synchronize a pulse of the laser beam to arrive at a position on the sample stage immediately after a pulse of charged particles; and

[0494] a second laser source and second focusing optics, wherein the second focusing optics are configured to synchronize laser beam pulses from the second laser source to ionize a plume of sample material from a location on the sample, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source,

[0495] wherein the charged particle source and the charged particle column and the second laser source and the second focusing optical device are configured so that the charged particle beam and the laser beam from the second laser source are directed to the same side of the sample stage; and the first laser source and the first focusing optical device are configured so that the laser beam from the first laser source is directed to the opposite side of the sample stage.

[0496] For example, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0497] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0498] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first side of the sample stage;

[0499] a first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the second side of the sample stage, wherein the first focusing optic is configured to synchronize the laser beam pulse to arrive at a position on the first side of the sample stage immediately after the charged particle pulse; and

[0500] A second laser source and a second focusing optic configured to direct a laser beam emitted by the second laser source toward the first side of the sample stage, wherein the second focusing optic is configured to synchronize the laser beam pulses from the second laser source to ionize a plume of sample material from the location, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source.

[0501] The apparatus typically comprises a mass detector, for example a TOF detector.

[0502] Thus, the present invention provides a sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0503] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0504] a charged particle source and a charged particle column for delivering a charged particle beam to a location on the first side of the sample stage;

[0505] a first laser source and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the second side of the sample stage, wherein the first focusing optic is configured to synchronize the laser beam pulse to arrive at a position on the first side of the sample stage immediately after the charged particle pulse; and

[0506] A second laser source and a second focusing optic configured to direct a laser beam emitted by the second laser source toward the first side of the sample stage, wherein the second focusing optic is configured to synchronize the laser beam pulses from the second laser source to ionize a plume of sample material from the location, the plume being generated by the charged particle pulses and the laser beam pulses from the first laser source.

[0507] The present invention also provides a method for analyzing a biological sample using the device described in this section. For example, the present invention provides a method comprising:

[0508] delivering a charged particle beam to a location on the sample;

[0509] irradiating the sample with a first laser beam pulse to generate a material plume comprising sample ions; and

[0510] The sample ions are detected by mass spectrometry.

[0511] In some cases, the method includes delivering a charged particle beam toward a location on the sample to sputter material from the sample; and wherein the method includes irradiating the sputtered sample material with a first laser beam pulse and ionizing the sputtered material to produce sample ions. That is, the method includes delivering a charged particle beam toward a location on the sample to sputter material from the sample; and wherein the method includes irradiating the sputtered sample material with a first laser beam pulse, thereby ionizing the sputtered material to produce sample ions. Sometimes, delivering the charged particle beam toward a location on the sample produces a sample ignition state; and wherein irradiating the sample at the location produces a sample energy pumping state at the location of the sample.

[0512] In some methods, the charged particle beam is transmitted from one side of the sample toward a location on the sample, and wherein the first laser beam pulse irradiates the sample from the same side of the sample. In some embodiments, the charged particle beam is transmitted from one side of the sample toward a location on the sample, and wherein the first laser beam pulse irradiates the sample from an opposite side of the sample. Sometimes, transmitting the charged particle beam to the location on the sample also includes sputtering material from the sample; and wherein the method includes irradiating the sputtered sample material with a second laser beam pulse and ionizing the sputtered material.

[0513] The first laser beam pulse and the second laser beam pulse irradiate the sample from the same side of the sample, or the first laser beam pulse and the second laser beam pulse irradiate the sample from opposite (opposite) sides of the sample.

[0514] As known to those skilled in the art, laser ionization of sputtered materials can be achieved through various ionization mechanisms, including single photon ionization, resonant and non-resonant multiphoton ionization, avalanche ionization, and cold avalanche ionization. In addition, different ionization mechanisms can work simultaneously, for example, multiphoton ionization and avalanche ionization.

[0515] Single photon ionization (SPI) refers to a process in which the absorption of one photon is sufficient to overcome the ionization potential of the sputtered material. However, ionization in this way requires very high energy UV or vacuum UV lasers, for example, excimer lasers or complex systems using nonlinear optical processes in gases. Therefore, the present invention provides methods and devices, wherein the first and / or second laser source is a high energy UV or vacuum UV laser.

[0516] Multiphoton ionization (MPI) involves the absorption of more than one photon to overcome the ionization potential, and this absorption can be a non-resonant or resonant step. Multiphoton ionization requires short, intense laser pulses. Lasers suitable for resonant systems include pulsed Nd:YAG lasers pumping two dye lasers, and lasers suitable for non-resonant laser systems include high power excimer or Nd:YAg lasers. Therefore, the present invention provides methods and apparatus, wherein the first and / or second laser source is a Nd:YAg laser pumping two dye lasers, a high power excimer laser or a Nd:YAG laser.

[0517] Avalanche ionization (AI) is the process by which electrons collide and ionize sputtered material, causing additional electrons to accelerate and collide with other sputtered material, thereby creating a chain reaction. In the present invention, the initial "seed" electron can be the result of any such ionization mechanism that is produced by the application of a laser pulse, for example, multiphoton ionization or single photon ionization. Some models suggest that avalanche ionization is not important when the laser pulse is shorter than 100 fs. However, various techniques have been demonstrated to persist for avalanche ionization with laser pulses shorter than 100 fs. The present invention provides methods of utilizing these techniques to ionize sputtered material by avalanche ionization using laser pulses shorter than 100 fs.

[0518] In the first technique, the electric field applied to the sample by a strong ionizing laser is used to reduce the effective energy threshold for impact ionization to occur, thereby allowing impact ionization to drive avalanche ionization even at short pulse lengths (less than 100 fs). This is a cold avalanche ionization mechanism. Suitable lasers can be, for example, Ti: Sapphire lasers (800 nm, 40-45 fs pulse length, energy 75 nJ, for example, Octavius ​​Ti: Sapphire lasers, available from Thorlabs), and it has been shown that avalanche ionization of fused silica can be achieved with 800 nm laser pulses as low as 40 fs from a regenerative amplifier (Rajeev, Gertsvolf et al., 2009, PRL 201). Therefore, the present invention provides methods and apparatus, wherein the first and / or second laser source is a Ti: Sapphire laser for ionizing sputtered material by avalanche ionization. In some aspects, the ignition (e.g., electron planting) mechanism after plasma development (e.g., avalanche ionization) may be similar for dielectric and biological samples. In the case of a biological sample, the sample format may be epoxy, which is a dielectric, but different from silicon oxide.

[0519] In the second technique, free carriers are injected into the sample to encourage a combination of exciton-induced multiphoton ionization and avalanche ionization. Free carriers can be injected into the dielectric from an EUV source generated by a high harmonic or attosecond pulse generator, and the sample can be ionized with laser pulses as short as 45 fs (800 nm laser) (Grojo, Gertsvolf et al., 2010, PR81). Thus, the present invention provides methods and apparatus wherein the first and / or second laser source is an EUV source.

[0520] Thus, the present invention provides methods and apparatus wherein the first and / or second laser is configured to ionize the sputtered material by avalanche ionization.

[0521] In this way, the present invention further addresses two major challenges in improving the resolution of IMC to the submicron scale. First, because laser pulses shorter than 100 fs can be used to achieve avalanche ionization, the risk of the area around the laser spot size being damaged due to thermal effects is reduced. In this way, the present invention maintains the spot area to a size below about 200 nm. Second, the present invention increases the possibility of multiphoton and avalanche ionization, thereby increasing the total ionization rate. In this way, the total amount of analyte in the ablated material provides a sufficient signal-to-noise ratio.

[0522] Components of sputtering-based sampling and ionization systems

[0523] Charged particle source

[0524] As described above, in the present invention, a charged particle source and a charged particle column may be used to deliver a charged particle beam to a location on a sample.

