High-speed modulation sample imaging device and method

Abstract

The present disclosure relates to systems and methods for high-speed modulated sample imaging. Disclosed herein are systems and methods for performing imaging mass cytometry, including labeling atoms by elemental (e.g., atomic) mass spectrometry. Various aspects include sampling systems with femtosecond (fs) lasers and / or laser scanning and methods using femtosecond (fs) lasers and / or laser scanning. Alternatively or additionally, various aspects include systems and methods for co-registering other imaging modalities with imaging mass cytometry.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims priority to U.S. Provisional Patent Application No. 62 / 729,241, filed September 10, 2018, and U.S. Provisional Patent Application No. 62 / 828,251, filed April 2, 2019, which are hereby incorporated by reference in their entireties for all purposes. Technical Field

[0003] The present invention relates to imaging samples after laser ablation using imaging mass spectrometry (IMS) and imaging mass cytometry (IMC) TM ) to image biological samples. Background Art

[0004] LA-ICP-MS (a form of IMS in which a sample is ablated by a laser, the ablated material is then ionized in an inductively coupled plasma, and the ions are detected by mass spectrometry) has been used for the analysis of a variety of substances, such as mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological materials[i].

[0005] Imaging of biological samples by IMC has been previously reported, enabling imaging at cellular resolution [ii, iii, iv] and more recently detailed imaging at subcellular resolution [v].

[0006] These methods of generating images by IMS and IMC are characterised by the movement of a stage supporting the sample so that the laser radiation can ablate different locations of the sample to produce pixels. However, the reliance on sample stage movement results in a relatively low pixel acquisition rate and therefore a relatively low throughput in terms of the sample area that can be investigated per unit time. There are fast stages that can move in the X and Y axes, with maximum speeds in the range of 100 mm / s. However, these stages still have the disadvantage of being subject to stage inertia, which means that time is spent accelerating the stage to its maximum speed during the imaging method. Stage inertia also means that stage movement cannot be used to quickly create arbitrary scan patterns.

[0007] It is an object of aspects of the present invention to provide further and improved apparatus and techniques for imaging samples. Summary of the Invention

[0008] Disclosed herein are systems and methods for performing imaging mass cytometry, including analysis of labeled atoms by elemental (e.g., atomic) mass spectrometry. Aspects include sampling systems and methods using femtosecond (fs) lasers and / or laser scanning. Alternatively or additionally, aspects include systems and methods for co-registering other imaging modalities with imaging mass cytometry.

[0009] In certain embodiments, the analyzer apparatus disclosed herein comprises two systems for performing extensive characterization of imaging elemental mass spectrometry.

[0010] The first is a sampling and ionization system. The system comprises a sample chamber, which is a component in which the sample is placed when it is analyzed. The sample chamber comprises a stage, which holds the sample (typically the sample is placed on a sample carrier, such as a microscope slide, for example a tissue section, a monolayer of cells or a single cell, such as a cell smear, in which a cell suspension has been dropped 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) and then convert it into ions, either as part of a process that results in the removal of material from the sample, or via a separate ionization system downstream of the sampling system. In order to produce elemental ions, hard ionization techniques are used.

[0011] The ionized material is then analyzed by a second system, a detector system. The detector system can take different forms, depending on the specific characteristics of the ionized sample material being determined, for example, a mass detector in a mass spectrometry-based analyzer device.

[0012] One aspect of the present invention provides an improvement over current IMS and IMC devices and methods by employing a laser scanning system in the sampling and ionization system. The laser scanning system directs laser radiation onto the sample to be ablated. Because the laser scanner can move faster than the sample stage due to its much lower or no inertia (i.e., has a faster response time), discrete spots on the sample can be ablated more quickly, thereby significantly increasing the area ablated per unit time without losing resolution. In addition, the rapid change in the spots to which the laser radiation is directed allows for the ablation of random patterns, for example, so that entire cells of uneven shape can be ablated by a rapid succession of bursts of laser radiation pulses / shots, which are then ionized and detected as a single cloud of material using a laser scanner system to target the bursts of laser radiation pulses / shots to locations on the sample, thereby enabling single cell analysis. These locations are typically adjacent locations or close to each other. A similar rapid cluster technique, namely cell LIFTing (laser-induced forward transfer), can also be employed in methods using desorption to remove sample material from a sample carrier. Adjacent locations of a plume of samples analyzed together as continuous events may be from within a single feature of interest, such as a specific cell.

[0013] In operation, a sample is placed in the device, sampled using a laser scanning system to produce ionized material (sampling may produce a gaseous / special material, which is then ionized by the ionization system), and the ions in the sample material are transferred to the detector system. Although the detector system can detect many ions, most of them will be ions of atoms that naturally make up the sample. In some applications, such as mineral analysis in geological or archaeological applications, this may be sufficient.

[0014] In some cases, such as when analyzing biological samples, the natural elemental composition of the sample may not provide adequate information. This is because all proteins and nucleic acids are generally composed of the same major constituent atoms, so while regions containing such proteins / nucleic acids can be distinguished from regions without such material, specific proteins cannot be distinguished from all other proteins. However, by labeling the sample with atoms that are not present in the material being analyzed, or at least not present in large quantities under normal conditions (e.g., certain transition metal atoms, such as rare earth metals; see the labeling section below for more details), specific characteristics of the sample can be determined. As with IHC and FISH, detectable labels can be attached to specific targets on or in the sample (such as fixed cells or tissue samples on a slide), particularly by using specific binding partners (SBPs) such as antibodies, nucleic acids, or lectins to target molecules on or in the sample. To detect ionized labels, a detector system is used because it will detect ions from atoms naturally present in the sample. By linking the detected signals to the known locations of the sample samples that produced these signals, an image of the atoms present at each location can be generated, including the native elemental composition and any labeled atoms (e.g., see References 2, 3, 4, 5). Insofar as the native elemental composition of the sample is depleted prior to detection, the image may only have labeled atoms. This technique allows for the analysis of many labels in parallel (also known as multiplexing), which has great advantages in biological sample analysis, with increased speed due to the application of the laser scanning system in the apparatus and methods disclosed herein.

[0015] Thus, aspects of the present invention provide an apparatus for analyzing a sample, such as a biological sample, the apparatus comprising:

[0016] (i) a sampling and ionization system for removing material from the sample and ionizing the material to form elemental ions, the system comprising a laser source, a laser scanning system, and a sample stage;

[0017] (ii) A detector that receives elemental ions from the sampling and ionization system and detects the elemental ions.

[0018] In some embodiments, the sampling and ionization system comprises a sampling system and an ionization system, wherein the sampling system comprises a laser source, a laser scanning system, and a sample stage, and wherein the ionization system is adapted to receive material removed from the sample by the sampling system and ionize the material to form elemental ions.

[0019] The laser scanning system imparts relative movement of the direction of the laser beam emitted by the laser source relative to the sample stage using one or more positioners (e.g., two positioners) along one or more non-parallel, and in some embodiments, orthogonal, axes (e.g., the Y and X axes). As described below, the positioners can take the form of mirror-based positioners (such as galvanometer mirrors, polygon scanners, MEMS mirrors, piezoelectric device mirrors) and / or solid-state positioners (such as AODs or EODs). The sample stage can also be moved to produce relative movement of the sample on the stage relative to the laser radiation beam. The sample stage can typically move the sample along the x- and y-axes, and can also move the sample along the z-axis, with its movement coordinated by a controller module with the movement of the positioners in the laser scanning system. For example, the stage can move the sample along a first direction, and this position can introduce relative movement into the laser beam along a second (i.e., non-parallel, such as substantially orthogonal) direction. As described above, IMS and IMC have been achieved with subcellular resolution, and laser scanning systems can be used at such resolutions. Thus, ablation can be performed with a spot size of less than 10 μm, less than 5 μm, less than 2 μm, about 1 μm, or less than 1 μm in diameter. For example, ionization of the sample material to produce elemental ions can be achieved using ICP, laser desorption / ionization (LDI), and / or by laser generation of a plasma, and detection using a TOF mass spectrometer.

[0020] In some aspects, the locator can be operated to scan features such as a single cell or a portion of a single cell (such as a nucleus, cytoplasm, cell membrane, or organelle). Features can be acquired in a single ablation plume. Features may not have regular boundaries (e.g., may not be square or circular). For example, many cells in a tissue do not conform to a regular shape. Thus, optical inspection methods can identify features to be acquired by laser scanning and analyzed by ICP-MS. In some aspects, an initial sampling of the mass label distribution in the sample can inform the area of interest, and then optical inspection (e.g., optical microscopy) is used to identify features (such as cells) for acquisition by laser scanning coupled to ICP-MS.

[0021] Laser scanning systems also enable new modes of operating IMS / IMC devices, involving more sophisticated sampling methods. Many of these modes allow for the ablation of a region / feature of interest using bursts of laser pulses, such as plumes generated by firing bursts of laser pulses at multiple known locations within the region / feature of interest, which can be analyzed as a continuous event. Therefore, as described below, in some embodiments, the device includes a camera to help locate the location containing the region / feature of interest.

[0022] Thus, aspects of the present invention provide a method of analyzing a sample, the method comprising:

[0023] (i) performing laser ablation of the sample on a sample stage, wherein laser radiation is directed onto the sample using a laser scanning system, and wherein ablation is performed at a plurality of known locations to form a plurality of plumes; and

[0024] (ii) Ionization and mass spectrometry of the plume, whereby the detection of atoms in the plume allows the construction of an image of the sample.

[0025] Aspects of the present invention also provide a method for performing mass cytometry on a sample comprising a plurality of cells, the method comprising:

[0026] (i) labeling a plurality of different target molecules in a sample with one or more different labeling atoms to provide a labeled sample;

[0027] (ii) performing laser ablation of the sample on a sample stage, wherein laser radiation is directed onto the sample using a laser scanning system, and wherein ablation is performed at a plurality of locations to form a plurality of plumes; and

[0028] (iii) ionizing and mass spectrometrically analyzing the plume, whereby detecting atoms in the plume allows for the construction of an image of the sample, optionally where the plurality of locations is a plurality of known locations.

[0029] In certain aspects, for example, when a single plume is generated from a single feature and analyzed by mass spectrometry, the feature is acquired as a continuous event.

[0030] Sometimes, the method also constructs an image of the sample.

[0031] Aspects of the present invention also provide a method for analyzing a sample, the method comprising:

[0032] (i) desorbing the bulk of sample material using laser radiation, wherein the laser radiation is directed onto the sample on the sample stage using a laser scanning system; and

[0033] (ii) Ionizing a bulk of sample material and detecting the atoms in the bulk by mass spectrometry.

[0034] Another method provided by aspects of the present invention is a method for performing mass cytometry on a sample comprising a plurality of cells, the method comprising:

[0035] (i) labeling a plurality of different target molecules in a sample with one or more different labeling atoms to provide a labeled sample;

[0036] (ii) desorbing the mass of sample material using laser radiation, wherein the laser radiation is directed onto the sample on the sample stage using a laser scanning system; and

[0037] (iii) Ionizing a bulk of sample material and detecting the atoms in the bulk by mass spectrometry.

[0038] A method may include a method for registering images, the method comprising the steps of obtaining a first image from a first tissue section of a tissue sample by an imaging modality other than imaging mass cytometry, obtaining a second image from a second tissue section of the tissue sample by imaging mass cytometry, and registering the first image with the second image. In certain aspects, the first image, or both the first image and the second image, may be provided by a third party. Imaging mass cytometry may be performed by LA-ICP-MS, optionally using a femtosecond laser and / or a laser scanning system.

[0039] Methods of imaging mass cytometry may include identifying a feature in a sample using an optical microscope, scanning radiation through the feature to generate a plume of material, and delivering the plume of material to a mass analyzer. The feature may be a single cell. The sample may include mass-tagged SBPs. The method may include analyzing more than 100 single cells per second. The radiation may be laser radiation. The method may further include ionizing the material using ICP. The mass analyzer may include a time-of-flight detector. Systems for performing such methods are also described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the optical device of the existing equipment configuration.

[0041] Figure 2 is a schematic diagram of an optical arrangement of an exemplary embodiment of aspects of the present invention.

[0042] Figure 3 is a schematic diagram of an optical arrangement of another exemplary embodiment of aspects of the present invention.

[0043] Figure 4 is a schematic diagram of an optical arrangement of another exemplary embodiment of aspects of the present invention.

[0044] Figure 5is a schematic diagram of an optical arrangement of another exemplary embodiment of aspects of the present invention, illustrating sampling by directing laser radiation through a sample carrier.

[0045] Figure 6 This figure illustrates the difference in resolution provided when imaging a sample using a consistent spot size. As the spot size increases, signals from different cells begin to bleed into each other. This figure serves to demonstrate the importance of an advance embodied in various aspects of the present invention, whereby signals can be acquired from single cells using rapid, arbitrary scanning of patterns to ablate individual cells or desorb entire cells via LIFTing.

[0046] Figure 7 A laser path is shown that combines stage movement with relative movement of the light beam using a laser scanning system that includes at least one positioner as described herein. The laser scanner system movement allows for ablation of certain cells by directing the laser radiation beam by scanning in the Y-axis as the stage moves in the X-axis (including correction for stage movement in the X-axis by the scanning system). The scanner deflects the beam from the stage path only when cells are present that the user of the device wishes to ablate are present.

[0047] Figure 8 An alternative scanning mode of operation is shown, whereby the scanner system is moved in a manner that enables the laser beam to be directed over a large area. Laser pulses are fired at the sample only when the laser scanner system is in a direction that focuses the laser beam on the area of interest to be ablated, such as a specific cell.

[0048] Figures 9a and 9b depict the laser scanner system in non-resonant ( Figure 8 a) and resonance ( Figure 8 b) Path movement in the trajectory.

[0049] Figure 10is a simulated illustration of a method of various aspects of the present invention for desorbing a mass of material comprising a single cell from a sample on a sample carrier. In the method, a cell of interest is identified at a location of interest in image (A). In image (B), the area surrounding the cell of interest is cleared by ablation, which not only removes cellular material near the cell of interest, but also removes any desorbed membrane present on the sample carrier. As disclosed herein, a laser scanner system can be used to quickly clear various ablation spots surrounding the cell of interest because the laser scanner system allows the laser radiation beam to be quickly deflected to an arbitrary position, thereby being able to ablate complex patterns that track the location of the cell membrane of the cell without desorbing the cell of interest itself from the sample carrier. The cell of interest with the cleared area is shown in image (C). After clearing, the cell of interest is desorbed from the sample using a series of laser radiation spots directed at the sample. In the exemplary method shown in the image, the laser is directed to a delivery position for delivering laser radiation pulses inwardly in a spiral radiation pattern to release the mass of sample material from the sample carrier (D). The pulses of laser radiation can be delivered directly or through the sample carrier (in a manner similar to the embodiment of the present invention). Figure 5 The operating mode shown) acts directly on the sample.

[0050] Figure 11 The figure is an exemplary schematic diagram of laser ablation mass cytometry, which includes a laser ablation source that can be connected to a sample injector (such as a tube) and mounted to deliver the sample to an inductively coupled plasma (ICP) source (also known as an ICP torch). The plasma of the ICP torch can evaporate and ionize the sample to form ions, which can be received by a mass analyzer (such as a time-of-flight or magnetic sector mass spectrometer).

[0051] Figure 12 is a schematic diagram of high-NA optical devices that can be integrated into the system described in this article.

[0052] Figure 13 This is a second harmonic generation (SHG) image of collagen tissue published online by the University of Minnesota College of Biological Sciences.

[0053] Figure 14 Nonlinear microscopy images of breast cancer tissue are shown.

[0054] Figure 15 A system integrating a nonlinear microscope according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0055] Thus, various types of analyzer devices including laser scanner systems may be used in practicing the present disclosure, several of which are discussed in detail below.

[0056] Analyzer equipment based on quality inspection

[0057] 1. Sampling and ionization systems

[0058] a. Laser ablation sampling and ionization system

[0059] Laser ablation-based analyzers typically consist of three components. The first is a laser ablation sampling system, which is used to generate a plume of gaseous and particulate material from the sample to be analyzed. The sample must be ionized (and atomized) before the atoms in the plume of ablated sample material (including any detectably labeled atoms, as described below) can be detected by a detector system—the mass spectrometer assembly (MS assembly; the third assembly). Therefore, the instrument contains a second component, an ionization system, which ionizes the atoms to form elemental ions, making them detectable by the MS assembly based on their mass-to-charge ratio. (Some ionization of the sample material may occur at the ablation point, but space charge effects lead to almost immediate charge neutralization.) The laser ablation sampling system is connected to the ionization system via a delivery conduit.

[0060] Laser ablation sampling system

[0061] Briefly, the components of a laser ablation sampling system include a laser source that emits a beam of laser radiation that is directed onto 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 so that the sample can be moved relative to the laser radiation beam so that different locations on the sample can be sampled for analysis (e.g., locations that are much closer to each other than locations that can be ablated due to relative movement in the laser beam than can be induced by the laser scanning system described herein). 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 based on its elemental composition (including labeled atoms, such as labeled atoms from elemental tags). As further explained below, in an alternative mode of action, the laser system of the laser ablation sampling system can also be used to desorb material from the sample.