[0525] Primary ion beam:

[0526] Typically, the source of charged particles used in secondary neutral mass spectrometry is a primary ion beam source. The primary ions can be any suitable ions for producing sputtering from the sample to be analyzed. Examples of primary ion sources are: dual plasma tubes, which produce oxygen (( 16 O - , 16 O2 + , 16 O2 - ), Argon ( 40 Ar + ), xenon (Xe + ), SF5 + or C 60 + Primary ions; surface ionization sources that produce 133 Cs + primary ions; and a liquid metal ion gun (LMIG) that produces Ga + Primary ions. Other primary ions include cluster ions, e.g., Au n + (n=1-5), Bi n q+ (n=1-7, q=1 and 12), C 60 q+ probes (q=1-3) and large argon clusters (Muramoto, Brison & Castner, 2012).

[0527] The choice of ion source depends on the type of ion bombardment used (i.e. static or dynamic) and the sample to be analyzed. Static involves the use of low primary ion beam currents (1 nA / cm 2), usually a pulsed ion beam. Due to the low current, each ion hits a new part of the sample surface, removing only a monolayer of particles (2nm). Therefore, static is suitable for imaging and surface analysis (Gamble & Anderton, 2016). Dynamic involves the use of high primary ion beam current (10mA / cm 2 ), usually a continuous primary ion beam, which results in rapid removal of surface particles. Therefore, dynamic depth profiling can be used. In addition, since more material is removed from the sample surface, dynamic SIMS gives better detection limits than static. Dynamic usually produces high image resolution (less than 100nm) (Vickerman & Briggs, 2013).

[0528] Oxygen primary ions enhance the ionization of electronegative elements (Malherbe, Penen, Isaure & Frank, 2016) and are used in the commercially available Cameca IMS1280-HR, whereas cesium primary ions are used to study electronegative elements (Kiss, 2012) and are used in the commercially available Cameca NanoSIMS 50.

[0529] In order to analyze the sample quickly, a high frequency of sputtering is required, for example, more than 200 Hz (i.e., more than 200 ion packets are directed toward the sample per second). Typically, the primary ion source generates a primary ion pulse at a frequency of 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 400-100 MHz, in the range of 1 kHz-100 MHz, in the range of 10 kHz-100 MHz, in the range of 100 kHz-100 MHz, or in the range of 1 MHz-100 MHz.

[0530] Thus, the present invention provides an apparatus wherein the source of charged particles is a primary ion beam.

[0531] Electron beam

[0532] Alternatively, in some embodiments of the invention, the charged particle source is an electron beam. An electron beam with an energy of 2 kV to 30 kV may be particularly suitable for interrogating a sample with a thickness of 30 nm.

[0533] A high intensity pulsed electron beam is used to cause ablation / sputtering. When the pulse of electron current is not sufficient for ablation, its effect can be used only as an ignition event as described above, followed by energy pumping by a laser pulse whose brightness level is set below the ablation level of the native material but above the energy pumping level required to ablate the already activated material. In the energy activation mode of ablation / sputtering, the electron energy can be reduced because the electrons are only used to inject charge carriers into the otherwise insulating material.

[0534] In some aspects, an electron beam can be focused to a smaller spot (at a higher resolution) than a laser beam. Lower resolution lasers can only ablate and / or ignite samples at the spot irradiated by the electron beam, even when it strikes samples outside the electron beam spot. For example, an electron beam can be focused to a spot of 200nm, 100nm, 50nm, 30nm, or less than 10nm, and a laser can be focused to a spot of 200nm, 300nm, 500nm, 800nm, 1um, 2um, or larger that overlaps the electron beam spot, or onto the ablation plume released by the electron beam.

[0535] In order to analyze the sample quickly, a high frequency of sputtering is required, for example, more than 200 Hz (i.e., more than 200 electron packets are directed toward the sample per second). Typically, the electron source generates electron pulses at a frequency of 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 pulses 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 pulses is in the range of 400-100 MHz, in the range of 1 kHz-100 MHz, in the range of 10 kHz-100 MHz, in the range of 100 kHz-100 MHz, or in the range of 1 MHz-100 MHz.

[0536] One advantage of using an electron beam as the source of charged particles is that the entire apparatus can be built on a platform comprising an electron microscope. Thus, the present invention provides an apparatus further comprising an electron microscope. Thus, the present invention provides an apparatus wherein the source of charged particles is an electron beam, wherein the electron beam is the source of electrons in the electron microscope.

[0537] Charged Particle Column

[0538] The charged particle column guides the charged particles toward the sample. The charged particle column includes: a mass filter for filtering impurities in the charged particle beam; a lens and an aperture to appropriately control the intensity and shape of the primary ion beam as needed; and a deflection plate to shape the primary ion beam and optionally raster scan the charged particle beam on the sample surface (Villacob, 2016). Ion lenses and other components for constructing a 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 suitable for scanning a charged particle beam at multiple positions on a sample stage.

[0539] Typically, the charged particle beam used for secondary ion generation herein has a spot size of less than 100 μm (i.e., the size of the charged particle beam when it strikes the sample), for example, less than 20 μm, less than 5 μm, less than 1 μm, or less than 500 nm, or less than 300 nm, less than 200 nm, less than 100 nm, less than 50 nm, or less than 30 nm. The distance referred to as the spot size corresponds to the longest internal dimension of the ion beam, for example, a circular beam is a beam with a diameter of 2 μm, a square beam corresponds to the length of the diagonal between opposite corners, a quadrilateral is the length of the longest diagonal, and so on. Beam shaping and beam masking can be used to provide spot shape and size.

[0540] When used to analyze biological samples, in order to analyze individual cells, the spot size of the ion beam used will depend on the size and spacing of the cells. For example, in the case where cells are closely packed with each other (e.g., in a tissue section), if single cell analysis is to be performed, the charged particle beam can have a spot size that is no larger than these cells. The size will depend on the specific cells in the sample, but typically the diameter of the ion beam spot will be less than 4 μm, for example, in the range of 0.1-4 μm, 0.25-3 μm, or 0.4-2 μm. Therefore, the charged particle beam spot may 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, for example, about 400 nm or less, about 300 nm or less. In a specific embodiment, the spot size is about 200 nm or less, about 100 nm or less. In order to analyze cells with subcellular resolution, the system uses a primary ion beam spot size that is no larger than these cells, and more specifically, a primary ion beam spot size that can ablate material with subcellular resolution. Sometimes single cell analysis can be performed using a spot size that is larger than the cell size, for example, when the cells are spread out on a slide with spaces between them. Therefore, the specific spot size used can be appropriately chosen based on the size of the cells being analyzed. In biological samples, cells are rarely all the same size, so if subcellular resolution imaging is required, the ion beam spot size should be smaller than the smallest cell if a constant spot size is maintained throughout the sputtering process.

[0541] Laser

[0542] In the present invention, the laser can be used to ablate material, or to assist in the sputtering of material by irradiating a location on the sample after a pulse of charged particles has been delivered toward that location (see, e.g. Figure 7 and Figure 8 and related descriptions). In addition, lasers can be used to ionize sputtered material that was previously sputtered by a charged particle beam (see, for example, Figure 6 and Fig. 9 In addition, lasers can be used in combinations of the above, for example, see Fig.10 and related descriptions.

[0543] As will be appreciated by those skilled in the art, the requirements for lasers used for ablation / assist material sputtering are different from the requirements for lasers used for ionizing materials. Typically, lasers used for assist material sputtering or ablation provide energy on the order of μJ for pulse lengths of 300 to 400 fs or greater. In contrast, lasers used to ionize sputtered material are more powerful, providing energy on the order of 1 mJ or greater 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 may use one laser for the first and second laser sources, and use a beam splitter to separate the lasers and provide the first and second laser sources.

[0544] A variety of different lasers can be used for SNMS, including the commercial lasers discussed above in connection with the lasers of the laser ablation sampling system, which are adapted to be able to sputter materials as needed. Typically, the laser operates at the maximum achievable intensity in order to maximize ionization. Various types of lasers that can be used in the present invention are described below. Femtosecond lasers as described above are also advantageous in specific SNMS applications. For example, in the above-mentioned embodiment of the present invention including a first and a second laser source, a single femtosecond laser can be used to provide two laser sources. The laser has a repetition rate of 1 MHz, a pulse width of 200 fs, and an energy pulse of 1 microjoule. Those skilled in the art will appreciate that a beam splitter can be used to separate the laser beam of the laser to provide the first and second laser sources of the claimed invention.