[0062] Especially for biological samples (cells, tissue sections, etc.), the samples are usually heterogeneous (although heterogeneous samples are known in other application areas of the present disclosure, i.e. samples of non-biological nature). A heterogeneous sample is a sample that contains regions composed of different materials, so that at a given wavelength, some regions of the sample can be ablated at a lower threshold energy density than other regions. Factors that affect the ablation threshold are the absorption coefficient of the material and the mechanical strength of the material. For biological tissue, the absorbance coefficient will play a major role because it will vary by several orders of magnitude with the wavelength of the laser radiation. For example, in a biological sample, when using nanosecond laser pulses, areas containing proteinaceous material will absorb more easily in the wavelength range of 200-230nm, while areas containing mainly DNA will absorb more easily in the wavelength range of 260-280nm.

[0063] Laser ablation can be performed at energy densities close to the ablation threshold of the sample material. Ablation in this manner often improves aerosol formation, which in turn helps improve the quality of the data after analysis. Typically, a Gaussian beam is used to obtain the smallest crater possible, thereby maximizing the resolution of the resulting image. The cross section of a Gaussian beam records an energy density curve with a Gaussian distribution. In this case, the energy density 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 energy density and the threshold energy density.

[0064] Therefore, to ensure that a reproducible amount of material is consistently removed with each ablation laser pulse, thereby maximizing 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 by the laser pulse to the target to the ablation threshold energy of the ablated material. This requirement presents a problem when ablating heterogeneous samples, where the threshold ablation energy varies throughout the sample, such as biological tissues where the ratio of DNA to protein material varies, or in geological samples where it varies with the specific mineral composition within 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 efficiently ablate the sample based on the sample composition at that location.

[0065] Laser system for laser ablation sampling system

[0066] Laser systems can be configured to generate laser radiation at a single wavelength or at multiple wavelengths (i.e., more than two). Typically, the wavelength of laser radiation in question is the wavelength with the highest intensity (the "peak" wavelength). If a system generates different wavelengths, they can be used for different purposes, for example, to target different materials in a sample (targeting in this context means that the selected wavelength is one that is well absorbed by the material).

[0067] 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 a first radiation wavelength that is discrete from a second radiation wavelength, this means that the first laser source does not produce radiation at the second wavelength in pulses of the first wavelength, or produces radiation at the second wavelength at only a very low level, e.g., less than 10% of the intensity at the first wavelength, such as less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In general, when laser radiation of different wavelengths is generated by harmonic generation or other nonlinear frequency conversion processes, then when reference is made herein to a specific wavelength, those skilled in the art will understand that there will be some degree of variation with respect to the specific wavelength in the spectrum generated by the laser. For example, a reference to X nm includes lasers that produce a spectrum within the range of X ± 10 nm (such as X ± 5 nm, e.g., X ± 3 nm).

[0068] Laser scanning system

[0069] The present invention provides improvements over current IMS and IMC devices and methods by employing a laser scanning system within the sampling and ionization system. The laser scanning system directs laser radiation onto the sample to be ablated. Compared to moving the sample stage relative to a fixed laser beam (due to the much lower or no inertia of the operating components of the scanning system), the laser scanner can re-point the laser focus position on the sample more quickly (due to the much lower or no inertia of the scanning system's operating components), thus enabling faster ablation of discrete spots on the sample. This faster speed can enable larger areas to be ablated and recorded as individual pixels, or the speed of laser spot movement can simply translate into, for example, an increase in pixel acquisition rate, or a combination of both. Furthermore, the rapid change in the spot position to which the pulses of laser radiation are directed allows for the ablation of random patterns, for example, enabling the ablation of entire cells of non-uniform shape through rapid succession of bursts of laser radiation pulses / emissions, using the laser scanner system to target the pulses / emissions to locations on the sample, which are then ionized and detected as a single cloud of material, thereby enabling single-cell analysis (see the section "Sample Chamber of Laser Ablation Sampling Systems" starting on page 28). In methods using desorption to remove sample material from a sample carrier, a similar rapid burst technique, namely cell LIFTing (laser induced forward transfer), can also be employed, as discussed in detail with respect to apparatus and methods from page 55 onwards.

[0070] In existing imaging mass cytometry systems, the stage can be moved to allow ablation of different pixels (ablation spots). Laser scanning using the positioner described herein (optionally combined with translation of the sample stage) can allow acquisition of pixels of arbitrary shape and size, such as rapid acquisition of features or portions of features. Pixels can be detected as continuous signals provided by transient ablation plumes.

[0071] Thus, aspects of the present invention provide an apparatus for analyzing a sample, such as a biological sample, the apparatus comprising:

[0072] (i) a sampling and ionization system for removing material from the sample and ionizing the material to form elemental ions, the system comprising a laser scanning system and a sample stage;

[0073] (ii) A detector that receives elemental ions from the sampling and ionization system and detects the elemental ions.

[0074] Using a scanning system to increase the acquisition rate has many advantages over other strategies for increasing the sample imaging rate. For example, a 100 μm × 100 μm area can be ablated with a single laser pulse using appropriately adapted equipment. However, this type of ablation leads to a number of problems. Ablating a large area of the sample at once with a single laser pulse causes the ablated material to break into large pieces that initially fly away at speeds close to the speed of sound. Instead of small particles and moving the material quickly away from the sample in the carrier gas flow (described in more detail below), large pieces may take longer to be entrained than small pieces (extending the flushing time of the sample chamber), fail to be entrained, or simply fly away from the sample or another part of the sample randomly. If a large piece of material flies away from the sample, any information in the form of detectable atoms (such as marker atoms) in that piece of material is lost. If the large piece of material lands on another part of the sample, the information will be lost from the ablated area. In addition, any detectable atoms in the large piece of material are now dependent on and may interfere with the signal to be acquired from another part of the sample. Larger ablation spot sizes can also complicate sample separation because differences in the biomaterial within the ablation spot (e.g., between cartilage and muscle) can also affect how the product breaks down, with some materials being entrained in the gas flow to a lesser extent than others. Furthermore, as described herein, in many applications, small spot sizes, on the order of microns, rather than hundreds of times larger, are preferred, and switching between laser spot sizes over multiple orders of magnitude (e.g., 100 μm versus 1 μm) can also present technical challenges. For example, a laser capable of ablating a spot size of 1 μm may not have the energy to ablate an area with a spot size of 100 μm in a single laser pulse, and therefore complex optics are required to facilitate the transition between 1 μm and 100 μm without significant loss of laser beam energy and loss of sharpness in the ablation spot.

[0075] Therefore, in addition to ablating 100 μm 2Instead of a single spot of 1 μm in diameter, the entire area can be rastered using 100×100 (i.e., 10,000) spots of 1 μm diameter to ablate the area. Of course, smaller ablation spot sizes do not suffer as much from the above problems—the particles produced by smaller ablation spots must themselves be much smaller. In addition, with smaller spots, the resulting smaller particles produced by ablation have a shorter and more deterministic flushing time from the sample chamber. When it is desired to resolve each smaller spot separately, this in turn has the following consequences: data can be acquired faster because the transients from each ablation laser pulse do not overlap (or overlap to an acceptable extent, as described below) when detected in the detector.

[0076] However, as mentioned above, moving the sample stage in 1 μm increments along one row and then to the next row is relatively slow due to inertia. Therefore, by using a laser scanner system to raster over the entire area, the relatively slow speed of the sample stage does not limit the rate at which the sample can be ablated without moving the sample stage, or moving the sample stage infrequently or at a constant speed.

[0077] Therefore, in order to enable rapid scanning, the laser scanning system must be able to rapidly switch the location at which the laser radiation is directed onto the sample. The time taken to switch the ablation location of the laser radiation is referred to as the response time of the laser scanning system. Thus, in some embodiments of aspects 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.

[0078] The laser scanning system can direct the laser beam in at least one direction relative to the sample stage on which the sample is positioned during ablation. 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 gradually move the sample in the X-axis, and the laser can be swept across the sample in the Y-axis (for a description of relative movement, see Figure 7 to Figure 9). When using a 1 μm spot size, movement in the X-axis might be in 1 μm increments. At a given position in the X-axis, a 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 laser radiation to different positions in the Y-axis much faster than the stage can be moved incrementally in the X-axis, the ablation rate can be significantly increased in this simple operation of the scanner.

[0079] In some aspects, the laser scanning system can be configured to scan in only one direction. For example, the laser scanning system may have only one positioner that is capable of scanning in only one direction. In this case, the sample stage can be moved to provide movement in a different direction that is not parallel to the laser beam direction.

[0080] In some aspects, the scanned area (e.g., a region of interest) can be increased by moving the sample stage while the laser beam is directed by the laser scanning system. Without sample stage movement, the area scanned by the laser beam may be limited by the size of the window through which the beam passes, such as a window on top of the laser ablated cell and / or a window of a portion of an injection tube within the laser ablated cell (chamber) that is located for absorbing the irradiated sample. Alternatively or additionally, without sample stage movement, the area covered by the laser beam may be limited by the need to position the portion of the sample affected by the laser beam near an aerosol absorption system (e.g., an injector tube) that transports the sample (e.g., the sample ablated, desorbed, or lifted by the laser beam) to the ionization system and / or mass detector. Thus, movement of the stage during laser scanning may increase the area scanned continuously. In some aspects, multiple regions of interest are scanned.

[0081] In some cases, the laser scanning system directs the laser beam in both the X and Y axes. 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 and Y axes, the sample stage can be moved at a constant speed in the X axis (thereby eliminating inefficiencies associated with the inertia of the sample stage during movement across each row, except for acceleration / deceleration at the beginning / end of the row), while the laser scanning system directs the laser radiation pulses up and down across the columns of the sample while compensating for the movement of the sample stage. To achieve this movement, a triangular wave control signal can be applied to the scanner in the X direction, while a sawtooth signal is applied in the Y direction. Alternatively, as will be understood 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. Alternatively, one of the scanner components can be rotated slightly to pre-compensate for a tilted scan pattern. In some embodiments, the controller of the laser scanning system causes the laser scanner system to move the beam in a figure-8 pattern as the sample stage moves.

[0082] If the laser used in the laser sampling system has a sufficiently high repetition rate (as described below), the laser radiation can be (re)directed significantly more quickly to different locations on the sample, allowing for faster ablation of large sample areas. For example, if fewer than five pulses per second can be directed to different locations on the sample, the time required to study the ablation of a 1 mm × 1 mm area at a spot size of 1 μm would exceed two days. At a rate of 200 Hz, this would be approximately 80 minutes, and analysis time is further reduced with increasing pulse frequency. However, samples are typically much larger. The average microscope slide on which a tissue section can be placed measures 25 × 75 mm. Ablation at a rate of 200 Hz would take approximately 110 days. However, this time can be significantly reduced if a laser scanning system is used. For example, the sample stage moves along the X-axis at a constant speed (1 mm / s), while the laser beam moves back and forth along the Y-axis with the laser scanning system. The laser scanning system can scan the position of the laser focus at a rate that matches the stage movement speed (500 Hz in this case). At this speed, a spacing of 1 μm is produced between adjacent lines in the raster pattern. Then, the degree of deflection of the laser radiation by the matching laser scanning system is selected based on the maximum laser repetition rate. Here, a peak-to-peak amplitude of 100 microns will be generated, which will require a laser repetition rate of 100 kHz. This is in contrast to the maximum 0.0004 mm of current equipment. 2 / s, the equipment can process 0.1mm 2 / s. Compared to the 110-day figure discussed above, it only takes about 5 hours to process a slide using the laser scanning system discussed in this paragraph.

[0083] Another application is the shaping of arbitrary ablation areas. If a high repetition rate laser is used, a beam of closely spaced laser pulses can be sent at the same time that the laser sends one pulse in one nanosecond. By rapidly adjusting the X and Y positions of the ablation spot during a burst of laser pulses, ablation pits of arbitrary shape and size (down to the diffraction limit of light) can be created. For example, the distance between the n and n+1 positions in the burst must not be greater than or 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), such as 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 diameter of the spot size. Specific methods using this technology are discussed in the methods section below, page 36.

[0084] Thus, in some embodiments, the laser scanning system includes a positioner to impart a first relative movement of the laser beam emitted by the laser with respect to the sample stage (eg, with respect to the Y-axis of the sample surface).

[0085] In some embodiments, the positioner of the laser scanning system is capable of applying a second relative movement of the laser beam relative to the sample stage, wherein the first relative movement and the second relative movement are not parallel, such as wherein the relative movements are orthogonal (e.g., the first movement direction is on the Y axis relative to the sample surface and the second movement direction is on the X axis relative to the sample surface).

[0086] In some embodiments, the laser scanning system further comprises a second positioner capable of applying a second relative movement of the laser beam relative to the sample stage, wherein the first relative movement and the second relative movement are not parallel, such as wherein the relative movements are orthogonal (e.g., the first movement direction is on the Y axis relative to the sample surface and the second movement direction is on the X axis relative to the sample surface).

[0087] Laser Scanning System Components

[0088] Any component that can quickly direct 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 those skilled in the art can select an appropriate positioner based on the specific application in which the device is to be used, as each positioner has inherent advantages and limitations. In some embodiments of aspects of the present invention, as described below, multiple positioners discussed below can be combined in a single laser scanning system. Positioners can generally be divided into positioners that rely on moving components to introduce relative movement into the laser beam (e.g., galvanometer mirrors, piezoelectric mirrors, MEMS mirrors, polygon scanners, etc.) and those that do not use a positioner (examples include acousto-optic devices and electro-optic devices). The types of positioners listed in the previous sentence can controllably deflect the laser radiation beam to various angles, resulting in translation of the ablation spot. The laser scanning system can include a single positioner, or it can include a positioner and a second positioner. In the description of "a positioner" and "a second positioner" as two positioners in a laser scanning system, there is no restriction on the order in which the laser radiation pulses strike the positioners on their path from the laser source to the sample.

[0089] -Galvanometer mirror positioner

[0090] A galvanometer motor, mounted on a shaft with a mirror, can be used to deflect laser radiation to different locations on the sample. Movement can be achieved using a fixed magnet and a moving coil, or a fixed coil and a moving magnet. The fixed coil and moving magnet arrangement produces a faster response time. Typically, sensors are included in the motor to sense the position of the shaft and mirror, providing feedback to the motor controller. A single galvanometer mirror can steer a laser beam into one shaft; therefore, using this technique, pairs of galvanometer mirrors can be used to achieve beam direction in both the X and Y axes.

[0091] One advantage of a galvanometer mirror is that it can achieve large deflection angles (much larger than a solid-state deflector), so the sample stage can be moved less frequently. However, since the moving components of the motor and the mirror have mass, they will be affected by inertia, so the time for the components to accelerate must be accommodated within the sampling method. Typically, a non-resonant galvanometer mirror is used. As the person skilled in the art will understand, a resonant galvanometer mirror can be used, but a device using only resonant components such as a positioner of a laser scanning system will not be able to have an arbitrary (also called random access) scanning pattern. Since it is based on a mirror, a galvanometer mirror deflector will reduce the quality of the laser radiation beam and increase the size of the ablation spot, so the person skilled in the art will again understand that this is the case that can best tolerate such effects on the beam.

[0092] Galvanometer-mirror-based devices can be prone to positioning errors due to sensor noise or tracking errors. Therefore, in some embodiments, each mirror is associated with a position sensor that feeds the mirror's position back to the galvanometer to refine the mirror's position. In some cases, the position information is relayed to another component, such as an AOD or EOD connected in series to the galvanometer mirror, to correct for the mirror's positioning errors.

[0093] Galvanometer mirror systems and assemblies are commercially available from various manufacturers, such as Thorlabs (New Jersey, USA), Laser2000 (United Kingdom), ScanLab (Germany), and Cambridge Technology (Massachusetts, USA).

[0094] In embodiments that include only galvanometer mirror-based positioners, the rate at which ablation laser pulses can be directed toward 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.

[0095] Thus, in some embodiments of aspects of the present invention, the laser scanner system includes one or more positioners that are galvanometer mirrors, such as galvanometer mirror arrays. Figure 1 、 Figure 2 、 Figure 3 and Figure 5 Discuss the setup of a mirror-based laser scanner.

[0096] Figure 1Figure 1 is a schematic diagram of the optical components of a conventional apparatus setup. Here, a laser source (e.g., a pulsed laser source, optionally including a pulse picker) 101 emits a laser beam, which is directed through an energy control module 102 and then beam shaping optics 103. The radiation beam then passes through beam / illumination combining optics 104, through focusing optics and objective lens 105, and is directed toward the sample. The sample is positioned on a glass side 107, located on a three-axis (i.e., x, y, z) translation stage 108 within a sample chamber 106. Figure 1 The setup also contains a camera 111 for observing the sample using the same focusing optics and objective 105. The 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 towards the sample.