[0545] In general, lasers that can be used for ionization include lasers that provide energy measured in microjoules per pulse, but they can produce high-frequency pulses. The laser is used to produce elemental ions from the sample material. The elemental ions produced can then be analyzed by a mass spectrometer in the device. The laser can be a picosecond laser or a femtosecond laser or a nanosecond laser. In some embodiments, the laser is a femtosecond laser.

[0546] Femtosecond lasers can be solid-state lasers. Passively mode-locked solid-state bulk lasers can emit high-quality ultrashort pulses with typical durations between 30 fs and 30 ps. Examples of such lasers include diode-pumped lasers, for example, lasers based on neodymium-doped or ytterbium-doped crystals. Titanium-sapphire lasers can be used for pulse durations below 10 fs, and in extreme cases down to about 5 fs (e.g., Octavius ​​Ti:Sapphire lasers, available from Thorlabs). In most cases, the pulse repetition rate is between 1 kHz and 500 MHz.

[0547] Femtosecond lasers can be fiber lasers. Various types of ultrafast fiber lasers, which can also be passively mode-locked, typically offer pulse durations between 50 and 500 fs, repetition rates between 0.10 and 100 MHz, and average powers between a few milliwatts and several watts (femtosecond fiber lasers are available from Toptica, IMRA America, Coherent).

[0548] Femtosecond lasers can be semiconductor lasers. Some mode-locked diode lasers can generate pulses with a duration of femtoseconds. Directly at the laser output, the pulse duration is usually at least a few hundred femtoseconds, but with external pulse compression, shorter pulse durations can be achieved.

[0549] In some embodiments, the laser is a nanosecond laser. The nanosecond laser can be a pump laser, for example, Quantel's Q-smart DPSS, Solar Laser LQ929's high power pulsed Nd:YAG laser, or Litron HighEnergy Pulsed's Nd:YAG laser. All of these lasers can produce deep UV radiation in the mJ range, and are therefore suitable for ionization and have short pulse durations.

[0550] It is also possible to passively mode-lock vertical external cavity surface emitting lasers (VECSELs); these are particularly interesting because they can provide a combination of short pulse duration, high pulse repetition rate and sometimes high average output power, but they are not suitable for high pulse energies.

[0551] In some embodiments, the laser is suitable for generating pulses with nanosecond, picosecond or femtosecond pulse durations. For example, the laser can have a duration of 500 fs or less, for example, 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 suitable for generating pulses with a duration of less than 1 ps.

[0552] In some embodiments, the laser is adapted to have a pulse repetition frequency of at least 100,000 Hz, e.g., 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 more.

[0553] In some embodiments, the laser is adapted to have a beam width (1 / e 2 ). The focus of the laser is where the energy of the beam is most concentrated, and therefore where maximum ionization is achieved.

[0554] In some embodiments, the laser is adapted to have a pulse energy between 1 nanojoule and 50 millijoules. Lasers used for auxiliary material sputtering or material ablation can be adapted to have a pulse energy between 1 nanojoule and 100 microjoules, for example, between 10 nanojoules and 100 microjoules, between 100 nanojoules and 10 microjoules, between 500 nanojoules and 5 microjoules, for example, about 1 microjoule, about 2 microjoules, about 3 microjoules, or about 4 microjoules. Lasers used for post-ionization can be adapted to have a pulse energy between 1 millijoules and 50 millijoules, for example, between 5 millijoules and 40 millijoules, between 10 millijoules and 30 millijoules, between 20 millijoules and 35 millijoules, or about 25 millijoules or 35 millijoules.

[0555] In some embodiments, the laser is adapted to have a pulse energy of about 1 microjoule, have a pulse repetition frequency of at least 10 MHz, and produce pulses having a duration of less than 100 fs, e.g., less than 50 fs, less than 45 fs, less than 25 fs, less than 20 fs, or less than 10 fs.

[0556] As will be appreciated by those skilled in the art, the present invention may be used with one or a combination of suitable lasers described herein. However, for the sake of completeness, a number of specific combinations of lasers that may be used with the present invention are as follows:

[0557]

[0558] Postionization requires a small volume (e.g., 5 μm 3The high energy density of laser radiation in the post-ionization volume (below). Because the post-ionization volume is small, this limits the amount of material that can be ionized at one time. If a large number of positive and negative charges are generated in a small volume, the movement of the ions formed will be controlled by the local field generated by the space charge induced by the ions and electrons. If there are too many charged ions in a small volume, the external field (for example, the field in the ion optical device present in the mass spectrometer to guide the resulting ions to the detector for detection) will not be able to effectively separate the positive and negative charges, and this ion cloud will eventually neutralize, thereby reducing the ionization efficiency. For example, an ion cloud with a diameter of 10μm containing 10,000 elemental charges produces an electrostatic potential of about 3V. Since a few eV is the energy that holds an electron to an atom, it is also the possible energy level of a free electron after ionization. Therefore, in a volume on the scale of 10 microns, the ion density of 10,000 ions approaches the limit where space charge behavior begins to dominate.

[0559] Therefore, by ensuring that the amount of sputtered material remains fairly small, this effect can be avoided. For example, ablation of material on the scale of a 10×10×10 nm cube to a 30×30×30 nm cube or similar volume represents the size of the highest amount of material that can be delivered into the post-ionization region of a few microns without producing strong spatial variations and ion neutralization. Since the system can only process a 30×30×30 nm cube per single event, this creates the opportunity to image with a spatial resolution of 30 nm or even 10 nm, to which the ion beam can be focused (as described herein).

[0560] The post-ionization beam is co-aligned with the sputtering beam to within micrometer accuracy, which is common in optical setups.

[0561] In some embodiments, the laser beam is directed at an angle to the sample and into a previously ablated region of the sample (see Figure 6 ). This configuration minimizes the interaction between the laser and the unablated sample. The laser beam can be focused to a tight focus that overlaps the ablation plume volume. Laser beam focusing can be performed at a high numerical aperture (NA) to facilitate fine focusing in the overlap region and to allow the laser energy to spread rapidly outside the overlap region to minimize the potential for damage to the sample surrounding the area being sampled.

[0562] because Figure 6Due to the limitations of ion optics that focus the primary beam and transmit the secondary beam for mass analysis, in some embodiments, the space for the laser beam optics may be limited. As a result, in some embodiments, the numerical aperture of the laser beam can be confined to one plane. For example, the laser beam can have a low NA in the drawing plane to avoid interference with the ion optics, and a high NA in a plane perpendicular to the drawing plane. This setup results in an elliptical focal spot extending in the low NA plane. The elliptical focal spot can increase the overlap with the sputtering / ablation plume. Therefore, in some embodiments, the laser of the post-ionization system has an elliptical focal spot.

[0563] In addition to spatial coordination, the sputtering caused by the primary ion or electron beam needs to be synchronized with the delivery of laser radiation to ionize the sputtered material. The speed at which the sputtered material leaves the target is typically on the scale of the speed of sound, i.e. 1000 m / s. Therefore, in order to ensure that the sputtering cloud and the laser beam are aligned with an accuracy of 1 micron, a time accuracy of 1 ns is required. Ion beam bunching and timing to 1 ns is typically achieved with accelerating voltages of the order of 5 to 20 kV. The same or similar bunching techniques can be applied to the primary beam. For example, such techniques are used in current TOF MS equipment.

[0564] Thus, in this mode of operation, a primary ion or electron beam sputters material from the sample and, shortly thereafter, the ejected material is post-ionized by a pulse of laser radiation.