[0097] Figure 2 is a schematic diagram of an optical arrangement of exemplary embodiments of various aspects of the present invention. Figure 1 The same elements are set up. A laser source (e.g., a pulsed laser source, optionally including a pulse picker) 201 emits a laser radiation beam, which is directed through an energy control module 202. Before the laser radiation beam is shaped and imaged by the beam shaping and imaging optics 203, a positioner - a mirror 212 such as a galvanometer mirror (or piezoelectric mirror, MEMS mirror or polygon scanner, as described below) - deflects the laser radiation beam. A single mirror in a galvanometer mirror based device allows the laser radiation beam to be scanned in one direction, for example in one direction relative to the Y axis of the sample. The deflection introduced by the mirror 212 is carried throughout the optics, resulting in ablation at different locations on the sample 207 depending on the position of the mirror. The mirror is coordinated by a movement and trigger controller 213. In Figure 2 In the setup, the controller 213 coordinates the mirror with the position on the sample stage 208 to determine a specific position on the sample after ablation by the laser radiation beam. The controller 213 is also connected to the laser source to coordinate the generation of laser pulses (so that the laser source generates pulses when the mirror 212 is in a defined position rather than when it moves between two positions). The radiation beam is then directed toward the sample by the beam / illumination combining optics 204 through the focusing optics and the objective lens 205. The sample is located on the glass side 207 and on the sample stage in the sample chamber 206, which is a three-axis (i.e., x, y, z) translation sample stage 208. Figure 2 The setup also includes a camera 211 for observing the sample using the same focusing optics and objective 205. An illumination source 209 emits visible light which is directed to the sample through illumination / inspection separation optics 210, beam / illumination combining optics 204 and focusing optics 205. Figure 5 An alternative arrangement is shown in . Here, Figure 5All components of Figure 2 Same, except the system operates to ablate the sample through the sample carrier. This arrangement may be preferred, for example, when it is necessary to impart additional kinetic energy into the ablated sample material to help remove material from the area proximal to the ablation spot.

[0098] Figure 3 is a schematic diagram of an optical device arrangement of another exemplary embodiment of aspects of the present invention. Figure 2 The same elements of the setup are used. However, instead of using a single mirror positioner, a pair of mirror positioners is used to introduce deflection into the laser radiation beam. As described elsewhere herein, the mirror pair can be arranged to provide scanning in two orthogonal directions (X and Y), which can compensate for movement of the sample on the sample stage. Figure 3 The other components correspond to Figure 2 Components marked with corresponding reference numerals in FIG (ie, 301 is a laser source (eg, a pulsed laser source, optionally including a pulse picker), as shown in FIG Figure 2 201 etc.).

[0099] -although Figures 1 to 5 The camera is displayed on the same side of the sample support (such as a glass slide), but configurations that enable transillumination are also within the scope of this application. For example, the translatable stage can be offset from the sample so that the sample support allows transillumination. Transillumination can provide improved optics for certain applications, but can compete with the injector that transfers the ablated material to the mass analyzer. Thus, the systems described herein may not allow transillumination. As used herein, a sample support can refer to any slide for holding a sample and / or a sample stage for holding a slide. Although glass slides are described in some examples, the slides can be any suitable material, such as a transparent material (e.g., glass, silicon, quartz, etc.).

[0100] Piezoelectric mirror positioner

[0101] Similarly, a piezoelectric actuator on a mirror-mounted shaft can be used as a positioner to deflect the laser radiation to different locations on the sample. Again, as a mirror positioner based on a moving component with mass, there will inherently be inertia, and thus the component will have an inherent time overhead in the movement of the mirror. Therefore, the present positioner will be understood by those skilled in the art to have application in certain embodiments where the nanosecond response time of the laser scanning system is not essential. Similarly, because it is based on a mirror, the piezoelectric mirror positioner will reduce the quality of the laser radiation beam and increase the size of the ablation spot, and again, those skilled in the art will understand that it is most suitable for situations that tolerate such effects on the beam.

[0102] In a piezo mirror based on a tilted tip mirror arrangement, the direction of the laser radiation directed towards the sample in the X and Y axes is provided in the form of a single component.

[0103] Piezoelectric mirrors are commercially available from suppliers such as Physik Instrumente (Germany).

[0104] Thus, in some embodiments of aspects of the present invention, the laser scanner system includes a piezoelectric mirror, such as a piezoelectric mirror array or a tilted tip mirror.

[0105] In embodiments that include only piezoelectric mirror-based positioners (such as a piezoelectric mirror array or a tilted tip mirror), the rate at which ablation laser pulses can be directed to the sample can be between 200Hz-1MHz, 200Hz-100kHz, 200Hz-50kHz, 200Hz-10kHz, 1kHz-1MHz, 5kHz-1MHz, 10kHz-1MHz, 50kHz-1MHz, 100kHz-1MHz, 1kHz-100kHz, or 10kHz-100kHz.

[0106] -MEMS mirror positioner

[0107] A third type of positioner that relies on the physical movement of the surface that directs the laser radiation onto the sample is a MEMS (micro-electromechanical system) mirror. The micro-mirrors in this assembly can be actuated by electrostatic, electromechanical and piezoelectric effects. Many advantages of this type of assembly derive from their small size, such as light weight, ease of positioning in the device, and low power consumption. However, since the deflection of the laser radiation still ultimately depends on the movement of the component in the assembly, the component will experience inertia. Again, since the MEMS mirror positioner is based on a mirror, it will reduce the quality of the laser radiation beam and increase the size of the ablation spot, so again, those skilled in the art will understand that this type of scanner assembly is therefore suitable for situations where such influences on the laser radiation can be tolerated.

[0108] MEMS mirrors are commercially available from suppliers such as Mirrorcle Technologies (California, USA), Hamamatsu (Japan), and Precisely Microtechnology, Inc. (Canada).

[0109] Thus, in some embodiments of aspects of the present invention, the laser scanner system includes a MEMS mirror.

[0110] In the case of a positioner based on a MEMS mirror only, the rate at which ablation laser pulses can be directed to the sample can be between 200Hz-1MHz, 200Hz-100kHz, 200Hz-50kHz, 200Hz-10kHz, 1kHz-1MHz, 5kHz-1MHz, 10kHz-1MHz, 50kHz-1MHz, 100kHz-1MHz, 1kHz to 100kHz or 10kHz-100kHz.

[0111] -Polygon Scanner

[0112] Another type of positioner that relies on the physical movement of the surface that directs the laser radiation onto the sample is a polygon scanner. Here, a reflective polygon or multifaceted mirror rotates on a mechanical axis, generating an angularly deflected scanning beam each time a flat facet of the polygon passes the incident beam. A polygon scanner is a one-dimensional scanner that can guide 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 requiring movement of the sample on the sample stage). In contrast to the back-and-forth motion of scanners based on, for example, galvanometer mirrors, once the end of a raster scan line is reached, the beam is directed back to the position at the beginning of the scan line. Depending on the application, the polygon can be regular or irregular. The spot size depends on the size and flatness of the facets, and the scan line length / scan angle depends on the number of facets. Very high rotational speeds, and thus high scanning speeds, can be achieved. However, this type of positioner does have drawbacks: lower positioning / feedback accuracy due to manufacturing tolerances and axial wobble of the facets, as well as potential wavefront distortion introduced by the mirrors. Once again, those skilled in the art will appreciate that this scanner assembly is therefore suitable for applications subject to such influences on the laser radiation.

[0113] Polygon scanners are commercially available from, for example, Precision Laser Scanning (Arizona, USA), II-VI (Pennsylvania, USA), Nidec Copal Electronics (Japan), and the like.

[0114] In a positioner that only includes a polygon scanner, the rate at which ablation laser pulses can be directed toward the sample can be between 200Hz-10MHz, 200Hz-1MHz, 200Hz-100kHz, 200Hz-50kHz, 200Hz-10kHz, 1kHz-10MHz, 5kHz-10MHz, 10kHz-10MHz, 50kHz-10MHz, 100kHz-10MHz, 1kHz-1MHz, 10kHz-1MHz, or 100kHz-1MHz.

[0115] -Electro-Optical Deflector (EOD) locator

[0116] Unlike the previously mentioned laser scanner system component types, EODs are solid-state components—that is, they contain no moving parts. Therefore, they do not experience mechanical inertia when deflecting laser radiation, resulting in very fast response times, on the order of approximately 1 nanosecond. They are also not subject to wear and tear like mechanical components. EODs are formed from an optically transparent material (such as a crystal) whose refractive index varies with an applied electric field, which is controlled by applying a voltage across the medium. Refraction of laser radiation is caused by the introduction of a phase delay across the beam's cross-section. If the refractive index varies linearly with the electric field, the effect is known as the Pockels effect. If it varies quadratically with the field strength, it is known as the Kerr effect. The Kerr effect is typically much weaker than the Pockels effect. Two typical EOD configurations are those based on refraction at optical prism interfaces and those based on refraction due to a refractive index gradient perpendicular to the propagation direction of the laser radiation. To apply the electric field to the EOD, electrodes are bonded to opposite sides of the optically transparent material serving as the medium. Bonding a set of opposing electrodes produces a one-dimensional scanning EOD. Bonding a second set of electrodes orthogonal to the first set of electrodes creates a two-dimensional (X, Y) scanner.

[0117] For example, the deflection angle of an EOD is smaller than that of a galvanometer mirror, but the angle can be increased if several EODs are placed in sequence, which can be increased if required for a given device. 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.

[0118] The angular accuracy of the EOD is very high and depends 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 (because the (constantly changing) electric field in the crystal is set up according to the speed of light in the material, rather than the speed of sound in the material; see and Bechtold, 2014, Physics Procedia 56:29–39).

[0119] Therefore, in some aspects of the present invention, the laser scanner system includes an EOD. In some cases, the EOD is an electrode having two sets of electrodes orthogonally connected to a refractive medium.

[0120] In embodiments including an EOD-based locator, the rate at which ablation laser pulses can be directed toward 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, 10-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz.

[0121] -Acousto-Optic Deflector (AOD) locator

[0122] This type of positioner is also a solid-state component. Its deflection is based on the propagation of sound waves through an optically transparent material, which causes a periodic variation in the refractive index. This variation in refractive index occurs due to compression and rarefaction (i.e., density changes) of the material as the sound waves propagate through it. The periodic variation in refractive index acts like a grating, diffracting a laser beam passing through the material.

[0123] An AOD is created by bonding a transducer (typically a piezoelectric element) to an acousto-optic crystal (e.g., TeO2). The transducer, driven by an electrical amplifier, introduces sound waves into a refractive medium. The crystal is typically cut at an angle at the opposite end and used with an absorbent material to prevent reflection of the sound waves back into the crystal. When the wave propagates through the crystal in one direction, this creates a one-dimensional scanner. A two-dimensional scanner can be created by placing two AODs orthogonally in a row, or by bonding two transducers to orthogonal crystal faces.

[0124] As for the EOD, the AOD has a smaller deflection angle than a galvanometer mirror, but its angular accuracy is very high compared to such mirror-based scanners, and its frequency drive crystal is digitally controlled and can typically be resolved to 1Hz. and Bechtold (2014) note that AODs generally do not suffer from the drift and temperature dependence issues common to galvanometer mirror-based scanners compared to analog controllers.

[0125] Exemplary materials for use as AOD refractive media include tellurium dioxide, fused silica, 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.

[0126] To change the deflection angle, the frequency of the sound introduced into the crystal must be changed, and it takes a finite amount of 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 some degree of delay. However, compared to positioners in laser systems based on moving parts, the response time is relatively fast.

[0127] Another characteristic of the AOD that can be exploited in certain situations is that the acoustic power applied to the crystal determines how much of the laser radiation is diffracted relative to the zero-order (i.e., undiffracted) beam. The undiffracted beam is typically directed to a beam dump. Thus, the AOD can be used to efficiently control (or modulate) the intensity and power of the deflected beam at high speed.

[0128] The diffraction efficiency of an AOD is typically nonlinear, so a plot of diffraction efficiency versus power can be plotted for different input frequencies. The mapped efficiency curve for each frequency can then be recorded as a formula or lookup table for subsequent use in the disclosed apparatus and methods.

[0129] Thus, in some aspects of the present invention, a laser scanner system comprises an AOD.

[0130] Figure 4 is a schematic diagram of an optical device arrangement of another exemplary embodiment of aspects of the present invention. Figure 2 However, instead of using a rotating mirror, a solid-state positioner (e.g., AOD or EOD) 412 is used to cause deflection into the laser radiation beam. Figure 2 Mirror-based positioner 212. As described elsewhere, 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 orthogonally in series. Figure 4 The other components correspond to Figure 2 Components marked with corresponding reference numerals in FIG. 4 (ie, 401 is a laser source (eg, a pulsed laser source, optionally including a pulse picker), as shown in FIG. Figure 2 201 etc.).

[0131] In embodiments including an AOD-based locator, the rate at which ablation laser pulses can be directed toward the sample can be between 200Hz-100MHz, 200Hz-10MHz, 200Hz-1MHz, 200Hz-100kHz, 200Hz-50kHz, 200Hz-10kHz, 1kHz-100MHz, 5kHz-100MHz, 10kHz-100MHz, 50kHz-100MHz, 100kHz-100MHz, 1MHz-100MHz, 10-100MHz, 1kHz-10MHz, 10kHz-10MHz, or 100kHz-10MHz.

[0132] -Combination of locators

[0133] In the previous paragraphs, two types of laser scanning system positioners were discussed: mirror-based positioners with 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 deflection angle range but a much faster response time. Therefore, in some embodiments of various aspects of the present invention, a 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, for example, the mirror-based component provides a large range but can accommodate the inertia of the mirror-based component. See, for example, Matsumoto et al., 2013 (Journal of Laser Micro / Nanoengineering 8:315:320).

[0134] Thus, for example, a solid-state positioner (i.e., an AOD or EOD) may be used to correct for errors in a mirror-based scanner assembly. In this case, the position sensor associated with the feedback of the mirror position to the solid-state assembly, as well as the deflection angle introduced into the laser radiation beam by the solid-state assembly, may be appropriately varied to correct for positional errors of the mirror-based scanner assembly.

[0135] An example of a combined system includes a galvanometer mirror and an AOD (where the AOD can be deflected in one or two directions (by using two AODs in series, or by bonding two actuators to orthogonal faces of the crystal of a single AOD)). The system can include two galvanometer mirrors to produce a two-dimensional scanning system in combination with the AOD (where the AOD can achieve deflection in one or two directions (by using two AODs in series, or by bonding two actuators to orthogonal faces of a single AOD crystal)). In such a system, the rate at which ablation laser pulses can be directed toward 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, 10-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz. Alternative examples of combined systems include a galvanometer mirror and an EOD (wherein the EOD can be deflected in one or two directions (by bonding two orthogonally arranged electrodes to the crystal)). The system can include two galvanometer mirrors to produce a two-dimensional scanning system in combination with an EOD (wherein the EOD can be deflected in one or two directions (by bonding two orthogonally arranged electrodes to the crystal)). In such systems, the rate at which ablation laser pulses can be directed toward 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, 10-100 MHz, 1 kHz-10 MHz, 10 kHz-10 MHz, or 100 kHz-10 MHz.

[0136] -Other optional components of the laser scanning system

[0137] To control the positioner of the laser scanning system, the laser scanning system may include a scanner control module (such as a computer or programming chip) that coordinates the movement of the positioner in the Y-axis and / or X-axis, as well as with the movement of the sample stage. In some cases, such as back and forth rastering, the appropriate pattern will be pre-programmed into the chip. However, in other cases, the control module may apply inverse dynamics to determine the appropriate ablation pattern to follow. Inverse dynamics may be particularly useful, for example, in generating arbitrary ablation patterns, so as to map the optimal ablation process between multiple and / or irregularly shaped cells to be ablated. The scanner control module may also coordinate the emission of pulses of laser radiation, for example by coordinating the operation of a pulse picker.

[0138] Sometimes, a positioner causes the laser radiation beam it guides to diverge. 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 adapted to compensate 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 comprising 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 comprising compensating for any dispersion caused by the first positioner and a second dispersion compensator for compensating for any dispersion caused by the second positioner. WO 03 / 028940 describes how to use another suitable AOD to compensate for the dispersion caused by an AOD positioner.

[0139] Sometimes, due to the movement of the positioner to direct the laser radiation to different locations, the focal length of the radiation beam may change relative to the position of the sample. This can be compensated in a number of ways. For example, a movable focusing lens can be moved so that the diameter of the spot size on the sample remains constant or nearly constant, regardless of the specific position on the sample to which the laser radiation is directed. Alternatively, an adjustable focusing lens can be used (commercially available from Optotune). Spot size changes can also be compensated for by changing the height of the sample stage in the z-axis. Both of these techniques rely on moving components, which however introduces a time overhead into the operation of the system. If an AOD is used with a Gaussian beam, the size of the ablation spot can be controlled by applying power to the crystal in the AOD, thereby rapidly modulating the first- and zeroth-order beam intensities.

[0140] laser

[0141] Generally, the wavelength and power of the laser used to ablate the sample can be selected according to conventional practices in cell analysis. The laser must have sufficient energy density to allow ablation to the desired depth without substantially ablating the sample carrier. 2 Laser energy densities between 3 and 4 J / cm are usually suitable, for example 2 or about 3.5J / cm 2 Initially, the laser would ideally be capable of producing pulses with such energy densities at a frequency of 200 Hz or greater. In some cases, a single laser pulse from such a laser should be sufficient to ablate the cellular material for analysis, such that the laser pulse frequency matches the frequency at which the ablation plume is produced. Typically, to be a laser useful for imaging biological samples, the laser should produce pulses having 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 below. In some embodiments of the present invention, in order to utilize the uses of the laser scanning system discussed above, the ablation rate (i.e., the rate at which the laser ablates a spot on the sample surface) is 200 Hz or greater, such as 500 Hz or greater, 750 Hz or greater, 1 kHz or greater, 1.5 kHz or greater, 2 kHz or greater, 2.5 kHz or greater, 3 kHz or greater, 3.5 kHz or greater, 4 kHz or greater, 4.5 kHz or greater, 5 kHz or greater, 10 kHz or greater, 100 kHz or greater, 1 MHz or greater, 10 MHz or greater, or 100 MHz or greater. The repetition rate of many lasers exceeds the laser ablation frequency, so appropriate components, such as pulse pickers, can be used to appropriately control the ablation rate. Thus, in some embodiments, the laser repetition rate is at least 1 kHz, such as at least 10 kHz, at least 100 kHz, at least 1 MHz, at least 10 MHz, about 80 MHz, or at least 100 MHz, optionally wherein the sampling system further comprises a pulse picker, such as wherein the pulse picker is controlled by a control module that also controls the movement of the sample stage and / or positioner of the laser scanning system. In other cases, a single spot can be ablated using multiple closely spaced pulse bursts (e.g., a train of 3 closely spaced pulses). For example, by using 100 bursts of 3 closely spaced pulses in each spot, a 10×10 μm area can be ablated; this is useful for lasers with limited ablation depth (e.g., femtosecond lasers) and can produce a continuous plume of ablated cellular material without sacrificing resolution. Thus, in some embodiments, the laser scanning system is adapted to ablate the sample using a method where each spot on the sample is ablated using three temporally close pulses (e.g., where the interval between pulses is less than 1 μs, such as less than 1 ns, or less than 1 ps).

[0142] As described herein, the laser can be an fs laser. For example, an fs laser in the near-infrared range can be operated at the second harmonic to provide laser radiation in the green range, or at the third harmonic to provide laser radiation in the UV range. Lower wavelengths (e.g., green or ultraviolet) can provide higher resolution (e.g., smaller spot size). The sample support needs to be transparent to the laser radiation as the laser radiation propagates through it and strikes the sample. Glass and silicon dioxide are transparent to green wavelengths, while silicon dioxide, but not glass, is transparent to UV. In order to achieve high resolution while allowing the use of glass slides, an IR fs laser can be operated at the second harmonic (e.g., with a conversion efficiency of approximately 50%) to provide green laser radiation. It is worth noting that commercially available objective lenses are generally best corrected in the green range. The resolution obtained by a green or UV fs laser can be a spot size less than or equal to 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, or 100 nm.

[0143] For example, the ablation frequency of the laser system is in the range of 200Hz-100MHz, 200Hz-10MHz, 200Hz-1MHz, 200Hz-100kHz, 500-50kHz, or 1kHz-10kHz. As mentioned above, the ablation frequency of the laser should match the scanning rate of the laser scanning system.

[0144] At these frequencies, if it is necessary to resolve each ablation plume individually (which, as discussed below, may not necessarily be necessary when firing a train of ablation pulses at the sample), the instrument must be able to analyze the ablated material rapidly enough so that there is no significant signal overlap between successive ablations. Preferably, the overlap between the signals originating from successive plumes is <10%, more preferably <5%, and ideally <2%. The time required to analyze the plume will depend on the flushing time of the sample chamber (see the Sample Chamber section below), the transit time of the plume aerosol into and out of 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 image of the sample, as discussed in more detail below.

[0145] In some embodiments, the laser source comprises a laser with a nanosecond or picosecond pulse duration or an ultrafast laser (pulse duration of 1 ps (10 -12 Ultrafast pulse durations offer numerous advantages because they limit the diffusion of heat away from the ablation zone, providing more precise and reliable ablation craters and minimizing the amount of debris generated by each ablation event.

[0146] In some cases, femtosecond lasers are used as the laser source. Femtosecond lasers are lasers that emit optical pulses with durations less than 1 ps. Such short pulses are typically generated using passive mode-locking techniques. Various types of lasers can be used to generate femtosecond lasers. Typical durations between 30 fs and 30 ps can be achieved using passively mode-locked solid-state bulk lasers. Similarly, various diode-pumped lasers, such as those based on neodymium-doped or ytterbium-doped gain media, operate in this context. Titanium-sapphire lasers with advanced dispersion compensation are even suitable for pulse durations below 10 fs, and in extreme cases, down to approximately 5 fs. In most cases, the pulse repetition rate is between 10 MHz and 500 MHz, although lower repetition rate versions exist, with repetition rates of several megahertz for higher pulse energies (available from, for example, Lumentum (California, USA), Radiantis (Spain), and Coherent (California, USA). Lasers of this type can be coupled with amplifier systems to increase the pulse energy.

[0147] There are also various types of ultrafast fiber lasers, which are also passively mode-locked in most cases and typically provide pulse durations between 50 and 500 fs and repetition rates between 10 and 100 MHz. Such lasers are commercially available from companies such as NKT Optoelectronics (Denmark; formerly Fianium), Amplitude Systems (France), and Laser-Femto (California, USA). The pulse energy of such lasers can also be increased by amplifiers, usually in the form of integrated fiber amplifiers.

[0148] Some mode-locked diode lasers can generate pulses of femtosecond duration. Directly at the laser output, the pulse duration is typically on the order of hundreds of femtoseconds (available, for example, from Coherent, California, USA).

[0149] In some cases, picosecond lasers are used. Many of the types of lasers discussed in the previous paragraphs can also be adapted to produce pulses with durations in the picosecond range. The most common light sources are actively or passively mode-locked solid-state bulk lasers, such as passively mode-locked neodymium-doped YAG, glass, or vanadate lasers. Similarly, picosecond mode-locked lasers and laser diodes are commercially available (e.g., NKT Optoelectronics (Denmark), EKSPLA (Lithuania)).

[0150] Nanosecond pulse duration lasers (gain switched and Q-switched) may also find utility in specialized equipment setups (Coherent (CA, USA), Thorlabs (NJ, USA)).

[0151] Alternatively, an externally modulated continuous wave laser can be used to generate pulses of nanosecond or shorter duration.

[0152] Typically, the spot size (i.e., at the sampling location) of the laser beam used for ablation in the laser systems discussed herein is 100 μm or less, such as 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less, such as about 3 μm or less, about 2 μm or less, about 1 μm or less, about 500 nm or less, or about 250 nm or less. This distance is called the spot size and 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 is the length of the diagonals between opposite corners, for a quadrilateral, it is the length of the longest diagonal, and so on. (As mentioned above, the diameter of a circular beam with a Gaussian distribution is defined as the diameter of the beam at which the energy density has been reduced to 1 / e of the peak energy density. 2 times the distance between points). As an alternative to a Gaussian beam, beam shaping and beam masks can be used to provide the desired ablation spot. For example, in some applications, a square ablation spot with a high-top energy distribution may be useful (i.e., a beam with a near-uniform energy density, as opposed to a Gaussian energy distribution). This arrangement reduces the dependence of the ablation spot size on the ratio between the energy density at the peak of the Gaussian energy distribution and the threshold energy density. Ablation close to the threshold energy density can provide more reliable ablation crater generation and control the generation of debris. Therefore, the laser system can include a beam mask and / or beam shaping components, such as diffraction optics, arranged in a Gaussian beam to recolor the beam and produce a laser focus of uniform or near-uniform energy density, such as an energy density that varies less than ±25% across the entire beam, such as less than ±20%, ±15%, ±10%, or less than ±5%. Sometimes, the laser beam has a square cross-sectional shape. Sometimes, the beam has a high-top energy distribution.

[0153] When used to analyze biological samples, the spot size of the laser beam used will depend on the size and spacing of the cells to analyze individual cells. For example, when cells are closely packed together (such as in a tissue section), the spot size of the one or more laser sources in the laser system can be no larger than the cells. This size will depend on the specific cells in the sample, but typically the laser spot diameter should be less than 4 μm, for example, 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, or between 300 nm and 1 μm. To analyze a given cell with subcellular resolution, the system uses a laser spot size no larger than the cells, more specifically, a laser spot size that can ablate material with subcellular resolution. Sometimes, single-cell analysis can be performed using a spot size larger than the cell size, for example, by spreading the cells on a slide with spaces between them. In this case, a larger spot size can be used, and single-cell characterization can be achieved because the additional ablation area surrounding the cell of interest does not contain 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 desired, the ablation spot 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 (i.e., the laser beam diameter at the beam focus) of 1 μm, but variations in the laser focus can be greater than +20% due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and variations 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 (inverted Cassegrain). Refractive objectives can also be used, as can combinations of reflective and 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 spot size of the laser, which can be used alone or in combination with the above-mentioned objectives, is to pass the beam through an aperture before focusing. Different beam diameters can be achieved by passing the beam through apertures of different diameters in a diameter array. In some cases, such as when the aperture is an iris aperture, there is a single aperture of variable size. Sometimes, the iris aperture is an iris aperture. 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.

[0154] For the sake of completeness, the standard lasers for LA with subcellular resolution known in the art (e.g. [5]) are excimer lasers or compound excimer lasers. Suitable results can be obtained using argon fluoride lasers (λ = 193 nm). These lasers have a pulse duration of 10-15 ns, which allows for sufficient ablation.

[0155] Overall, the frequency and intensity of the laser pulses 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, rapid, high-resolution imaging is now possible.

[0156] If the laser system emits laser radiation at more than two wavelengths, this may be achieved by using more than two laser sources, wherein each laser source is adapted to emit laser radiation at a wavelength that is different from the wavelength of laser radiation emitted by the other laser sources in the laser system.

[0157] Thus, a laser system can include a first laser source that emits laser radiation having a wavelength of 213 nm and a second laser source that emits laser radiation having a wavelength of 266 nm (such that the first laser source primarily ablates protein material and the second laser source primarily ablates DNA material). If ablation at a third wavelength of laser radiation is desired, a third laser source is used in the laser system, and so on.

[0158] Sometimes, a laser system for emitting laser radiation at multiple wavelengths comprises a single laser source adapted to emit laser radiation at multiple wavelengths (i.e., one laser emits laser radiation at multiple wavelengths; the laser system may include other laser sources). Some laser sources use wavelength conversion methods, such as harmonic or sum frequency generation, supercontinuum generation, optical parametric amplifier or oscillator (OPA / OPO) technology, or a combination of several technologies, to emit laser radiation at the desired wavelength, as is standard in the art. For example, an Nd YAG laser produces laser radiation at a wavelength of 1064 nm, which is referred to as the fundamental frequency. This wavelength can be converted to a shorter wavelength (if desired) by harmonic generation methods. The fourth harmonic of this laser radiation will be at 266 nm (1064 nm ÷ 4), while the fifth harmonic will be at 213 nm. Thus, the fourth harmonic can target a high absorption band of DNA material, while the fifth harmonic can target a high absorption band of proteins. In many laser arrangements, the generation of the fifth harmonic is based on the generation of the fourth harmonic. Thus, although lower harmonics (having longer wavelengths) are typically filtered out in lasers, the fourth harmonic will already be present in a laser that produces a fifth harmonic output. Therefore, removing the appropriate filters enables emission of laser radiation at multiple wavelengths. Examples of such lasers are commercially available from Coherent, RP Optics, Lee Lasers, and the like.

[0159] Another useful harmonic frequency pair is the fourth and third harmonics of a laser with a fundamental wavelength of about 800 nm. Here, the fourth and third harmonics have wavelengths of 200 nm and 266 nm, respectively. Examples of such lasers are commercially available (Coherent Corporation, Spectra Physics).

[0160] In some cases, the first wavelength of laser radiation and the second wavelength of laser radiation are generated by the same laser source, and the wavelengths are not generated through harmonic generation, but are generated by a laser having a broad emission spectrum. The emission spectrum of the laser can be at least 10 nm, at least 30 nm, at least 50 nm, or at least 100 nm. White light lasers or supercontinuum lasers can generate light at multiple wavelengths.

[0161] Laser ablation focus

[0162] In order to achieve maximum efficiency in ablating the material in the sample by the laser, the sample should be located in an appropriate position relative to the laser focus, for example at the focal point, since the focal point is where the laser beam has the smallest diameter and the most concentrated energy. This can be achieved in a number of ways. The first way is that the sample can be moved on the axis of the laser directed at it (i.e., up and down the laser path / toward and away from the laser source) to a desired point where the light intensity is sufficient to achieve the desired ablation. Alternatively or in addition, a lens can be used to move the focus of the laser and thus have the effective ability to ablate material at the sample location, for example by zooming out. One or more lenses are located between the laser and the sample stage. A third way is to change the position of the laser, which can be used alone or in combination with one or both of the above two ways.

[0163] To help the user of the system place the sample in the most appropriate position for ablating material from the sample, a camera can be directed towards the stage that holds the sample (discussed in detail below). Accordingly, the present disclosure provides a laser ablation sampling system that includes a camera that is directed towards the sample stage. The image detected by the camera can be focused to the same point where the laser is focused. This can be achieved by using the same objective lens for both the laser ablation and the optical imaging. By aligning the two focal points, the user can ensure that the laser ablation will be most effective when the optical image is in sharp focus. Precise movement of the stage to focus the sample can be achieved by using piezoelectric activators, such as those provided by PhysikInstrumente, Cedrat-technologies, Thorlabs, and other suppliers.

[0164] In another mode of operation, the laser ablation is directed to the sample via the sample carrier. In this case, the sample support should be chosen so that it is (at least partially) transparent to the laser radiation frequency used to ablate the sample. Figure 5Through-sample ablation is shown in FIG. Under certain circumstances, through-sample ablation can be advantageous because this mode of ablation can provide additional kinetic energy to the plume of material ablated from the sample, moving the ablated material further away from the sample surface, thereby facilitating the flow of the ablated material away from the sample for analysis at the detector. Similarly, desorption-based methods can be employed to remove chunks of sample material by mediating laser radiation through a carrier. The additional kinetic energy provided to the chunk of desorbed material can help eject the chunk from the sample carrier, thereby facilitating entrainment of the chunk in the carrier gas flowing through the sample chamber.

[0165] In order to achieve 3D imaging of a sample, the sample or a limited area thereof can be ablated to a first depth that does not completely pass through the sample. Afterwards, the same area can be ablated again to a second depth, and so on to a third, fourth, and other depths. In this way, a 3D image of the sample can be established. In some cases, it may be preferable to ablate all areas for ablation to the first depth before continuing to ablate at the second depth. Alternatively, repeated ablations can be performed at the same point to ablate different depths before proceeding to the next location in the area for ablation. In both cases, the imaging software can deconvolute the signal obtained at the MS to the position and depth of the sample. In some aspects, a high-speed laser (e.g., a femtosecond laser) can provide short and intense laser pulses that can more cleanly ablate each spot, thereby allowing resampling at the spot without damaging the sample (e.g., very little heat is spread around the original sample spot). When a separate pixel is obtained for each laser spot, constructing a 3D image may take a lot of time. Thus, laser scanning of a region of interest (e.g., such as a cell) as described can allow rapid resampling of the ROI at a second depth.

[0166] Laser system optics for various operating modes

[0167] As a matter of conventional arrangement, the optical components can be used to direct laser radiation, optionally having different wavelengths, to different relative positions. The optical components can also be arranged to direct laser radiation, optionally having different wavelengths, onto the sample from different directions. For example, one or more wavelengths can be directed onto the sample from above, and one or more wavelengths of laser radiation (optionally, different wavelengths) can be directed from below (i.e., through a substrate carrying the sample, such as a microscope slide, also known as a sample carrier). This allows the same device to adopt multiple operating modes. Therefore, the laser system can include an arrangement of optical components that is arranged to direct laser radiation, optionally having different wavelengths, onto the sample from different directions. Thus, the optical components can be arranged so that the arrangement directs laser radiation (optionally, having different wavelengths) onto the sample from opposite directions. In this context, "opposite" directions are not limited to laser radiation directed perpendicularly onto the sample from above and below (which are 180° opposite), but include arrangements in which laser radiation is directed onto the sample at angles other than perpendicular to the sample. The laser radiation directed onto the sample from different directions does not need to be parallel. Sometimes, when the sample is on a sample carrier, the reflector arrangement may be arranged to direct laser radiation of a first wavelength directly onto the sample and to direct laser radiation of a second wavelength to the sample through the sample carrier.

[0168] Directing laser radiation through a sample carrier onto the sample can be used to ablate the sample. However, in some systems, directing laser radiation through the carrier can be used in a "LIFTing" mode of operation, as discussed in more detail below with respect to desorption-based sampling systems (although those skilled in the art will appreciate that ablation and LIFTing can be performed by the same apparatus, so that a laser ablation sampling system referred to herein can also be used as a desorption-based sampling system). The NA (numerical aperture) of the lens used to focus the laser radiation onto the sample from a first direction may be different from the NA (numerical aperture) of the lens used to focus the laser radiation (optionally at a different wavelength) onto the sample from a second direction. Lifting operations (e.g., where the laser radiation is directed through a sample carrier) typically employ a spot size of larger diameter than when ablation is performed.

[0169] High NA objective and opposite side ablation

[0170] In certain aspects, the sample chamber of the present methods and systems can include a high NA objective (e.g., lens). For example, Figure 12 The sample chamber 1206 shows a high NA objective lens 1205. The laser radiation 1216 is focused by the high NA objective lens onto the sample 1215 on the sample support 1207, and the sample material is then transported to the mass analyzer. The high NA objective lens can be an air lens, an oil immersion lens, or a solid immersion lens. Thus, the medium 1207 can be air (or low pressure vacuum), oil, or a solid transparent material. Figure 12 As shown, the laser radiation 1216 and the high NA objective lens can be located on the opposite side of the sample support 1207 from the sample 1215.

[0171] When an immersion lens is used (e.g., when the immersion lens is located on the side of the slide opposite the sample), the sample can be an ultrathin sample, such as a tissue section having a thickness of 300 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 75 nm or less, 50 nm or less, or 30 nm or less. Such tissue sections (particularly tissue sections having a thickness of 100 nm or less) can be prepared in a manner similar to or identical to that used for electron microscopy. For example, the tissue can be embedded in a resin (e.g., epoxy, acrylic, or polyester) prior to ultrathin sectioning.