[0565] To further promote the consistent sputtering / ejection of material from the target, in some cases, the target can be irradiated with a laser pulse synchronized with the primary ion pulse (instead of or in addition to the above-mentioned post-ionization). For materials that are not exposed to the primary ion or electron beam, the energy of the laser pulse should be set below the ablation threshold. In the region where the primary ion beam or electron beam interacts with the target, the state of matter is similar to plasma, and because it is confined to the nanoscale, it is referred to as nanoplasma in this article. Therefore, the beam can interact with the nanoplasma and pump additional energy into the volume, resulting in the consistent release of the material. This release mode can be regarded as an ignition state (provided by the primary ion beam or electron beam that interacts with the target and produces the nanoplasma), followed by an energy pumping state, in which the laser pumps additional energy into the nanoplasma, promoting its ejection.

[0566] This mode of operation helps overcome the limitation of relatively slow sputtering of primary ion or electron beams. Since the sputtering rate of a primary ion / electron beam is limited by the total number of primary ions / electrons bombarding a given area. The ability to focus the primary ion / electron beam is limited due to ion / electron current and space charge effects. This, combined with the need to time the primary ion / electron beam to only sputter material when the post-ionization laser is turned off, limits the sputtering rate. Using laser radiation to enhance sputtering can overcome this limitation because fewer primary particles are required to seed the sample material with the excitation energy required by the laser beam to affect sputtering / ablation.

[0567] In the case where the laser is used for post-ionization and enhanced sputtering, the same laser is also suitable for pumping energy into the nanoplasma to promote material release. For example, a single femtosecond laser can meet both needs of the device. The laser can have a repetition rate of 1 MHz, a pulse width of 200 fs, and an energy of 1 microjoule per pulse (i.e., an average power of 1 W). Such laser pulses can be separated (for example, using a beam splitter), and a portion of them is used for energy pumping of the nanoplasma. The remaining portion can be used for post-ionization. A compression stage can be applied to the post-ionization beam to reduce the amount of energy per pulse required for post-ionization.

[0568] In some embodiments, post-ionization of the laser beam is performed by pumping energy into the nanoplasma on the surface. Thus, the depumping and post-ionization processes are combined into a single process activated by the laser pulse delivered to the nanoplasma on the target.

[0569] Furthermore, pumping energy into the plasma on the target can be achieved by tuning the laser wavelength to the absorption band of the plasma while avoiding the absorption band of the unionized target material. In this way, the bombardment of the primary ion / electron beam is used to change the state of the material in the target from not absorbing the pump light to absorbing the pump light. The duration of the pump light can be from picoseconds to a few nanoseconds and down to a few femtoseconds. At the same time, the picosecond pulses are short enough to avoid significant diffuse broadening of the plasma volume in the sample.

[0570] In some embodiments, laser radiation used for energy pumping is delivered through the sample carrier (see Figure 8 ). For example, a primary electron beam or a primary ion beam can be focused to a spot on the scale of 10 to 30 nm, while laser focusing is limited by diffraction effects and may stay on the scale of 100 to 1000 nm.

[0571] Devices known in the art can be used to introduce a delay between laser pulses. Thus, in some embodiments, the device includes an optical delay line to introduce a delay between laser pulses. An example of an optical delay line suitable for use in the present invention is any optical delay line available from ThorLabs.

[0572] Variable delay lines can be used to synchronize the arrival of the ionization pulse with respect to the primary ion or electron packet, thereby enabling the same laser to operate in either post-ionization or pumping mode, depending on the relative timing of the primary ion or electron packet and the laser pulse.

[0573] Sample chamber

[0574] 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. It includes a stage that supports the sample. The stage can be a translation stage, movable in an xy or xyz axis. The sample chamber will also include an outlet through which material removed from the sample by the laser radiation can be directed. The outlet is connected to a detector so that the sample ions can be analyzed.

[0575] In some cases, the sample chamber is maintained at 133322-13.3Pa, such as 1333.22-133.322Pa. In some cases, the sample chamber is maintained under vacuum. Thus, in some cases, the sample chamber pressure is less than 50000Pa, less than 10000Pa, less than 5000Pa, less than 1000Pa, less than 500Pa, less than 100Pa, less than 10Pa, less than 1Pa, about 0.1Pa or less than 0.1Pa, such as 0.01Pa or less. For example, the partial vacuum pressure can be about 50-2000Pa, 100-1000Pa or 200-700Pa, and the vacuum pressure is less than 10Pa, 1Pa or 0.2Pa. Suitable gases include argon, helium, nitrogen and mixtures thereof.

[0576] As will be appreciated by those skilled in the art, the choice of whether the sample pressure is at atmospheric pressure, at partial vacuum pressure, or at vacuum depends on the particular analysis being performed. For example, at atmospheric pressure, sample handling is easier and softer ionization can be applied. In addition, the presence of gas molecules may be desirable so that collisional cooling phenomena can occur, which may be of interest when the label is a macromolecule, which is not expected to break, for example, a molecular fragment comprising a label atom or a combination thereof. Alternatively, in the case where laser radiation is used to post-ionize the material, the presence of gas molecules (e.g., at partial vacuum pressure) may be advantageous. For example, collisional cooling can allow cooling of nanoplasmas generated on the sample surface (e.g., after charged particle bombardment or laser irradiation of the sample to produce an energy pumping state) and expansion of the ablated material plume, and before re-ionization in a post-ionization system. Collisions can also allow at least partial charge reduction, reducing space charge effects and / or improving the ability of ion optics to guide ions. The charge state is reduced to only one charge per ion, making it easier to read the signals of different mass labels.

[0577] Maintaining the sample chamber under vacuum prevents collisions between the generated sample ions and other particles in the chamber.

[0578] The main difference between the sample chamber of the SNMS system and that of the laser ablation based system is that the sample chamber is kept under vacuum to prevent collisions between the sample ions and other particles within the sample chamber, which could result in loss of ion charge, similar to the case of the laser ablation based and released sample chamber. Loss of secondary ions would reduce the sensitivity of the device.

[0579] Ion Microscope

[0580] Sample ions are captured from the sample via an electrostatic lens located near the sample, which is called an immersion lens (or extraction lens) in the art. The immersion lens removes secondary ions directly from the location of the sample. This is usually achieved by having a large voltage difference between the lens and the sample. Depending on the polarity of the sample relative to the immersion lens, the immersion lens captures positive or negative secondary ions. The polarity of the sample ions captured by the immersion lens is independent of the ion polarity of the charged particle beam.

[0581] The sample ions are then transferred to the detector via one or more additional electrostatic lenses (referred to in the art as transfer lenses). The transfer lens focuses the secondary ion beam onto the detector. Typically, in a system with multiple transfer lenses, only one transfer lens is used in a given analysis. Each lens can provide a different magnification of the sample surface. Typically, there are additional ion manipulation components between the immersion lens and the detector, such as one or more apertures, mass filters, or deflector plate sets. The immersion lens, transfer lens, and any other components together constitute an ion microscope. Components for producing ion microscopes are available from commercial suppliers, such as Agilent.

[0582] camera

[0583] The system may also include a camera. A camera system is discussed above with respect to the laser ablation sampling system, and features of the camera described above may also be present in the secondary ion production system unless incompatible (e.g., it may be connected to an optical microscope, such as a confocal microscope, but it is not possible to focus the primary ion beam through the same optics through which the light is directed to the camera because one beam is of ions and the other is of photons).

[0584] c. Sampling and ionization system based on dual pulse laser

[0585] Similar to certain devices, systems and techniques discussed in Section 1.b above, electron planting to produce a "sample ignition state" can be achieved at a certain location by a laser radiation pulse. Thereafter, a second laser pulse with a laser spot diameter larger than that location but a flux lower than the ablation threshold of the sample can be directed toward the sample to cause ablation only at the location pointed to by the first laser pulse, because the energy required to ablate the pre-planted location is lower than the surrounding area. Therefore, a tightly focused, low-energy first pulse can be used to plant electrons, and a higher-energy, less focused pulse can be used to cause ablation at the location where the electrons are planted. Therefore, the present invention provides another way of high-resolution imaging based on this technology.