[0172] The NA of a high NA objective lens can be greater than 0.5, greater than 0.7, greater than 0.9, greater than 1.0, greater than 1.2, or greater than 1.4. It is worth noting that NAs greater than 1.0 can be achieved using a medium such as oil or a solid transparent material that has a higher refractive index than air or a vacuum (e.g., greater than 1.0). High NA optics can provide spot sizes of less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, or less than 100 nm.

[0173] In some aspects, the wavelength of the laser radiation focused by the high NA objective lens is below 1 μm, such as in the green or UV range. As described herein, the laser can be an fs laser. For example, an fs laser in the near-infrared range can be operated at the second harmonic to provide laser radiation in the green range, or at the third harmonic to provide laser radiation in the UV range. Lower wavelengths (such as green or UV) can provide higher resolution (e.g., smaller spot size). The sample support needs to be transparent to the laser radiation as the laser radiation propagates through it and strikes the sample. Glass and silica are transparent to green wavelengths, and silica slides, but not glass, are transparent to UV. In order to maximize resolution while allowing the use of glass slides, an IR fs laser can be operated at the second harmonic (e.g., with a conversion efficiency of approximately 50%) to provide green laser radiation. It is worth noting that commercially available objectives typically have optimal correction in the green range.

[0174] Sample chamber of laser ablation sampling system

[0175] When a sample undergoes laser ablation, it is placed in a sample chamber. The sample chamber contains a stage that holds the sample (typically on a sample carrier). During ablation, material in the sample forms a plume, and the gas flow through the sample chamber from the gas inlet to the gas outlet carries away the plume of atomized material, including any labeled atoms located at the ablation site. The gas carries the material to the ionization system, which ionizes the material for detection by the detector. The atoms in the sample (including the labeled atoms) can be distinguished by the detector, and their detection reveals the presence of multiple targets in the plume, thereby determining which targets are present at the ablated site on the sample. The sample chamber thus serves a dual purpose: it holds the solid sample being analyzed and serves as the starting point for transferring the atomized material to the ionization and detection systems. This means that the gas flow through the chamber affects how the ablated plume of material will spread as it travels through the system. A measure of how well the ablated plume spreads is the sample chamber flushing time. This value is a measure of the time it takes for the gas flowing through the sample chamber to remove the material ablated from the sample from the sample chamber.

[0176] The spatial resolution of the signal generated from laser ablation in this manner (i.e., when ablation is used for imaging rather than specifically for removal, as described below) depends on the following factors: (i) the spot size of the laser, since the signal is integrated over the total area being ablated; and the relationship between the speed at which the plume is generated and the movement of the sample relative to the laser; and (ii) the speed at which the plume can be analyzed relative to the speed at which the plume is generated, so as to avoid overlap of signals from consecutive plumes as described above. Therefore, if plumes need to be analyzed individually, being able to analyze them in the shortest possible time minimizes the likelihood of plume overlap (which in turn allows plumes to be generated more frequently).

[0177] Therefore, a sample chamber with a short flushing time (e.g., less than 100 ms) is advantageous for use with the apparatus and methods disclosed herein. A sample chamber with a long flushing time will limit the speed at which images are produced, or result in overlap between signals originating from consecutive sample points (e.g., reference vi, where the signal duration exceeds 10 seconds). Therefore, the aerosol flushing time is a key limiting factor in obtaining high resolution without increasing the total scan time. Sample chambers with flushing times ≤ 100 ms are known in the art. For example, reference vii discloses a sample chamber having a flushing time of less than 100 ms. In reference viii (see also reference ix), a sample chamber is disclosed having a flushing time of less than 30 ms, thereby allowing high ablation frequencies (e.g., greater than 20 Hz), so that rapid analysis can be performed. Another such sample chamber is disclosed in reference x. The sample chamber of reference x includes a sample capture chamber configured to be operably arranged near a target, the sample capture chamber comprising: a capture cavity having an opening formed on a surface of the capture cavity, wherein the capture cavity is configured to receive a target material ejected or generated from a laser ablation site through the opening, and a guide wall exposed within the capture cavity, the guide wall being configured to guide a carrier gas within the capture cavity from an inlet to an outlet, so that at least a portion of the target material received within the capture cavity can be transferred to the outlet as a sample. The volume of the capture cavity in the sample chamber of reference x is less than 1 cm 3 and can be less than 0.005cm 3 Sometimes the sample chamber flushing time is 25 ms or less, such as 20 ms or less, 10 ms or less, 5 ms or less, 2 ms or less, 1 ms or less, 500 μs or less, 200 μs or less, 100 μs or less, 50 μs or less, or 25 μs or less. For example, the sample chamber flushing time can be 10 μs or more. Typically, the sample chamber flushing time is 5 ms or less.

[0178] For the sake of completeness, plumes in the sample may sometimes be generated more frequently than the sample chamber flush time, and the resulting image may exhibit smearing accordingly (e.g., if it is deemed that the highest possible resolution is not required for a particular analysis). While this may not be ideal for high-resolution imaging, as discussed herein, bursts of pulses are directed toward the sample (e.g., the pulses are all directed toward features / regions of interest, such as cells), and it is not important that the material in the plume, which is detected as a series of events, overlaps with the signal from a particular plume. In fact, here, the plumes from each individual ablation event within the burst actually form a single plume, which is then subsequently detected.

[0179] 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 (in some embodiments of the invention, the sample stage and laser beam can move simultaneously), for example, the sample stage moves at a constant speed, and as the sample moves on the sample stage, the laser scanning system directs the laser light on a matching scan across the sample; for example, the sample stage moves in the X-axis and the laser scanning system sweeps in the Y-axis, with the main vector of movement of the laser scanning system being orthogonal to the direction of travel of the stage (taking into account any movement in the laser scanner to account for the movement of the stage). When an operating mode is used that requires the laser radiation to pass through the sample carrier to reach the sample, such as in the LIFTing method discussed herein, the stage holding the sample carrier should also be transparent to the laser radiation used.

[0180] Thus, the sample can be positioned on a side of a sample carrier (e.g., a glass slide) that faces the laser radiation when the laser radiation is directed onto the sample, such that the ablation plume is released and captured on the same side as the laser radiation is directed onto the sample. Alternatively, the sample can be positioned on a side opposite to the laser radiation when the laser radiation is directed onto the sample (i.e., the laser radiation passes through the sample carrier before reaching the sample), and the ablation plume is released and captured on the side opposite to the laser radiation.

[0181] Control of the movement of the sample stage in an apparatus according to aspects of the present invention may be coordinated by the same control module that coordinates the movement of the laser scanner system and optionally controls the emission of laser radiation pulses (e.g., a trigger controller for a pulse picker).

[0182] A characteristic of the sample chamber is its wide range of motion. Within the sample chamber, specific portions of discrete regions of the sample are ablated. Within this range, the sample can be moved relative to the laser (the laser beam is directed onto the sample along the z-axis) in the x and y (i.e., horizontal) axes, with the x and y axes being perpendicular to each other. By moving the stage within the sample chamber and fixing the position of the laser within the instrument's laser ablation sampling system, more reliable and accurate relative positioning can be achieved. The greater the range of motion, the greater the distance between the discrete ablation regions. The sample is moved relative to the laser by moving the stage on which the sample rests. Thus, the sample stage has a range of motion within the sample chamber in the x and y axes of at least 10 mm, such as 20 mm, 30 mm, 40 mm, 50 mm, or even 75 mm. This range of motion sometimes allows analysis of the entire surface of a standard 25 mm x 75 mm microscope slide within the chamber. Of course, in addition to enabling a wide range of motion, the motion must be precise to achieve subcellular ablation. Therefore, the stage can be configured to move the sample on the x and y axes in increments of less than 10 μm, such as 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, such as 1 μm ± 0.1 μm. Commercially available microscope stages can be used, for example, those available from Thorlabs, Prior Scientific, and Applied Scientific Instrumentation. Alternatively, a motorized stage can be constructed from components based on a positioner that provides the required range of movement and suitable precision movement, such as the SLC-24 positioner from Smaract. The moving speed of the sample stage also affects the analysis speed. Therefore, the sample stage has an operating speed greater than 1 mm / s, such as 10 mm / s, 50 mm / s, or 100 mm / s.

[0183] Naturally, when the sample stage in the sample chamber has a wide range of motion, the size of the sample must be appropriately adjusted to accommodate the movement of the stage. Therefore, the size of the sample chamber depends on the size of the sample involved, which in turn determines the size of the movable sample stage. Exemplary dimensions of the sample chamber include an internal 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. A skilled person will be able to select appropriate dimensions based on the teachings herein. The internal dimensions of a sample chamber for analyzing biological samples using a laser ablation sampler must be larger than the range of motion of the sample stage, for example, by at least 5 mm, such as at least 10 mm. This is because if the walls of the chamber are too close to the edge of the stage, the flow of carrier gas through the chamber can cause a plume of ablated material to be displaced from the sample and into the ionization system, which can cause turbulence. The turbulence disturbs the ablated plume, so that the plume of material begins to spread out after being ablated and carried away to the device's ionization system, rather than remaining as a compact cloud of ablated material. The broad peak of 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 poor spatial resolution unless the ablation rate is slowed to the point where it is no longer experimentally relevant.

[0184] As described above, the sample chamber includes a gas inlet and a gas outlet for bringing the material to the ionization system. However, as determined by one skilled in the art, it may include other ports that serve as inlets or outlets to direct the flow of gas within the chamber and / or provide a gas mixture to the chamber suitable for performing a particular ablation procedure.

[0185] camera

[0186] In addition to identifying the most effective positioning of the sample for laser ablation, including a camera (e.g., such as a charge-coupled device (CCD)-based image sensor camera, an active pixel sensor-based camera), or any other light-detecting device in the laser ablation sampling system allows for a variety of further analysis and techniques. A CCD is a device used to detect light and convert it into digital information that can be used to produce an image. In a CCD image sensor, there is an array of capacitors that detect light, with each capacitor representing a pixel on the determined image. These capacitors can convert the incident photons into electrical charges. These charges are then read using the CCD, and the recorded charges can be converted into an image. An active pixel sensor (APS) is an image sensor consisting of an integrated circuit containing an array of pixel sensors, each of which includes a photodetector and an active amplifier, such as a CMOS sensor.

[0187] A camera can be incorporated into any of the laser ablation sampling systems 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 of a particular morphology), or fluorescent probes specific for antigens, intracellular receptors, or structures. In certain embodiments, the fluorescent probe is a histochemical stain or antibody that also contains a detectable metal tag. Once such cells are identified, laser pulses can be directed to these specific cells to ablate material for analysis, for example in an automated manner (wherein the system can both identify and ablate features / areas of interest (such as cells)) or a semi-automated process (wherein the user of the system (e.g., a clinical pathologist) identifies the features / areas of interest and the system then ablates in an automated manner). This can significantly increase the speed with which analysis is performed because the entire sample does not need to be ablated to analyze a specific cell, but rather the cell of interest can be specifically ablated. This creates efficiencies in the method of analyzing biological samples in terms of the time spent performing the ablation, particularly in terms of the time spent interpreting the data from the ablation, and in terms of constructing images based on the ablation. Constructing an image from the data is one of the more time-consuming parts of the imaging process, so by minimizing the data collected from the relevant parts of the sample, the overall analysis speed can be increased.

[0188] The camera can record images from a confocal microscope. A confocal microscope is a form of optical microscope with many advantages, including the ability to reduce interference from background information (light) away from the focal plane. This can be achieved by eliminating defocused light or glare. A confocal microscope can be used to assess the cell morphology of an unstained sample, or whether the cell is a discrete cell or a part of a cell mass. Typically, the sample is specifically labeled with a fluorescent marker (such as an antibody by a label or a nucleic acid by a label). These fluorescent markers can be used to stain specific cell populations (e.g., expressing certain genes and / or proteins) or specific morphological features on the cell (such as the nucleus or mitochondria), and when irradiated with light of an appropriate wavelength, these sample areas can be clearly identified. Therefore, some systems described herein can include a laser for exciting a fluorophore in the label, which is used to label the sample. Alternatively, an LED light source can be used to excite a fluorophore. Non-confocal (e.g., wide-field) fluorescence microscopes can also be used to identify certain areas of a biological sample, but have a lower resolution than a confocal microscope.

[0189] As an example technique combining fluorescence and laser ablation, the nuclei of cells in a biological sample can be labeled with antibodies or nucleic acids conjugated to fluorescent moieties. Thus, by stimulating the fluorescent marker and then using a camera to observe and record the location of the fluorescence, the ablation laser can be directed specifically to the nucleus or to an area that does not contain nuclear material. Separating a sample into nuclear and cytoplasmic regions will find particular application in the field of cell chemistry. By using an image sensor (such as a CCD detector or an active pixel sensor, such as a CMOS sensor), and by using a control module (such as a computer or programmed chip) to correlate the location of the fluorescence with the x, y coordinates of the sample, and then directing the ablation laser to that location, the process of identifying features / regions of interest and then ablating them can be fully automated. As part of this process, the first image captured by the image sensor may have a low objective lens magnification (low numerical aperture), allowing measurements to be taken over a large area of the sample. Subsequently, an objective lens with a higher magnification can be used to focus on specific features of interest that have been determined to be fluorescing through higher magnification optical imaging. These features recorded as fluorescing can then be ablated with the laser. Using a lower numerical aperture lens first has the further advantage that the depth of field is increased, thus meaning that features buried in the specimen can be detected more sensitively than if a higher numerical aperture lens were used for screening from the outset.

[0190] In methods and systems using fluorescence imaging, the emission path of the fluorescence from the sample to the camera may include one or more lenses and / or one or more filters. By including filters adapted to pass a selected spectral bandwidth from one or more fluorescent markers, the system is adapted to handle chromatic aberration associated with emission from the fluorescent markers. 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 reduce the amount of emission light of undesirable wavelengths that reaches the camera is to specifically exploit 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 described in WO2005 / 121864.

[0191] In this coupling of optical techniques and laser ablation sampling, higher resolution optical images are advantageous because the accuracy of the optical image then determines the accuracy with which the ablation laser can be guided to ablate the sample.

[0192] Thus, in some embodiments disclosed herein, the apparatus of the present invention includes a camera. The camera can be used online to identify features / regions of a sample (e.g., specific cells) that can then be ablated (or desorbed by LIFTing - see below), such as by firing a burst of pulses at the feature / region of interest to ablate or desorb a block of sample material from the feature / region of interest. With the burst of pulses directed toward the sample, the material in the resulting plume can be detected as a continuous event (the plumes from each individual ablation actually form a single plume that is then subsequently detected). While each cloud of sample material formed by the aggregated plume at 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 ablating different features / regions of interest to allow sample material from the nth feature / region to be detected before commencing ablation of the (n+1)th feature / region.

[0193] In another mode of operation that combines fluorescence analysis and laser ablation sampling, instead of analyzing the fluorescence across the entire slide before targeting the laser ablation to those locations, the laser pulse can be fired at a spot on the sample (at low energy 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 is analyzed by the detector as described below. This has the advantage that the rastered analysis mode can be maintained, but speed can be increased because the fluorescence can be pulsed and tested and the results obtained from the fluorescence immediately (rather than the time spent analyzing and interpreting the ion data from the detector to determine whether the area is of interest), again allowing only the important sites to be targeted for analysis. Therefore, when this strategy is applied 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) irradiating 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, guiding the laser to ablate the location to form a plume; (v) subjecting the plume to inductively coupled plasma mass spectrometry, and (vi) repeating steps (ii)-(v) for one or more other known locations on the sample, so that detecting the labeled atoms in the plume can construct an image of the sample in the ablated area.

[0194] In some cases, the sample or sample carrier can be modified so as to include optically detectable (e.g., by optical or fluorescence microscopy) portions at specific locations. The fluorescence locations can then be used to locate the sample in the device. The use of such marker locations is useful, for example, so that the sample can be visually inspected "offline" - that is, inspected in a device other than the device of the present invention. The optical image of the highlighted features / regions of interest and the sample can be marked with features / regions of interest corresponding to specific cells, for example by a physician, before such optical image and the sample are transferred to the device according to aspects of the present invention. Here, by reference to the marker locations in the annotated optical image, the device of aspects of the present invention can identify the corresponding fluorescence locations using a camera and calculate an ablation and / or desorption (LIFTing) plan for the location of the laser pulse accordingly. Therefore, in some embodiments, aspects of the present invention include a directional controller module capable of performing the above steps.

[0195] In some cases, the selection of features / regions of interest may be performed using the apparatus of aspects of the present invention based on an image of the sample taken by a camera of the apparatus of aspects of the present invention.

[0196] Nonlinear microscopy

[0197] Another imaging technique is two-photon excitation microscopy (also known as nonlinear or multiphoton microscopy). This technique typically uses near-infrared light to excite fluorophores. Each excitation event absorbs two infrared photons. IR minimizes scattering in tissue. In addition, due to multiphoton absorption, background signals are strongly suppressed. The excitation spectra of the most commonly used fluorophores are in the 400-500 nm range, and the lasers used to excite two-photon fluorescence are in the near-infrared range. If a fluorophore absorbs two infrared photons simultaneously, it absorbs enough energy to excite into an excited state. The fluorophore then emits a single photon, the wavelength of which depends on the type of fluorophore used, which can then be detected.

[0198] When using a laser to excite fluorophores for fluorescence microscopy, sometimes the laser is the same laser used to ablate material from a biological specimen, but the power used is insufficient to cause ablation of material from the specimen. Sometimes the fluorophore is excited by a wavelength of light that is then used by the laser to ablate the specimen. In other cases, a different wavelength can be used, for example, by generating different harmonics of the laser in addition to the harmonic used to ablate the specimen, or by utilizing different harmonics generated in a harmonic generation system as described above. For example, if the fourth and / or fifth harmonics of an Nd:YAG laser are used, the fundamental or the second through third harmonics can be used for fluorescence microscopy.

[0199] Imaging mass cytometry systems integrated with nonlinear microscopy can provide one or more of two-photon fluorescence, second harmonic generation (SHG), three-photon fluorescence (3PF), third harmonic generation (THG), and / or coherent anti-Stokes Raman scattering (CARS). In certain aspects, samples can be prepared for imaging using one or more forms of nonlinear microscopy, such as with contrast agents or with fluorophore-labeled SBPs. Samples can be further prepared with mass-labeled SBPs.

[0200] In second harmonic generation (SHG), the signal is most intense in collagen-containing tissues and has been shown to provide rich information about the type of collagen in the laser focus and its 3D orientation. This information cannot be obtained by other microscopy techniques. In third harmonic generation, the signal is generated uniquely in the sample in the presence of interfaces between different materials. For example, the signal is generated at the cell membrane, which means that it can be used to improve the accuracy of cell segmentation. In two-photon excited fluorescence, the signal behaves very similarly to "normal" fluorescence, except that since the signal is not generated outside the laser focus, the signal-to-noise ratio of the resulting image is generally much better. In stimulated Raman scattering or coherent anti-Stokes Raman scattering (SRS, CARS), the signal is generated by the concentration of specific chemicals (intrinsic or introduced) and optically active vibrating bonds that resonate at specific frequencies. For example, recent studies have shown 30-fold SRS imaging of a range of engineered chemicals. Another strong application of this signal is the detection of high lipid concentrations, such as cell walls or intracellular lipid droplets.

[0201] Figure 13 This is a second harmonic generation (SHG) image of collagen tissue published online by the University of Minnesota School of Biological Sciences.

[0202] Figure 14 Nonlinear microscopy images of breast cancer tissue posted online by the Biophotonics Imaging Laboratory at the University of Illinois are shown. Breast cancer tissue was imaged using a variety of nonlinear microscopy signals. SHG can be seen highlighting the extracellular matrix (primarily composed of collagen) and exposing its structure and orientation. THG can be seen highlighting cell interfaces and the concentration of lipids (i.e., lipid droplets). Two-photon and three-photon excited fluorescence images show autofluorescence from fluorescent stains or intrinsic fluorophores introduced into the tissue. Coherent anti-Stokes Raman scattering (CARS, a technique similar to SRS) shows the concentration of specific chemicals that may be intrinsic to the tissue or introduced by the researcher. Each of these signals can be of significant benefit to researchers and can be highly complementary to the information from imaging mass cytometry.

[0203] like Figure 15As shown, the system incorporating a nonlinear microscope may include other elements described in other embodiments and figures. For example, the system may include a collection objective 1514, a spectroscopic optical device 1516, and an integrating detector 1515, such as a photomultiplier tube. Although a nonlinear microscope may benefit from transillumination (e.g., for detection of certain features), a system incorporating a nonlinear microscope may not be able to provide transillumination. For example, directional optical devices on one side of the sample support 1507 (including both the illumination optics and the collection objective 1514, the spectroscopic optical device 1516, and the integrating detector 1515) can allow more injectors located above the sample to directly inject the ablation plume into the mass analyzer. As described herein, such injectors may be short and straight.

[0204] Figure 15 A diagram of a setup that can be used to capture nonlinear microscopy signals in imaging mass cytometry is shown. Multiple different nonlinear microscopy signals can be detected, such as three signals detected by three integrating detectors 1515. These signals can include, for example, second harmonic generation, third harmonic generation, and / or two-photon excited fluorescence. If stimulated Raman scattering (SRS) or CARS is to be added to the setup, the laser source also needs to be modified because two coherent, synchronized laser beams with a well-defined wavelength difference can generate SRS or CARS signals. Thus, an imaging mass spectrometry system integrated with SRS or CARS can include a laser source 1501 that provides two coherent laser beams with a defined wavelength difference. Specifically, the laser source 1501 can generate secondary pulses that are coherent and co-propagating with the main pulse and have a specific wavelength offset compared to the main pulse. In CARS, the laser source can be tuned to the chemical transition frequency of a specific target (e.g., a class of molecules). An imaging mass cytometer with an integrated CARS microscope includes a notch filter.

[0205] Sampling and analysis methods based on laser scanning

[0206] As noted above in the discussion of the laser scanner system itself, the system allows for rapid scanning of the laser beam across the sample, thereby increasing the speed at which the sample can be ablated and analyzed, and is also capable of ablation of arbitrary shapes, thereby enabling specific single areas to be ablated, including irregularly shaped cells, without ablating material from adjacent areas / cells.

[0207] Thus, aspects of the present invention provide a method of analyzing a sample, such as a biological sample, comprising:

[0208] (i) performing laser ablation on the sample, wherein laser radiation is directed onto the sample on a sample stage using a laser scanning system, and wherein ablation is performed at a plurality of locations to form a plurality of plumes; and

[0209] (ii) ionizing and mass spectrometrically analyzing the plume, whereby detection of atoms in the plume allows construction of an image of the sample, optionally where the plurality of locations is a plurality of known locations.

[0210] Aspects of the present invention also provide a method for performing mass cytometry on a sample comprising a plurality of cells, the method comprising:

[0211] (i) labeling a plurality of different target molecules in a sample with one or more different labeling atoms to provide a labeled sample;

[0212] (ii) performing laser ablation on the sample, wherein laser radiation is directed onto the sample on a sample stage using a laser scanning system, and wherein ablation is performed at a plurality of locations to form a plurality of plumes; and

[0213] (iii) ionizing and mass spectrometrically analyzing the plume, whereby detecting atoms in the plume allows for the construction of an image of the sample, optionally where the plurality of locations is a plurality of known locations.

[0214] Exemplary methods of labeling samples, suitable labels, and other related teachings are provided below in the Labeling section.

[0215] Many applications can be uniquely enabled or enhanced by the laser scanning methods and systems described herein.

[0216] Biological samples may have small and / or irregular features (e.g., cells on the micron scale) and may benefit from analysis over a wide field of view. As used herein, features may include tissue regions, single cells, subcellular components, cell membranes, cell-cell interfaces and / or extracellular matrices, and different tissues or cells within a slice or image (e.g., healthy tissue, tumors, lymphocytes (such as tumor infiltrating lymphocytes), muscles (such as skeletal muscle or smooth muscle), epithelia (such as vasculature) and / or connective tissue (such as matrix or fiber)). Such features can be acquired (e.g., selectively acquired) by laser scanning as described herein. In conventional IMC, analyzing such features over a wide field of view (e.g., in millimeters or centimeters) and / or across multiple samples may take hours or days, where each pixel is approximately 1 um in size and needs to be distinguished from surrounding pixels. In the present method and system, laser scanning (optionally combined with stage movement) can allow rapid acquisition of individual features. In some aspects, a system and / or method enables a cell acquisition rate greater than 10, 50, 100, 200, 500, 1000, 2000, or 5000 cells per second. Features can be automatically identified by optical microscopy (e.g., bright field and / or fluorescence microscopy) and sampled by laser modulation, as described herein. In some aspects, contrast agents can improve the identification of these features.

[0217] In certain aspects, a method and / or system can sample over a wide field of view to identify regions of interest (ROIs). Specifically, the presence of mass tags can be detected by rapidly scanning with an fs laser, removing only a thin layer of the sample and leaving the remainder of the intact mass-tagged sample intact (suitable for further analysis). Sampling from spaced (non-adjacent) spots can allow for initial interrogation of the spatial distribution of mass tags and identification of regions of interest for more in-depth sampling (e.g., pixel-by-pixel or repeated scanning). During this initial interrogation, the laser can be scanned and the stage moved continuously. Thus, a large field of view and / or a large number of samples (e.g., totaling more than one square centimeter) can be rapidly (e.g., in less than one hour, 30 minutes, 10 minutes, or five minutes) initially interrogated to identify ROIs for further investigation by IMC.

[0218] In some aspects, a sample of suspended cells (such as peripheral blood mononuclear cells (PBMC), non-adherent cell cultures or decomposed cells from intact tissue or adherent cell cultures) can be provided for analysis as a cell smear. These cells can be stained with SBP labeled with mass in a suspension and applied to a surface (such as a slide) for analysis by the present method and system. The cell smear can be provided on a support together with element standard particles for calibration and / or normalization. Alternatively or additionally, a cell smear can be provided along with assay barcode beads to detect free analytes in a biological sample. For example, a cell smear comprising PBMC and an assay barcode bead combined with free analytes can be provided, and the free analytes are from the same blood sample as the PBMC. In some aspects, the surface can have a capture site, such as a micron-sized hole, for retaining cells and / or beads.

[0219] The beads of the assay barcode can be individually detectable and can be micron-sized. Such beads can contain an assay barcode on its surface or inside it that identifies the SBP on the bead surface. The unique combination of assay barcode isotopes can identify the SBP on the bead surface, thereby distinguishing each assay barcode bead with different SBPs by the assay barcode. The assay barcode beads can be mixed with a biological fluid (e.g., cell supernatant, cell lysate, or serum) and combined with free analytes (e.g., cytokines) in the sample. The reporter SBP bound to the reporter mass tag can be bound to the analyte of the SBP on the cell surface. The same reporter mass tag can be used for the beads of the assay barcode because the assay barcode can distinguish analytes.

[0220] In some aspects, a control cell sample, such as a homogenous cell line or PBMC, can be applied to a slide (for example, as a cell smear, a tissue section or as an adherent cell). The control cell sample can be used for the change in normalization sample processing, such as dyeing. The control cell sample can be from a previously characterized sample (for example, and with a known marker expression level) and / or can be used on multiple slides with other samples. The control cell sample can be used for normalization and / or quantification and / or for classification, and the change in sample dyeing can be controlled. For example, although element standards can be used for calibration, normalization and / or quantification of mass labels to address fluctuations in instrument sensitivity, the control cells dyed together with the sample of interest can allow normalization to address the change in sample dyeing. Control cells with a previously defined population of interest (for example, PBMC) can be used to classify cells of similar populations in one or more samples of interest. The control cell can have one or more marker atoms (such as sample barcodes) that can identify cells as control cells.

[0221] The control cell sample can be a paraffinized cell sample, such as when the sample of interest (e.g., on the same slide) is also a paraffinized sample. In some aspects, the control cell sample can be a paraffinized cell line on a sample slide for tracking the reproducibility of sample processing. Alternatively, the control cell sample can be a frozen tissue section, such as when the sample of interest (e.g., on the same slide) is also a frozen tissue sample. In either case, the control cell sample can be processed together with the sample of interest, including a staining step. Alternatively or additionally, the control cell sample can be pre-stained. For example, a pre-stained control cell sample can be a control cell sample stained together with the sample of interest to determine whether the staining is similar (and optionally normalize changes from other aspects of staining and / or sample preparation).

[0222] The interior of the assay barcode bead can include an assay barcode, such as a distinguishable combination of metal isotopes. The interior of the bead can be any of a variety of suitable structures, such as a solid metal core, a metal chelate polymer interior, a nanocomposite interior, or a mixed interior. The solid metal core can be formed by subjecting a mixture (e.g., a solution) of one or more metal elements and / or isotopes to high heat and / or high pressure. The nanocomposite structure can include a combination of nanoparticles / nanostructures (e.g., a matrix) (e.g., each containing different physical properties and contributing one or more assay barcode elements / isotopes and / or providing support for other nanoparticles containing assay barcode elements / isotopes). The interior of the bead can include a polymer that entraps the assay barcode metal and / or chelates the assay barcode metal (e.g., through a side group such as DOTA, DTPA, or a derivative thereof). Suitable polymer backbones can be branched (e.g., hyperbranched) or form a matrix. In some aspects, the polymer can be formed in emulsion form or by controlled living polymerization. In certain aspects, the interior of the assay bead can present an inert surface (e.g., such as a solid metal surface) that needs to be functionalized (e.g., by polymerization across the surface) prior to attachment to an assay biomolecule (e.g., an oligonucleotide or antibody). The surface of the assay bead can comprise a polymer, a spatially separated assay biomolecule (e.g., an SBP) and / or a linker (e.g., a PEG linker) that increases colloidal stability, one or more functional groups for attachment to (or to which) assay biomolecules and / or sample barcodes.

[0223] When barcoding a sample, cell smears and / or cells assayed for barcoded beads from multiple samples can be combined. The sample barcode can contain multiple isotopes that are not used for staining (i.e., not associated with the mass tag of the SBP). The sample barcode can include one or more small molecules or SBPs that transfer the sample barcode isotopes to the cells or beads. A unique combination of isotopes is applicable to the beads and / or cells of each sample. When the cells or beads are analyzed by mass cytometry (e.g., LA-ICP-MS), the unique combination of barcode isotopes can identify the original sample of the cell or bead. The samples may come from different sources and / or may be subjected to different processing and / or staining conditions. In certain aspects, live cell barcodes (e.g., tellurium-based barcodes based on thiol reactivity, or antibodies labeled with elements targeting widely expressed surface markers) can be used, which can also increase the benefits of barcoding live cells in the sample (e.g., fresh blood). The method can be performed simultaneously with stimulation or other treatment of live cells (e.g., PBMCs). In some cases, the sample barcode can barcode living cells. In some cases, the sample barcode can be harmless to living cells, such as non-toxic to living cells.

[0224] In some cases, barcode reagent can be provided in a pre-configured form by preparing a barcode reagent with a unique combination of many detection barcodes and sample barcodes. In this type of case, each unique barcode reagent can be stored in different containers, such as in the different holes of an orifice plate. In one example, an orifice plate can be set up so that all holes along a specific column (or row) share the same mensuration barcode, while all holes along a specific row (or column) share the same sample barcode. In another example, an orifice plate can be set up so that each filled hole comprises a barcode reagent, which has the various combinations of specific unique sample barcode and a large number of mensuration barcodes. Therefore, the first hole can comprise all barcode reagents with the first sample barcode but each having different mensuration barcodes, and the second hole can comprise all barcode reagents with the second barcode but each having different mensuration barcodes. In some cases, pre-configured barcode reagents may need to manufacture tens of thousands of groups of unique beads.

[0225] For automated staining, a biological sample (eg, comprising cells) on a surface can be stained by flowing a mass-tagged SBP over the cell surface (eg, using an automated flow system).

[0226] In some embodiments, the plumes produced by performing laser ablation are individually ionized and mass spectrometrically analyzed. In such cases, each plume will represent a discrete pixel of the image. However, in other cases, bursts of laser radiation pulses are directed to different locations on the sample in rapid succession, such that the plume from each location is not analyzed individually, but rather is ionized and mass spectrometrically analyzed as a single mass of sample material. Such methods can be used to ablate an entire cell as a single event on a detector. Thus, in some cases, in the above-described methods of aspects of the present invention, bursts of laser radiation pulses are directed to closely spaced areas on the sample, and the plumes produced by the bursts of laser radiation pulses are ionized and detected as continuous events (i.e., the plumes overlap). The use of lasers such as femtosecond lasers and rapidly moving laser scanning arrays (e.g., based on AOD and / or EOD) will allow the ablation of arbitrary shapes (such as single cells) using multiple ablation spots of 1 μm diameter within the pulse duration of a laser with a nanosecond pulse duration. Thus, in some embodiments, the method is performed using a spot size of less than 3 μm, less than about 2 μm, or less than about 1 μm for each laser pulse. The burst of laser radiation comprises at least three laser pulses, wherein the duration between each laser pulse is less than 1 ms, such as less than 500 μs, less than 250 μs, less than 100 μs, less than 50 μs, less than 10 μs, less than 1 μs, less than 500 ns, less than 250 ns, less than 100 ns, less than 50 ns, or less than about 10 ns. The burst of laser radiation may comprise at least 10, at least 20, at least 50, or at least 100 laser pulses. To achieve such a short time between laser pulses, a high repetition rate laser is required, whose repetition rate is suitable for the timing interval, such as those discussed above in the "Laser" section of the apparatus of various aspects of the present invention. For example, for a pulse burst where each pulse is approximately 10 ns apart, the laser should have a repetition rate of 100 MHz (ie 1 s ÷ 10 ns).

[0227] In some embodiments, the laser scanning system imparts a first relative movement of the laser radiation beam used for ablation relative to the sample (e.g., the Y axis). In some embodiments, the laser scanning system imparts a first relative movement and a second relative movement of the laser radiation beam used for ablation relative to the sample (e.g., the Y axis and the X axis), wherein the first relative movement and the second relative movement are orthogonal. In some embodiments, a single positioner in the laser scanning system imparts both movements simultaneously (e.g., an EOD with orthogonal electrode groups connected). In other words, the first positioner imparts the first relative movement and the second positioner imparts the second relative movement. This arrangement can be seen, for example, using a pair of galvanometer mirrors, or using two orthogonally placed AODs. Thus, in some embodiments, the method includes controlling at least one first positioner and, if present, an optional second positioner to impart the first relative movement and the optional second relative movement in the laser radiation beam used to ablate the sample.