[0586] The behavior of optics is well known, namely, that the minimum spot size produced by an optical system is directly proportional to the wavelength of the light and inversely proportional to the numerical aperture of the objective. Since the wavelength of visible light is approximately 500nm and the numerical aperture of a typical objective rarely exceeds 1.0, the focused spot size of this light ends up being just under 1 micron. Therefore, by operating with a shorter wavelength laser, the light can be focused to a smaller spot on the sample. For example, an excimer laser can produce VUV light with a wavelength of approximately 200nm. In addition, modern lithography tools use EUV light with a wavelength of 13.5nm. Unfortunately, generating VUV and EUV light pulses with enough energy for ablation requires large, complex, and expensive equipment.

[0587] The present invention solves the problem of reducing the ablation spot by pre-seeding the spot to be ablated with a small amount of UV, EUV or XUV light. This pre-seeding additionally produces free electrons in the dielectric material. This change in the material properties lowers the threshold for subsequent pulse ablation. The energy required for pre-seeding is many orders of magnitude lower than the energy required for direct ablation. This makes the equipment used to generate such pulses relatively economical. Since the wavelength of the pre-seeding pulses is shorter, they can be focused to a diameter of 100 or 30nm. Shortly after the pre-seeding is completed, pulses with more common wavelengths (e.g., IR or visible light) are sent to the same sample. Energy is stored more efficiently in the pre-seeded area. Therefore, the pre-seeded area can be ablated, while other areas covered by infrared light will not be ablated. Therefore, the size of the ablation spot is controlled by the diffraction limit imposed by the UV, VUV, EUV wavelengths. This limit is much lower than the ablation resolution of visible or infrared light applied alone.

[0588] In addition, the present invention can lead to ionization of ablated materials. The ion composition of ablated materials can be analyzed and imaging mass spectrometry or imaging mass spectrometry flow cytometry workflows can be provided. The ablated material will be ionized in the plasma, resulting in the generation of a large number of elemental ions. When using MaxPar reagents or similar reagents, the efficiency of ionization and ion sampling from the plasma can become high enough to facilitate single copy detection of antibodies. Here, ions can be directly extracted via an ion microscope for analysis, as described below.

[0589] When operating at such a high spatial resolution (e.g., 30nm), there are several analytical problems. One problem is the total acquisition rate versus the field of view (or region of interest) dimension. The existing Hyperion runs at 200 pixels / s with a pixel size of 1 micron. This results in a requirement of approximately 5000 seconds to record an image of a biologically relevant ROI of 1×1mm scale. If the pixel size is reduced by 30 times (approximately 30nm), the time to record an image from a similar area will increase to 500,000 seconds. Obviously, this is an impractically long time. The present invention solves this problem by operating ionization sampling in at least a partial vacuum (see further detailed discussion of the sample chamber below), where the pixel rate can be as high as 1Mpixels / s, meaning that an area of ​​1×1mm will be acquired in just 100 seconds. Multiple consecutive slices can be analyzed to provide a 3D image of a biological scale of approximately 0.03mm. This would require 1000 slices (30nm each) and approximately 1 day of experimentation. This is a very high speed considering the multi-parameter and high spatial resolution nature of the data obtained. If the acquisition rate is increased to 10Mpixels / s, it would allow such images to be collected in 2.5 hours. At a spatial resolution of 100nm, sample analysis would also be about 30 times faster.

[0590] As mentioned above, using MaxPar reagents (mass-tagged antibody reagents, described below) can compensate for the sensitivity loss due to smaller ablation by having approximately 100 atoms per copy of the antibody. Ion sampling and transmission above 5% enables detection of a single antibody copy. When an object is imaged with a pixel size of 30nm, only very few antibodies can be found in such a pixel. Therefore, achieving single copy detection for imaging at such a small scale becomes critical.

[0591] The plasma produced by the dual pulse technology of the present invention can solve the problem of efficient ionization and sampling. Small-scale ablation leads to small-scale plasma, which in turn reduces the possibility of plasma neutralization during ion sampling. In addition, the density of the ablated solid material leads to an initial plasma pressure of 10,000 atm, which in turn leads to a local thermal equilibrium model of the plasma. The local thermal equilibrium allows the generation of an optimal plasma temperature to promote nearly 100% efficient ionization of the labeled atoms from the mass tags to form elemental ions that can be controlled via the parameters of the second pulse.

[0592] Devices based on dual-pulse laser ablation systems typically include three components. The first component is a first laser source for planting electrons in a sample (which will be contained on a sample stage in the devices and systems disclosed herein). The second component is a second laser source for ablating the sample in which electrons have been planted by the first laser source. (These two components together constitute a dual-pulse laser sampling and ionization system.) The third component is a detector component for detecting ionized material, such as a mass detector. The laser sources are typically pulsed. In some cases, the first (pre-planting) pulse can be obtained from the same laser that delivers the second (ablation) pulse, for example, by harmonic generation. The positions for directing the laser beam onto the sample stage mentioned in this section refer to the configuration of the components in the devices and systems, but those skilled in the art will understand that when the device / system is used to analyze a sample, the laser radiation from the first and second laser sources will impinge on the sample being analyzed.

[0593] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0594] Sample table;

[0595] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the sample stage; and

[0596] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the sample stage.

[0597] The apparatus typically comprises a mass detector, for example a TOF detector.

[0598] Therefore, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0599] Sample table;

[0600] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the sample stage; and

[0601] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the sample stage.

[0602] In some embodiments, a first focusing optical device configured to direct a laser beam emitted by a first laser source toward a sample stage is configured to direct the laser beam emitted by the first laser source toward a position on the sample stage, and a second focusing optical device configured to direct a laser beam emitted by a second laser source toward the sample stage is configured to direct the laser beam emitted by the second laser source toward the position on the sample stage.

[0603] For example, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0604] Sample table;

[0605] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a position on the sample stage; and

[0606] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and second focusing optics configured to direct a laser beam emitted by the second laser source toward a location on the sample stage.

[0607] The apparatus typically comprises a mass detector, for example a TOF detector.

[0608] For example, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0609] Sample table;

[0610] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a position on the sample stage; and

[0611] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and second focusing optics configured to direct a laser beam emitted by the second laser source toward a location on the sample stage.

[0612] Fig.13is a schematic diagram of a configuration of an exemplary embodiment of the present invention, which shows the reduction of the ablation spot size by two pulse ablation. A support 20 holds a thin slice of a sample 30. In some embodiments (e.g., nanofabrication applications), the support and the sample can be integral. A pulse of UV or VUV or EUV or even XUV light 40 is focused 50 on the sample 30 by an objective 60. Special objectives for UV, VUV, EUV optics are known in the art and are generally based on a reflective optical configuration. For example, a pulse of EUV light generates seeds of free electrons in the focal spot. Since the sample is generally non-conductive, pre-seeding with free electrons changes the properties of the material at the focal spot of the laser. A second light pulse 70 is sent to the same area, including the location where the electrons were pre-seeded, to provide energy to develop a plasma at the pre-seeded location. The second light pulse 70 is sent by another objective 80. Due to the significantly longer wavelength of the second pulse, the focal spot 90 of the second pulse is much larger than the focal spot of the first pulse. The process of electron multiplication in the sample during the second pulse is called avalanche ionization. In practice, many phenomena contribute to the preferential excitation of the pre-seeded area. The approach of defining the ablation area by pre-seeding with, for example, EUV pulses is effective as long as the energy required for preferential excitation is significantly below the threshold for direct ablation.

[0613] Accordingly, the present invention provides apparatus and systems for analyzing a sample (e.g., a biological sample), wherein a second focusing optical device is configured to synchronize a second pulse of laser radiation from a second laser source to arrive at a position on a sample stage immediately after a pulse of laser radiation from a first laser source.

[0614] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0615] Sample table;

[0616] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the sample stage; and

[0617] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and second focusing optics configured to direct the laser beam emitted by the second laser source toward the sample stage;

[0618] Wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source.

[0619] The apparatus typically comprises a mass detector, for example a TOF detector.