[0228] As described above, the AOD can also be used to modulate the intensity of the laser radiation beam. Thus, in some embodiments of the methods of the various aspects of the present invention disclosed above, the method includes the step of controlling the intensity of the laser radiation beam via the AOD. Furthermore, in an experimental arrangement that includes both a mirror-based positioner and a solid-state positioner, the solid-state positioner can be controlled to correct for positional errors or noise-induced inaccuracies in the position at which the mirror-based positioner directs the laser radiation onto the sample.

[0229] One advantage of the present invention is that the laser scanning system allows the sample stage to be moved at a constant speed in one direction (e.g., X), and then the laser scanning system ablates above and below the center line of the X-axis movement of the ablation stage, such as Figure 79 . Furthermore, in laser scanning systems that allow movement in both the X and Y axes, scanning can compensate for movement of the sample stage along the sample's X axis. Thus, in some embodiments, the sample being analyzed is positioned on the sample stage. In some cases, the sample stage moves at a constant speed relative to the laser scanning system in a first direction, thereby imparting a first relative movement to the sample relative to the laser scanning system (e.g., the X axis), and the laser scanning system imparts a second relative movement (e.g., the Y axis). In other words, the stage can move the sample in a first direction, and this position can introduce relative movement into the laser beam in a second direction (i.e., non-parallel, such as substantially orthogonal, e.g., orthogonal). In some cases, the laser scanning system compensates for relative movement of the sample stage, thereby maintaining a regular, rectilinear raster pattern for the ablation spots on the sample (i.e., spots generated by a single scan of the laser scanning system with the Y axis not offset relative to each other on the X axis). Thus, in some embodiments, the sample stage is movable in at least the x-axis, and the positioner is adapted to introduce deflection in at least the y-axis into the path of the laser beam onto the sample stage. In some embodiments, the positioner is further adapted to introduce a deflection in the x-axis into the path of the laser beam to the sample stage; or (ii) the apparatus comprises a second positioner adapted to introduce a deflection in the x-axis into the path of the laser beam to the sample stage; optionally, wherein the positioner of the laser scanning system is controlled by a control module that also controls movement of the sample stage. In these embodiments, the sample stage is movable in the x- and y-axes as well as the z-axis.

[0230] However, it is not necessary for the laser scanning system to perform a full scan across the entire amplitude possible in the system. Rather, an arbitrary ablation pattern can be ablated to ablate only specific features of interest, such as individual cells.

[0231] In order to be able to identify the area that should be ablated, the identification of cells of interest generally involves an examination of the visual image of the cells. For example, in order to simplify the analysis, in a cell smear, it is necessary to analyze individual cells that exist as discrete cells on the smear (i.e., not binaries, trisomers, or higher number of cell clusters), and this determination can be easily completed by visual inspection of the sample. As described below, in certain embodiments disclosed herein, markers in the sample can be examined that are apparent by examining the cells in the visible light range. Sometimes, the cell morphology identified under a confocal microscope is sufficient to identify the cells of interest. In other cases, the sample can be stained with one or more histochemical stains or one or more SBPs coupled to a fluorescent marker (in some cases, it can be an SBP that is also conjugated to a labeled atom). 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 (such as the nucleus or mitochondria), and when illuminated with light of the appropriate wavelength, these regional samples can be clearly identified. In some cases, the lack of a specific type of fluorescence from a specific region may be characteristic. For example, a first fluorescent marker targeting a cell membrane protein can be used to broadly identify cells, but then a second fluorescent marker targeting the ki67 antigen (encoded by the MKI67 gene) can distinguish between proliferating cells and non-proliferating cells. Therefore, by targeting cells that lack fluorescence from the second marker fluorescence, non-replicating cells can be specifically targeted for analysis. Therefore, in some embodiments, the system described herein may include a laser for exciting the fluorophore in the label for marking the sample. Alternatively, an LED light source can be used to excite the fluorophore. Non-confocal (e.g., wide field) fluorescence microscopes can also be used to identify certain areas of biological samples, but have a lower resolution than confocal microscopes.

[0232] When fluorescence microscopy is performed using laser-excited fluorescence microscopy, in some embodiments, the laser is the same laser that generates the laser radiation used to ablate material from a biological sample (and for LIFTing), but at an energy density insufficient to cause ablation or desorption of material from the sample. In some embodiments, the fluorophores are excited by the wavelength of the laser radiation used for sample ablation or desorption. In other embodiments, different wavelengths can be used, for example, by utilizing different harmonics of a laser to obtain laser radiation of different wavelengths. The laser radiation that excites the fluorophores can be provided by a different laser source than the ablation and / or lift-off laser source.

[0233] By using an image sensor (such as a CCD detector or an active pixel sensor, such as a CMOS sensor), the process of identifying features / regions of interest and ablating them can be fully automated using a control module (such as a computer or programmed chip) to correlate the location of fluorescence with the x,y coordinates of the sample, and then directing the ablation laser to the area surrounding that location before lifting the cells at that location. As part of this process, in some embodiments, the first image captured by the image sensor may have a low objective magnification (low numerical aperture), allowing measurements to be taken over a large area of the sample. Thereafter, switching to an objective with a higher magnification can be used to focus on specific features of interest, which have been identified as being of interest through higher magnification optical imaging, such as if the sample is stained with a fluorescent labeling agent and therefore fluoresces. These features registered as being of interest (e.g., fluorescing) can then be ablated / desorbed. Using a lower numerical aperture lens initially has the further advantage of an increased depth of field, thereby meaning that features buried in the sample can be more sensitively detected than if screening were performed using a higher numerical aperture lens from the outset.

[0234] The analysis to identify features / regions of interest can be performed by the apparatus of aspects of the present invention, or can be performed externally to the apparatus. For example, a slide can be analyzed remotely from the apparatus of aspects of the present invention by a physician or histologist, and information about the locations on the slide that should be ablated can be fed back to the apparatus.

[0235] Thus, in some embodiments, the above methods include the steps of identifying one or more features of interest and the locations of the one or more features of interest on the sample. For example, some methods of aspects of the present invention include the following steps:

[0236] (i) identifying one or more features of interest on the sample;

[0237] (ii) recording the location information of one or more features of interest on the sample;

[0238] (iiI) performing laser ablation of the sample, wherein laser radiation is directed onto the sample on a sample stage using a laser scanning system, using positional information of one or more features of interest to form a plurality of plumes; and

[0239] (iv) Ionization and mass spectrometry of the plume are performed, thereby detecting the atoms in the plume to allow an image of the sample to be constructed.

[0240] Some methods of aspects of the present invention include the following steps:

[0241] (i) labeling a plurality of different target molecules in a sample with one or more different labeling atoms to provide a labeled sample;

[0242] (ii) identifying one or more features of interest on the sample;

[0243] (ii) recording the location information of one or more features of interest on the sample;

[0244] (iiI) performing laser ablation of the sample, wherein laser radiation is directed onto the sample on a sample stage using a laser scanning system, using positional information of one or more features of interest to form a plurality of plumes; and

[0245] (iv) Ionization and mass spectrometry of the plume are performed, thereby detecting the atoms in the plume to allow an image of the sample to be constructed.

[0246] For example, embodiments of aspects of the present invention may include identifying the location of a feature of interest, such as a cell, and directing a burst of laser pulses to sample all or a portion of the cell. As described herein, the burst of laser pulses is directed by a laser scanning system to multiple known locations within the feature of interest, and the plume generated by the burst of laser pulses can be detected as a single event.

[0247] In some cases, the position information can be in the form of absolute measurements of the position of the feature of interest on the sample carrier. In other cases, the position information of the feature of interest can be recorded in a relative manner. For example, after irradiation with UV light having a number of fluorescent features, a visual image of the sample can be recorded. The position of the feature of interest can be recorded as position information relative to the pattern of fluorescent features. The relative position information is used to identify the location to be ablated, thereby reducing errors caused by imprecise positioning of the sample in the device. Methods for calculating the position of the feature of interest relative to such a reference pattern are standard to those skilled in the art, for example by using a barycentric coordinate system.

[0248] In some cases, a feature of interest, such as a cell in a biological sample, may be surrounded by other biological material, such as an intracellular matrix or other cells that may affect the ablation of the cell of interest. Here, ablation using a laser scanner system can be used to clear material surrounding the cell of interest, allowing bursts of laser pulses to ablate the target cell in continuous events or with subcellular resolution. Sometimes, no data is recorded for ablation to clear the area surrounding the feature of interest (e.g., a cell of interest). Sometimes, data is recorded by ablating the surrounding area. Useful information that can be obtained from the surrounding area includes which target molecules, such as proteins and RNA transcripts, are present in the surrounding cells and intercellular environment. This may be of particular interest when imaging solid tissue samples, in which case direct cell-to-cell interactions are common, and which proteins are expressed in the surrounding cells, etc., may be helpful in understanding the state of the cell of interest.

[0249] Thus, in some embodiments disclosed herein, the method includes using positional information of a feature of interest to ablate cells, including first performing laser ablation to remove sample material surrounding the feature of interest before ablating the cells of interest. In some embodiments, the feature is identified by examining an optical image of the sample, optionally wherein the sample has been labeled with a fluorescent marker and illuminating the sample under conditions such that the fluorescent marker fluoresces.

[0250] Otherwise, typically in this method, laser ablation is performed in a manner previously described, for example in Giesen et al. (2014) and WO2014169394, with modifications relevant thereto (e.g., it is not necessary to use ICP to ionize the sample material, nor is it necessary to use a TOF MS detector). For example, as described below, the method can also be performed, but with OES detection instead of mass spectrometry detection.

[0251] Method disclosed herein can also be provided as a computer program product, and this computer program product includes the non-transitory machine-readable medium storing an instruction, and this instruction can be used for programming a computer (or other electronic device) to perform process as described herein.Machine-readable medium can include but is not limited to hard disk drive, floppy disk, optical disc, CD-ROM, DVD-ROM, ROM, RAM, EPROM, EEPROM, magnetic card or optical card, solid-state storage device or other types of medium / computer-readable medium suitable for storing electronic instructions.Therefore, aspects of the present invention also provide a kind of machine-readable medium, and this machine-readable medium includes the instruction for performing method disclosed herein.

[0252] delivery catheter

[0253] In certain aspects, a transfer conduit (also referred to as an injector) forms a link between the laser ablation sampling system and the ionization system and allows for the transfer of a plume of sample material generated by laser ablation of the sample from the laser ablation sampling system to the ionization system. A portion (or all) of the transfer conduit can be formed, for example, by drilling through a suitable material to form a lumen (e.g., a lumen having a circular, rectangular, or other cross-section) for passage of the plume. The inner diameter of the transfer conduit sometimes ranges from 0.2 mm to 3 mm. Sometimes, the inner diameter of the transfer conduit can vary along its length. For example, one end of the transfer conduit can be tapered. The length of the transfer conduit sometimes ranges from 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 lumen of the transfer conduit is straight along the entire distance (or substantially the entire distance) from the ablation system to the ionization system. Other times, the lumen of the transfer conduit is not straight along the entire distance and changes direction. For example, the delivery conduit can be gradually rotated 90 degrees. This configuration allows the plume generated by sample ablation in the laser ablation sampling system to initially move in a vertical plane, with the axis at the delivery conduit entrance pointing straight up and moving horizontally as it approaches the ionization system (for example, ICP torches are typically oriented horizontally to take advantage of convection cooling). The delivery conduit can be straight and at a distance of at least 0.1 cm, at least 0.5 cm, or at least 1 cm from the entrance aperture through which the plume enters or is formed. In general, the delivery conduit is adapted to minimize the time it takes to transport material from the laser ablation sampling system to the ionization system.

[0254] One or more gas streams can deliver the ablation plume to the ionization system. For example, helium, argon, or a combination thereof can deliver the ablation plume to the ionization system. In some cases, separate gas streams can be provided to the sample chamber and the injector, which mix when entraining the ablation plume into the injector. In some cases, only one gas stream is used, such as when the injector inlet is activated within the sample chamber.

[0255] Delivery catheter inlet and / or aperture

[0256] The delivery conduit may be an inlet in the laser ablation sampling system (particularly within the sample chamber of the laser ablation sampling system; therefore, it also represents the primary gas outlet from the sample chamber). The inlet of the delivery conduit receives sample material ablated from the sample in the laser ablation sampling system and delivers the sample material to the ionization system. In some cases, the inlet of the laser ablation sampling system is the source of all gas flowing along the delivery conduit to the ionization system. In some cases, the inlet of the laser ablation sampling system that receives material from the laser ablation sampling system is an aperture in the wall of the conduit, along which a second "delivery" gas flows from a separate delivery flow inlet (e.g., as disclosed in WO2014146724 and WO2014147260). In this case, the delivery gas forms a large proportion, and in many cases, the majority of the gas flowing 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, and a gas flow out of the chamber is generated through the inlet of the delivery conduit. The gas stream can capture a separate plume of ablated material and entrain the plume as it enters a delivery conduit (e.g., by having the aperture of the delivery conduit be tapered, referred to herein as a sample cone) and exit the sample chamber into a conduit passing over the chamber. The conduit also has gas flowing into it from a separate delivery stream inlet (indicated by the delivery stream arrow on the left hand side of the figure). The assembly comprising the delivery stream inlet, the laser ablation sampling system inlet, and starting from the delivery conduit, which carries the ablated sample material toward the ionization system, can also be referred to as a flow cell (as in WO2014146724 and WO2014147260).

[0257] The transport flow performs at least three roles: it 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; it forms a "protection zone" above the sample surface and ensures that ablation occurs in a controlled environment; and it increases the flow rate in the transport conduit. Typically, the trapping gas has a lower viscosity than the transport gas. This helps confine the plume of sample material to the trapping gas in the center of the transport conduit and minimizes the spread of the sample material plume downstream of the laser ablation sampling system (because the transport rate is more stable and nearly flat in the center of the flow). The gas can be, for example, but not limited to, argon, xenon, helium, nitrogen, or a mixture of these gases. A common transport gas is argon. Argon is particularly suitable for preventing the plume from spreading before it reaches the walls of the transport conduit (and, in devices where the ionization system is an argon-based ICP, it also helps increase instrument sensitivity). The trapping gas is preferably helium. However, the trapping 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.

[0258] As described in WO2014169394, the use of a sample cone minimizes the distance between the target and the inlet of the laser ablation sampling system in the delivery catheter. This reduced distance between the sample and the point of the cone through which the capture gas can pass improves capture of the sample material while reducing turbulence and reduces the spread of the plume of ablated sample material. Therefore, the inlet of the delivery catheter is the aperture at the tip of the sample cone. The cone extends into the sample chamber.

[0259] An optional modification to the sample cone is to make it asymmetric. When the cone is symmetric, the airflow from all directions at the exact center cancels out, so the total airflow along the sample surface at the axis of the sample cone is zero. By making the cone asymmetric, a non-zero velocity is generated along the sample surface, which helps flush plume material from the sample chamber of the laser ablation sampling system.

[0260] In practice, any modification to the sample cone that results in a non-zero vector gas flow at the axis of the cone along the sample surface may be employed. For example, an asymmetric cone may include a notch or a series of notches adapted to produce a non-zero vector gas flow at the axis of the cone along the sample surface. An asymmetric cone may include an orifice in the side of the cone adapted to produce a non-zero vector gas flow at the axis of the cone along the sample surface. The orifice will unbalance the gas flow around the cone, thereby again producing a non-zero vector gas flow at the axis of the cone along the sample surface at the target. The side of the cone may include more than one orifice, and may include one or more notches and one or more orifices. The edges of the notches and / or orifices are typically smoothed, rounded, or chamfered to prevent or minimize turbulence.

[0261] Depending on the choice of capture and delivery gases and their flow rates, different orientations of the cone asymmetry will be suitable for different situations, and it is within the ability of the skilled person to appropriately identify the combination of gas and flow rate for each orientation.

[0262] As used in various aspects of the present invention, all of the above adaptations 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 contain one of a plurality of apertures, etc.

[0263] 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 near the sample, for example, by manipulating the sample in 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 can be: a) adjustable; b) sized to prevent disturbance of the ablated plume as it enters the delivery conduit; and / or c) approximately equal to the cross-sectional diameter of the ablated plume.

[0264] tapered catheter

[0265] In a tube with a smaller inner diameter, the same gas flow rate moves at a higher speed. Therefore, by using a tube with a smaller inner diameter, a plume of ablated sample material carried in a gas flow can be transported more quickly over a defined distance at a given flow rate (e.g., from a laser ablation sampling system to an ionization system in a delivery conduit). One of the key factors in enabling rapid analysis of a single plume is how much the plume has diffused between the time the plume is generated by ablation and the time its constituent ions are detected in the mass spectrometer component of the instrument (the transient time on the detector). 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 on the detector. Shorter transient times mean that more plumes can be generated and analyzed per unit time, resulting in higher quality and / or faster images.

[0266] The cone can include a portion of the delivery conduit where the inner diameter of the conduit gradually changes along the length of the delivery conduit (i.e., the inner diameter of the tube is a cross-section taken through the tube that decreases along the portion of the end from the portion toward the inlet (at the laser ablation sampling system end) to the outlet (at the ionization system end). Typically, the region near the conduit where ablation occurs has a relatively wide inner diameter. The larger volume of the conduit before the cone helps to confine the material produced by ablation. As the ablated particles fly away from the ablation spot, they travel at high speed. Friction in the gas slows these particles, but the plume can still spread out in the sub-millimeter to millimeter range. Sufficient distance is left from the wall, which helps to control the plume near the center of the flow.