[0620] Therefore, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0621] Sample table;

[0622] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the sample stage; and

[0623] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and second focusing optics configured to direct the laser beam emitted by the second laser source toward the sample stage;

[0624] Wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source.

[0625] In some embodiments, a first focusing optical device configured to direct a laser beam emitted by a first laser source toward a sample stage is configured to direct the laser beam emitted by the first laser source toward a position on the sample stage, and a second focusing optical device configured to direct a laser beam emitted by a second laser source toward a position on the sample stage is configured to direct the laser beam emitted by the second laser source toward a position on the sample stage.

[0626] Thus, the present invention provides apparatus and systems for analyzing a sample (eg, a biological sample) wherein first and second focusing optics are configured to direct laser radiation to opposite sides of a sample stage.

[0627] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0628] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0629] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first face of the sample stage; and

[0630] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the second face of the sample stage;

[0631] Wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source.

[0632] The apparatus typically comprises a mass detector, for example a TOF detector.

[0633] Therefore, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0634] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0635] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first face of the sample stage; and

[0636] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the second face of the sample stage;

[0637] Wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source.

[0638] The present invention also provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0639] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0640] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the second side of the sample stage; and

[0641] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the first face of the sample stage;

[0642] Wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source.

[0643] The apparatus typically comprises a mass detector, for example a TOF detector.

[0644] Therefore, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0645] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0646] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the second side of the sample stage; and

[0647] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the first face of the sample stage;

[0648] Wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source.

[0649] Fig.14 Another preferred embodiment of the present invention is shown. Here, 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 (e.g., a biological sample) in which the first focusing optics and the second focusing optics are configured to direct the laser radiation to opposite sides of the sample stage.

[0650] Thus, the present invention provides an apparatus for analyzing a sample (e.g., a biological sample), comprising:

[0651] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0652] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first face of the sample stage; and

[0653] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the first face of the sample stage;

[0654] wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source;

[0655] And wherein the first focusing optics and the second focusing optics use the same objective lens to focus radiation from the first and second laser sources.

[0656] The apparatus typically comprises a mass detector, for example a TOF detector.

[0657] Therefore, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0658] A sample stage, comprising a first surface and a second surface, the first surface and the second surface are opposite to each other, and wherein the first surface is adapted to accommodate a sample;

[0659] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward a first face of the sample stage; and

[0660] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and a second focusing optic configured to direct the laser beam emitted by the second laser source toward the first face of the sample stage;

[0661] wherein the second focusing optics is configured to synchronize a second pulse of laser radiation from the second laser source to arrive at a position on the sample stage immediately after a pulse of laser radiation from the first laser source; and

[0662] Therein, the first focusing optics and the second focusing optics use the same objective lens to focus radiation from the first laser source and the second laser source.

[0663] Fig.15 Another embodiment is shown in which the sample (30) is placed on a material that is at least semi-transparent to the first pulse and even more transparent to the second pulse. This material can be in the form of a metal mesh used in electron microscopy. This geometry allows the ablated plasma to expand into a cloud 100 composed of ions, electrons and neutral material. The ions here can be sampled into a mass spectrometer for imaging mass spectrometry and imaging mass spectrometry flow cytometry applications.

[0664] Thus, in some embodiments of the dual-pulse laser-based devices 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 the laser radiation from the first laser source and even more transparent to the radiation from the second laser source. Alternatively, as explained elsewhere herein, and reiterated here for completeness, when the laser radiation is directed through the sample stage to the sample on the first side, the sample stage should include a gap through which the laser radiation can pass to reach the sample (via a sample carrier). In the case where the sample stage includes 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 the radiation from the first laser source and even more transparent to the radiation from the second laser source.

[0665] Fig.16 Another embodiment is shown which facilitates efficient sampling of ablated ions. Here, a first laser pulse 40 is sent from a first side. The first laser pulse is formed by an objective lens 60 having an opening in the middle to allow ions 100 generated after ablation to pass through.

[0666] Therefore, in some embodiments of the dual-pulse laser based devices and sampling and ionization systems disclosed herein, the objective lens of the first focusing optics has an opening in the middle to allow ionized material from the sample to pass through the opening.

[0667] Components of a dual laser sampling and ionization system

[0668] First laser source - laser for pre-implanted electronics

[0669] As described above, the requirement for achieving high-resolution imaging is that the wavelength of the laser radiation can be focused to a focal spot with a diameter of less than 100 nm. Therefore, the wavelength of the laser radiation emitted by the first laser source must be ultraviolet (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 less than 200 nm, less than 175 nm, less than 150 nm, less than 125 nm, less than 100 nm, less than 75 nm, less than 50 nm, less than 25 nm, less than 20 nm or 15 nm. For example, in some embodiments, the laser radiation emitted by the first laser source is between 10 nm-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.

[0670] According to the above discussion, the pulse energy of the first laser source is selected so that it plants electrons in the material, but does not ablate the material. In some embodiments, the pulse energy of the first laser source is in the range of picojoules to femtojoules, for example, between 1 picojoule-100 femtojoules, between 10 picojoules-100 femtojoules, or between 100 picojoules-1 femtojoules.

[0671] The duration of the pulse must be short enough to minimize the diffusion of the seeded electrons before the second laser pulse that causes sample ablation. 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 below 10 ps. Thus, in some embodiments, 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.

[0672] First focusing optics

[0673] The first focusing optics directs radiation from the first laser source toward the sample and focuses it onto the sample. To achieve high resolution imaging, the first focusing optics must focus the laser radiation onto the sample to a spot size of less than about 100 nm, for example, less than 75 nm, less than 50 nm, or about 30 nm.

[0674] In some embodiments, the objective of the first focusing optics is a reflective objective.

[0675] Second laser source - laser for ablating the pre-seeded sample location

[0676] The requirements for the second laser source in the system vary depending on its function. Pulses of laser radiation from the second laser source provide the energy to form the plasma on the sample and to control its temperature.

[0677] Thus, the wavelength of the laser radiation emitted by the second laser source can be infrared light or visible light. If visible light is emitted by the second laser source, it can be focused more tightly, so less energy is required for each pulse ablation. In some embodiments, the laser radiation emitted by the second laser source is above 400nm, above 500nm, above 600nm, above 700nm, above 800nm, or above 1μm. For example, in some embodiments, the laser radiation emitted by the second laser source is between 400nm-100μm, for example, between 200nm-100μm, between 200nm-10μm, between 400nm-10μm, between 400nm-1μm, between 400nm-1μm, between 400nm-900nm, between 400nm-800nm, between 400nm-700nm, between 400nm-600nm, or between 500nm-600nm.

[0678] According to the above discussion, the pulse energy of the second laser source is selected so that it ablates only the sample material that has been pre-implanted with electrons in the laser spot, that is, the pulse flux is below the ablation threshold of the material. In some embodiments, the pulse energy of the second laser source is in the nanojoule range, for example, between 1 nanojoule-1 μJ, between 10 nanojoule-500 nanojoules, between 50 nanojoules-250 nanojoules, or about 100 nanojoules.

[0679] The duration of the pulse must be short enough to minimize the diffusion of the seeded electrons before the second laser pulse that causes sample ablation. 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 below 10 ps. Thus, in some embodiments, the duration of the pulse 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, 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.

[0680] Second focusing optics

[0681] The second focusing optic directs radiation from the second laser source toward the sample and focuses it onto the sample. High resolution imaging is achieved by tight focusing of the first short wavelength pulses, so the spot size can be larger than the first pulses. Nevertheless, the relatively small spot size can minimize sample radiation around the planting location. Therefore, in some embodiments, the second focusing optic focuses the laser radiation onto the sample with a spot size of about 2 μm or less, for example, less than 1 μm, less than 750 nm, less than 500 nm, less than 250 nm, less than 200 nm, less than 150 nm, or about 100 nm.

[0682] In some embodiments, the objective of the second focusing optics is a refractive or reflective objective, for example, a lens with NA greater than 0.7, greater than 0.8, or greater than 0.9. In some embodiments, the objective of the second focusing optics is also the objective of the first focusing optics as described above.

[0683] Synchronization of the first laser source and the second laser source

[0684] The apparatus of the present invention may use different lasers for the first laser source and the second laser source. Here, a technical program in the art (e.g., a programming module including instructions) can be used to coordinate the transmission of a first pulse from the first laser source to the sample and the transmission of a second pulse from the second laser source to the sample so that the pre-planting and ablation of the pre-planted area proceed as discussed above. In some embodiments, the first pulse and the second pulse are synchronized so that the time from the start of the pulse from the first laser source to the end of the pulse from the second laser source has a duration of less than 50 ps, ​​for example, shorter than 25 ps. In some embodiments, the time is shorter than 10 ps, ​​for example, shorter than 5 ps, shorter than 2 ps, or shorter than 1 ps.

[0685] In some embodiments, the apparatus and system use one laser as a first laser source and a second laser source and use a beam splitter to separate the laser light and provide the first laser source and the second laser source.

[0686] For example, a single laser engine that generates infrared pulses can be used. The pulses can be converted into visible light by a second or third harmonic generator. To produce short wavelength radiation for the first pulse, the laser can be coupled to a high harmonic generation stage. Optical delay lines can be used to control the time intervals between the first pulse and the second pulse, and outside the second pulse and the third pulse.

[0687] Thus, 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, wherein one harmonic generator is adapted to generate UV, VUV, EUV, or XUV laser radiation from a single laser as the first laser source (as discussed above for the first laser source), and the other harmonic generator generates infrared or visible wavelength laser radiation from a single laser as the second laser source (as discussed above for the second laser source).

[0688] Thus, 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 harmonic generator is adapted to generate UV, VUV, EUV, or XUV laser radiation from a single laser as a first laser source (as discussed above for the first laser source), and the other harmonic generator is adapted to generate infrared or visible wavelength laser radiation from a single laser as a second laser source (as discussed above for the second laser source); and wherein the optical delay line is adapted to transmit 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, such that the end of the second pulse is shorter than 50 ps, ​​e.g., shorter than 25 ps, shorter than 10 ps, ​​e.g., shorter than 5 ps, shorter than 2 ps, or shorter than 1 ps, after the start of the first pulse.

[0689] Optionally, the output of a harmonic generator suitable for producing UV, VUV, EUV or XUV laser radiation may be filtered to include the wavelength range of interest for pre-seeding the area to be ablated.

[0690] Magnetic sector instruments may be more suitable for high speed recording at 1 Mpixel and above. For recording rates of 100 kpixels / s, a time-of-flight mass analyser is suitable and may be simpler to design and manufacture.

[0691] Because this type of ion source produces a very tight ion spot, the characteristics of the ion beam in this technique make the ion beam suitable for use in many types of mass analyzers (both currently known and yet to be invented).

[0692] General Laser Considerations

[0693] Femtosecond lasers can be solid-state lasers. Passively mode-locked solid-state bulk lasers can emit high-quality ultrashort pulses with a typical duration between 30 fs and 30 ps. Examples of such lasers include diode-pumped lasers, for example, lasers based on neodymium-doped or ytterbium-doped crystals. Titanium-sapphire lasers can be used for pulse durations below 10 fs, and in extreme cases can be as low as about 5 fs (e.g., Octavius ​​Ti: Sapphire lasers, available from Thorlabs). In most cases, the pulse repetition rate is between 1 kHz and 500 MHz.

[0694] Femtosecond lasers can be fiber lasers. Various types of ultrafast fiber lasers, which can also be passively mode-locked, typically offer pulse durations between 50 and 500 fs, repetition rates between 0.10 and 100 MHz, and average powers between a few milliwatts and a few watts (femtosecond fiber lasers are available from Toptica, IMRA America, Coherent). Femtosecond fiber lasers are particularly suitable for this application. The lasers are reasonably priced when the energy output is less than 1 uJ. The repetition rate of the laser pulses is greater than 1 MHz, which may result in an acquisition rate of 1 Mpixel / s. The pulse duration of such lasers is about 200 fs, which is well within the upper limit of 10 ps determined by the diffusion broadening of the plasma region.

[0695] Femtosecond lasers can be semiconductor lasers. Some mode-locked diode lasers can generate pulses with femtosecond durations. Directly at the output of the laser, the pulse duration is typically at least a few hundred femtoseconds, but with external pulse compression, shorter pulse durations can be achieved.

[0696] In some embodiments, the laser is a nanosecond laser. The nanosecond laser can be a pump laser, for example, a Quantel Q-smart DPSS, a Solar Laser LQ929 high power pulsed Nd:YAG laser, or a Litron high energy pulsed Nd:YAG laser. All of these lasers can produce deep UV radiation in the mJ range and are therefore suitable for ionization and short pulse durations.

[0697] It is also possible to passively mode-lock vertical external cavity surface emitting lasers (VECSELs); these are particularly interesting because they can provide a combination of short pulse duration, high pulse repetition rate and sometimes high average output power, but they are not suitable for high pulse energies.

[0698] Postionization in sampling systems and devices based on dual-pulse laser ablation

[0699] Fig.17 An embodiment is shown 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 material on a small scale, similar to Fig.13 A third pulse 110 is sent to overlap the plume of ablated material. This is used to provide further ionization of this material. This step may be necessary to suppress neutralization effects that may occur in the ablation plume. The ions are then extracted through an opening (not shown) in the objective lens 60.

[0700] Postionization requires high energy density from laser radiation in a small volume, e.g., 5 μm 3Below. Because the post-ionization volume is small, it limits the amount of material that can be ionized at one time. If a large number of positive and negative charges are generated in a small volume, the motion of the ions formed will be dominated by the local fields generated by the space charge induced by the ions and electrons. If there are too many charged ions in a small volume, the external field (for example, the field of the ion optics present in the mass spectrometer to guide the resulting ions toward the detector for detection) will not be able to effectively separate the positive and negative charges, and this ion cloud will eventually neutralize, thereby reducing the ionization efficiency. For example, an ion cloud with a diameter of 10μm containing 10,000 elemental charges produces an electrostatic potential of about 3V. Since a few eV is the energy that holds an electron to an atom, it is also the possible energy level of a free electron after ionization. Therefore, in a volume on the scale of 10 microns, the ion density of 10,000 ions approaches the limit where space charge behavior begins to dominate.

[0701] Therefore, by ensuring that the amount of ablated material remains fairly small, this effect can be avoided. For example, ablated material on the scale of 10×10×10 nm cube to 30×30×30 nm cube or similar volumes represents the highest amount of material that can be delivered into the post-ionization region of a few microns, whose size does not produce strong spatial variations and ion neutralization. Since the system can only process a 30×30×30 nm cube at each single event, this creates the opportunity to image at a spatial resolution of 30 nm or even 10 nm, to which the ion beam can be focused (as described in this article).

[0702] The post-ionization laser beam / radiation is co-aligned with the beam of the second laser source within micrometer accuracy, which is typically obtained in the optical setup.

[0703] In some embodiments, the post-ionizing laser beam / radiation is directed at the sample at an angle and into the previously ablated region of the sample ( Figure 6 This configuration minimizes the interaction between the laser radiation used for post-ionization and the unablated sample. The laser beam can be focused to a tight focus that overlaps the volume of the ablation plume. Laser beam focusing can be performed at a high numerical aperture (NA) to facilitate precise focusing in the overlap region and to allow the laser energy to spread rapidly outside the overlap region to minimize the potential for damage to the sample surrounding the sampled region.

[0704] In some embodiments, the numerical aperture of the post-ionization laser beam / radiation can be confined in one plane. This arrangement results in an elliptical focal spot extending in the low NA plane. The elliptical focal spot can increase the overlap with the sputtering / ablation plume. Therefore, in some embodiments, the laser of the post-ionization system has an elliptical focal spot.

[0705] In addition to spatial coordination, the plume generated by the combined action of the first and second laser sources described above needs to be synchronized with the delivery of laser radiation to ionize the ablated material. The velocity of the ablated material leaving the target is typically in the range of the speed of sound, i.e., 1000 m / s. Therefore, in order to ensure that the ablation plume and the post-ionizing laser radiation / beam are aligned with an accuracy of 1 micron, a time accuracy of 1 ns is required. In some aspects, the speed can be between 1 km / s and 10 km / s, for example, between 2 km / s and 5 km / s, depending on the temperature and composition of the ablated material. As described herein, ablation can be classified as atmospheric pressure, partial vacuum pressure, or vacuum pressure.

[0706] Thus, in this mode of operation, the first and second laser sources act together to ablate material from the sample and shortly thereafter the ejected material is post-ionized by a pulse of laser radiation from the third laser source.

[0707] The nanoplasma produced by the action of the first laser and the second laser contains elemental ions, but in the case of post-ionization, these ions are not directly extracted. Instead, the ejected plume is allowed to expand, during which at least some charge neutralization occurs, because the plasma at that time is at high pressure and therefore dense, which means that collisional cooling and charge reduction occur very rapidly. As mentioned above, the expansion speed of the nanoplasma plume is about 1000-5000m / s. Therefore, after a few picoseconds, the plasma produced by the ablation of, for example, a 10nm diameter spot on the sample will have expanded by an order of magnitude, for example, to 100nm. 3 The additional or reionization of the plume after a 10-100 times size expansion of the original nanoplasma volume means that the components of the original plume are now significantly more dispersed, making collisions much less likely (and therefore charge neutralization less likely) when ionized to restore the micron-scale plasma, so a higher proportion of elemental ions can be extracted from the plume. Higher sensitivity is achieved through more efficient extraction of ions, including elemental ions from the labeled atoms.

[0708] Thus, in some embodiments, dual pulse laser based sampling and ablation systems and apparatus include a third laser source configured to ionize a plume of sample material ablated from the sample.

[0709] Therefore, the present invention provides a dual-pulse laser sampling and ionization system for analyzing a sample (e.g., a biological sample), comprising:

[0710] Sample table;

[0711] a first laser source configured to seed electrons in the sample; and a first focusing optic configured to direct a laser beam emitted by the first laser source toward the sample stage;

[0712] a second laser source configured to ablate the sample material pre-seeded with electrons by the first laser source; and second focusing optics configured to direct the laser beam emitted by the second laser source toward the sample stage;

[0713] a third laser source configured to ionize a plume of sample material ablated from the sample; and third focusing optics configured to direct a laser beam emitted by the third laser source toward a volume in which the ablated sample material forms the plume;

[0714] wherein the second focusing optics is configured to synchronize a second laser radiation pulse from the second laser source to arrive at a position on the sample stage immediately after a first laser radiation pulse from the first laser source; and

[0715] Therein, the third focusing optics is configured to synchronize the third laser radiation pulse to arrive at the volume above the location on the sample stage immediately after the second laser radiation pulse.

[0716] The third laser provides fast pulses, which may be less than 30 ps, ​​less than 10 ps, ​​less than 1 ps, less than 500 fs, or less than 100 fs, for example, between 1 fs and 10 ps, ​​between 1 fs and 1 ps, between 1 fs and 500 ps, ​​between 100 fs and 500 fs. In some embodiments, a first focusing optic configured to direct a laser beam emitted by a first laser source toward a sample stage is configured to direct the laser beam emitted by the first laser source toward a position on the sample stage, and a second focusing optic configured to direct a laser beam emitted by a second laser source toward the sample stage is configured to direct the laser beam emitted by the second laser source toward a position on the sample stage.

[0717] The volume discussed above with respect to the laser radiation from the third laser source refers to the volume above the sample. This is the volume where the ablation plume is formed when material is ejected from the sample. The above is the terminology used in this scenario relative to the sample, which is a sample on a horizontal plane. If the sample is held in a vertical plane so that the ablated plume is ejected along the horizontal axis, the volume reached by the laser radiation from the third laser source will be on the lateral side of the sample. In some embodiments, the volume is 5 μm 3 Below, for example, 2.5 μm 3 Below, 2μm 3 Below, 1μm 3 Below, or 100nm 3 In some embodiments, the volume is 5 μm 3 Below, for example, 2.5 μm 3 Below, 2μm 3 Below, 1μm 3 Below, or 100nm 3In some embodiments, the volume extends less than 2 μm, eg, less than 1 μm, or less than 100 nm from the sample surface.

[0718] As described above, the first laser source and the second laser source may represent separate lasers, or may be derived from the same single laser. Likewise, the third laser source may be a separate laser, or may be derived from (i) the same single laser as the first laser source; (ii) the same single laser as the second laser source, or (iii) the first, second, and third laser sources may all be derived from the same single laser.

[0719] In cases where different lasers are used, technical procedures in the art (e.g., a programming module comprising instructions) can be used to coordinate the delivery of the first pulse and the second pulse to the sample so that ablation of the p...

Claims

1. A device for analyzing a biological sample, comprising: Sample table; a charged particle source configured to deliver a charged particle beam to a location on the sample stage; as well as a first laser source and a first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, Wherein the charged particle source, the first laser source and the first focusing optics are configured such that the charged particle beam and the laser beam are directed towards opposite sides of the sample stage.

2. The device according to claim 1, wherein: The first focusing optics are configured to synchronize a laser beam pulse to arrive at the location on the sample stage immediately after a charged particle pulse.

3. The device according to claim 1, wherein: The first focusing optics are configured to synchronize laser beam pulses to ionize a plume of material sputtered by the charged particle pulses.

4. The apparatus of claim 1 further comprising a second laser source and a second focusing optics, wherein: The second focusing optics are configured to synchronize laser beam pulses from the second laser source to ionize a plume of material sputtered by the charged particle pulses.

5. The device according to claim 4, wherein: The first laser source and the first focusing optics and the second laser source and the second focusing optics are configured such that a laser beam from the first laser source and a laser beam from the second laser source are directed to the same side of the sample stage.

6. The device according to claim 4, wherein: The first laser source and the first focusing optics and the second laser source and the second focusing optics are configured such that a laser beam from the first laser source and a laser beam from the second laser source are directed toward opposite sides of the sample stage.

7. The device according to claim 6, wherein: The first laser source or the second laser source is configured to ionize a material by avalanche ionization.

8. The device according to claim 1, wherein: The charged particle source is a primary ion beam source or an electron beam source.

9. The device according to claim 1, wherein: The first laser source is a pulsed laser source.

10. The device according to claim 9, wherein: The first laser source is adapted to have a pulse energy between 1 nanojoule and 100 microjoules.

11. The device according to claim 1, wherein: The charged particle beam is pulsed.

12. The apparatus of claim 1, comprising a charged particle beam scanning system adapted to scan a plurality of locations on the sample stage with a charged particle beam.

13. The apparatus of claim 1 further comprising an electron microscope.

14. The device according to claim 13, wherein: The charged particle source is an electron beam source, wherein the electron beam source is an electron source in the electron microscope.

15. The apparatus according to claim 1, wherein: The sample stage is transparent or the sample stage includes a cutout portion.

16. The apparatus of claim 1 comprising a variable delay line.

17. The apparatus of claim 1, further comprising a sample chamber and a pump.

18. The apparatus of claim 1, further comprising a mass spectrometer.

19. An apparatus for analyzing a biological sample, comprising: Sample table; a charged particle source configured to deliver a charged particle beam to a location on the sample stage; as well as a first laser source and a first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage, Wherein, the first laser source is a pulsed laser source, and The first laser source is adapted to have a pulse energy between 1 nanojoule and 100 microjoules.

20. An apparatus for analyzing a biological sample, comprising: Sample table; a charged particle source configured to deliver a charged particle beam to a location on the sample stage; a first laser source and first focusing optics configured to direct a laser beam emitted by the first laser source toward the sample stage; as well as a second laser source and a second focusing optics, wherein the second focusing optics is configured to synchronize laser beam pulses from the second laser source to ionize a material plume sputtered by the charged particle pulses, and the first laser source or the second laser source is configured to ionize material by avalanche ionization.

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