[0267] Because the wide ID portion is only short (on the order of 1-2 mm), there is no significant effect on the total transient time if the plume spends more time in the longer portion of the narrower ID delivery catheter. Therefore, the larger ID portion is used to capture the ablation products, and the smaller ID catheter is used to quickly deliver these particles to the ionization system.

[0268] 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 circular tube and a known flow. Generally, a Reynolds number greater than 4000 will indicate turbulent flow and should therefore be avoided. A Reynolds number greater than 2000 will indicate a transitional flow (between non-turbulent and turbulent flow) and may therefore also need to be avoided. For a given mass of gas flow, the Reynolds number is inversely proportional to the diameter of the conduit. 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 a 4 liter per minute helium flow in the conduit has a Reynolds number greater than 4000.

[0269] Rough or even angular edges in the transition between the constant diameter portion of the delivery conduit and the cone may cause turbulence in the gas flow and are generally avoided.

[0270] sacrificial flow

[0271] At higher flow rates, the risk of turbulence in the conduit increases, particularly when the delivery conduit has a small inner diameter (e.g., 1 mm). However, if light gases such as helium or hydrogen are used instead of the argon traditionally used as the delivery gas flow, high-speed delivery (up to 300 m / s or more) can be achieved in delivery conduits with a small inner diameter.

[0272] High-speed delivery poses a problem because it can cause a plume of ablated sample material to pass through the ionization system without achieving an acceptable level of ionization. The ionization level may decrease as the increased cool gas flow reduces the plasma temperature at the torch tip. If the plume of sample material is not ionized to an appropriate level, information from the ablated sample material is lost because its composition (including any labeled atoms / elements) cannot be detected by the mass spectrometer. For example, in an ICP ionization system, the sample may pass through the plasma at the torch tip so quickly that the plasma ions do not have sufficient time to interact with the sample material and ionize it. This problem caused by high-flow, high-speed delivery in a narrow-bore delivery conduit can be addressed by introducing a sacrificial flow system at the delivery conduit's outlet. The sacrificial flow system is adapted to receive the gas flow from the delivery conduit and forward only a portion of the flow (the central portion of the flow containing any ablated sample material plume) to the injector leading to the ionization system. To facilitate diffusion of gas from the delivery conduit within the sacrificial flow system, the delivery conduit outlet can be flared.

[0273] The flow sacrificial system is located close to the ionization system, so the length of the tube (e.g., the injector) leading from the flow sacrificial system to the ionization system is short (e.g., ˜1 cm; compared to the length of the delivery conduit, which is typically on the order of tens of centimeters, e.g., ˜50 cm). Therefore, since the relatively slow portion of the entire delivery system is much shorter, 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.

[0274] In most arrangements, it is undesirable, or in some cases impossible, to significantly increase the diameter of the tube (e.g., injector) that passes from the flow sacrificial system to the ionization system as a way to reduce the gas velocity at the volume flow rate. For example, where the ionization system is an ICP, the conduit from the flow sacrificial system forms the injector in the center of the ICP torch. When a wider inner diameter injector is used, the signal quality is reduced because the plume of ablated sample material cannot be injected so precisely into the center of the plasma (the center of the plasma is the hottest and therefore the most efficient part of the plasma). Injectors with an inner diameter of 1 mm or narrower are strongly preferred (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 to be ionized in a relatively small volume in three dimensions (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 most 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 from the sacrificial flow system to the ionization system are also used in equipment with non-ICP ionization systems. As mentioned above, if the plume in the sample material is not ionized to an appropriate level, information from the ablated sample material is lost - because the components (including any marker atom / element labels) cannot be detected by the mass spectrometer.

[0275] Pumping can be used to help ensure a desired split ratio between the sacrificial flow and the flow entering the ionization system inlet. Therefore, sometimes, the sacrificial flow system includes a pump attached to the sacrificial flow outlet. A controlled flow restriction can be added to the pump to control the sacrificial flow. Sometimes, the sacrificial flow system also includes a mass flow controller adapted to control the flow restriction.

[0276] In cases where expensive gases are used, the gas pumped out of the sacrificial 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 helium losses in the system (i.e., when the helium enters the ionization system and is ionized). Therefore, a gas purification system is sometimes connected to the sacrificial outlet of the sacrificial flow system.

[0277] Ionization system

[0278] To generate elemental ions, a hard ionization technique is used that vaporizes, atomizes, and ionizes the atomized sample.

[0279] Inductively coupled plasma torch

[0280] Typically, an inductively coupled plasma (ICP) is used to ionize the material to be analyzed before it is passed to a mass detector for analysis. It is a plasma source in which the energy is provided by an electric current generated by electromagnetic induction. The ICP is maintained in a torch that can consist of multiple concentric tubes (e.g., three), the innermost tube being called the injector.

[0281] Figure 11 is an exemplary schematic diagram of a laser ablation mass cytometer, which includes a laser ablation source that can be connected to a sample injector (such as a tube) and is installed to deliver a sample to an inductively coupled plasma (ICP) source (also known as an ICP torch). The plasma of the ICP torch can vaporize and ionize the sample to form ions that can be received by a mass analyzer (such as a time-of-flight or sector magnetic mass spectrometer). The laser ablation source can include a laser and a sample chamber. The laser ablation source can include a positioner as described herein. In some aspects, the laser ablation source can be Figures 1 to 5 The system of any one of the foregoing. The injector can be coupled to a sample chamber of a laser ablation source.

[0282] [i] Tanner et al., Cancer Immunol Immunother (2013) 62:955–965.

[0283] [ii] Hutchinson et al. (2005) Anal. Biochem. 346: 225-33.

[0284] [iii] Seuma et al. (2008) Proteomics 8:3775-84.

[0285] [iv] Giesen et al. (2011) Anal. Chem. 83:8177-83.

[0286] [v] Giesen et al. (2014) Nature Methods. 11:417–422.

[0287] [vi] Kindness et al. (2003) Clin Chem 49: 1916-23.

[0288] [vii] Gurevich and (2007) J. Anal. At. Spectrom., 22:1043-1050.

[0289] [viii] Wang et al. (2013) Anal. Chem. 85: 10107-16.

[0290] [ix] WO 2014 / 146724.

[0291] [x] WO 2014 / 127034.

[0292] The injector can be coupled to the sample chamber described herein. The injector can include an inlet or aperture located above the sample support so that material released from the sample by laser ablation can be carried into the injector. The sample chamber can include one or more gas inlets for carrying the ablation plume into the injector, and the injector can include a transport gas inlet (e.g., a shield gas inlet) for transporting the ablation plume captured in the injector to the ICP torch. In some aspects, the system can include a single gas source.

[0293] The injector can have an inlet and an outlet on the outside of the sample chamber, or can have an inlet on the inside of the sample chamber. For example, when the injector is located on the same side of the sample (or sample support) as the laser radiation, the injector can include a window through which the laser radiation passes, and an aperture through which the laser radiation passes and the resulting laser ablation plume captured by the injector is then passed to the ICP torch. Alternatively, the injector can extend through a lens, window, or other optical device to perform laser ablation. In another example, the laser radiation can be opposite the sample (or sample chamber) from the injector and can pass through the sample support. When the laser radiation strikes the sample through the sample support, the injector may include an inlet near the laser ablation site, opposite the side of the laser radiation. In some aspects, the inlet or aperture of the injector can be in the form of a sample cone (e.g., with its narrow end oriented toward the laser ablation site).

[0294] The injector can be rigid and can extend in a straight line from the laser ablation site to the ICP torch. The injector can be short and can be less than 20, less than 10, less than 5 cm, or less than 3 cm in length. A straight and / or short injector can reduce the time to transfer the laser ablation plume to the ICP torch and / or can reduce the spread of the laser ablation plume, thereby allowing more different laser ablation plumes to be analyzed per second. In some aspects, optics such as laser ablation optics, illumination optics, and an image sensor (e.g., a CCD or CMOS) can be placed away from the injector (e.g., on the opposite side of the sample support from the injector). As described above, the injector can transfer the ablation plume to the ICP-MS system over a short distance.

[0295] Various aspects of the fluidics and / or optics can be configured to allow a short and / or straight path from the injector aperture or inlet to the ICP-MS system. For example, some or all of the optics can be positioned relative to the sample support from the injector. Alternatively or additionally, the injector can pass through optical elements, such as one or more lenses and / or mirrors.

[0296] An induction coil, providing the electromagnetic energy that sustains 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 accelerated in the alternating electromagnetic field. They then collide with and ionize argon atoms. In steady state, the plasma consists primarily of argon atoms, with a small fraction of free electrons and argon ions.

[0297] The ICP can be retained in the torch because the gas flow between the two outermost tubes keeps the plasma away from the walls of the torch. A second argon flow introduced between the injector (central tube) and the intermediate 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.

[0298] The ICP may include an injector having an inner diameter of less than 2 mm and greater than 250 μm for introducing material from a sample into the plasma. The diameter of the injector refers to the inner diameter of the injector at the end closest to the plasma. Extending away from the plasma, the injector may have a different diameter, such as a wider diameter, where the difference in diameter is achieved by a gradual increase in diameter or by the injector tapering along its length. For example, the inner diameter of the injector may be between 1.75 mm and 250 μm, such as a diameter between 1.5 mm and 300 μm, a diameter between 1.25 mm and 300 μm, a diameter between 1 mm and 300 μm, a diameter between 900 μm and 300 μm, a diameter between 900 μm and 400 μm, for example a diameter of around 850 μm. Injectors with an inner diameter of less than 2 mm have significant advantages over injectors with larger diameters. One advantage of this feature is that using a narrower injector reduces transients in the signal detected in the mass detector when a plume of sample material is introduced into the plasma (a plume of sample material is a cloud of specific, gaseous material that has been removed from the sample by the laser ablation sampling system). Thus, the time taken from the introduction of the plume of sample material into the ICP for ionization until the analysis of the resulting ions detected in the mass detector is reduced. The reduced time taken to analyze the plume of sample material allows more of the plume of sample material to be detected in any given period of time. Similarly, an injector with a smaller inner diameter allows sample material to be introduced more accurately into the center of the inductively coupled plasma, where more efficient ionization occurs (compared to an injector with a larger diameter, which may introduce more sample material toward the edge of the plasma, where ionization is less efficient).

[0299] ICP torches (Agilent, Varian, Nu Instruments, Spectro, Leeman Labs, PerkinElmer, Thermo Fisher, etc.) and injectors (e.g., from Elemental Scientific and Meinhard) are available.

[0300] Contralateral ablation (opposite side ablation)

[0301] As described above, radiation (e.g., laser radiation) can pass through the sample support to impinge on the sample. The radiation can be generated by an fs laser, such as an ultraviolet, infrared, or green laser. When the laser is ultraviolet, the sample support can be quartz or silicon dioxide. When the laser is infrared or green, the sample support can be glass. A green fs laser can allow for a glass support (e.g., a glass slide), which is preferable from a cost perspective while still achieving high resolution.

[0302] Other ionization techniques

[0303] electron ionization

[0304] 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 of 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 at the electron trap, and a magnetic field applied parallel to the direction of electron travel causes the electrons to travel in a spiral path. The gas phase sample is guided through the electron ionization chamber and interacts with the electron beam to form ions. Electron ionization is considered a difficult ionization method because the process often results in fragmentation of sample molecules. Examples of commercially available electron ionization systems include the Advanced Markus Electron Ionisation Chamber.

[0305] Optional additional components for laser ablation-based sampling and ionization systems

[0306] ion deflector

[0307] A mass spectrometer detects ions when they strike a detector surface. The collision of ions with the detector causes electrons to be released from the detector surface. These electrons multiply as they pass through the detector (the first released electron knocks out other electrons in the detector, which then strike a secondary plate, further increasing the electron count). The number of electrons striking the detector anode generates a current. The number of electrons striking the anode can be controlled by varying the voltage applied to the secondary plate. This current is an analog signal that can then be converted by an analog-to-digital converter into a count of ions hitting the detector. When the detector operates within its linear range, the current can be directly related to the number of ions. However, there is a limit to the number of ions that can be detected at one time (expressed as the number of ions detected per second). Above this limit, the number of electrons released by ions striking the detector no longer correlates with the number of ions. This, therefore, places an upper limit on the detector's quantitative capabilities.

[0308] When ions strike the detector, its surface becomes damaged by contamination. Over time, this irreversible contamination damage results in fewer electrons being released when ions strike the detector, ultimately necessitating detector replacement. This is known as "detector aging" and is a well-known phenomenon in MS.

[0309] Therefore, detector life can be extended by avoiding the introduction of excessive ions into the MS. As mentioned above, when the total number of ions impinging on the MS detector exceeds the upper detection limit, the signal is less informative than when the number of ions is below the upper detection limit, because the ions are no longer quantitative. Therefore, it is desirable to avoid exceeding the upper detection limit, as this will cause the detector to age faster and not produce useful data.

[0310] Analyzing large numbers of ions by mass spectrometry involves specific challenges not found in normal mass spectrometry. Specifically, typical MS techniques involve introducing a low and constant level of material into the detector, which should not approach the upper detection limit or cause accelerated aging of the detector. On the other hand, techniques based on laser ablation and desorption can analyze relatively large amounts of material within a very short time window of MS: for example, the ions extracted from a cell-sized patch of a tissue sample are much larger than the small packets of ions typically analyzed in MS. In effect, this is a deliberate almost overloading of the detector, analyzing the ion pileup caused by ablation or lift-off. Between two analytical events, the signal is in a baseline state (the signal is close to zero because no ions from the labeled atoms are deliberately introduced into the MS from the sampling and ionization system; some ions will inevitably be detected because the MS is not a perfect vacuum).

[0311] Therefore, in the apparatus described herein, there is a risk of accelerated detector aging, as ions from ionized sample material packets labeled with a large number of detectable atoms may exceed the upper detection limit and damage the detector without providing useful data.

[0312] To address these issues, the device can include an ion deflector located between the sampling and ionization system and the detector system (mass spectrometer), the ion deflector being operable to control the ions entering the mass spectrometer. In one arrangement, when the ion deflector is turned on, ions received from the sampling and ionization system are deflected (i.e., the path of the ions has been changed so that they do not reach the detector), but when the deflector is turned off, the ions will not be deflected and reach the detector. The manner in which the ion deflector is deployed will depend on the arrangement of the sampling and ionization system and the MS of the device. For example, if the path of ions entering the MS is not directly aligned with the path of ions leaving the sampling and ionization system, an appropriately arranged ion deflector will be turned on by default to direct ions from the sampling and ionization system into the MS. When an event is detected that is caused by the ionization of an ionized sample material package that is believed to be likely to overload the MS (see below), the ion deflector is turned off so that the remaining ionized material from the event is not deflected into the MS but can simply impact internal surfaces of the system, thereby extending the useful life of the MS detector. After preventing ions from the destructive event from entering the MS, the ion deflector is returned to its original state, thereby allowing ions from subsequently ionized packets of sample material to enter the MS and be detected.

[0313] Alternatively, in an arrangement where (under normal operating conditions) ions emerging from the sampling and ionization system are directed unchanged before entering the MS, the ion deflector is closed and ions from the sampling and ionization system pass through it for analysis in the MS. To prevent damage if a potential detector overload is detected, in this configuration the ion deflector is opened and ions are diverted away from entering the detector to prevent damage.

[0314] Ions that enter the MS through ionization of sample material (such as a plume of material produced by laser ablation or desorption) do not all enter the MS at once, but rather enter the MS in the form of a peak with a frequency that follows a probability distribution curve around a maximum frequency: starting from the baseline, initially a small number of ions enter the MS and are detected, then the frequency of ions increases to a maximum, and then the number of ions decreases again and falls back to the baseline. Events that could damage the detector can be identified because it is not the ion frequency at the leading edge of the peak that increases slowly, but rather the number of ions hitting the detector that increases rapidly.

[0315] During the analysis of ions in the ionized sample material packet, the ion flow that strikes the TOF MS detector (a special type of detector discussed below) is not continuous. The TOF contains a pulser that periodically releases ions into the flight chamber of the TOF MS in the form of pulse groups. By releasing the ions all at a known time, a time-of-flight mass spectrum can be determined. The time between the release of the ion pulses used to determine the time-of-flight mass spectrum is called the extraction or push of the TOF MS. The time of the push is in the order of microseconds. Therefore, the signal from one or more ion packets of the sampling and ionization system covers many pushes.

[0316] Thus, when the ion count reading jumps from the baseline to a very high count value during a push (i.e., the first portion of ions originating from a particular sample material packet being ionized), it can be predicted that the bulk of ions produced by the ionization of the sample material packet will be larger, thus exceeding the upper limit of detection. At this point, the ion deflector can be operated to ensure that the majority of the unwanted ions are directed away from the detector (by activating or deactivating, depending on the system configuration, as discussed above).

[0317] Suitable quadrupole-based ion deflectors are available in the art (eg, from Colutron Research and Dreebit).

[0318] b. Desorption-based sampling and ionization systems

[0319] A desorption-based analyzer typically comprises three components. The first is a desorption system that is used to produce a block of sample material from the sample for analysis. Before the atoms in the desorbed block of sample material (including any detectable labeled atoms discussed below) can be detected, the sample must be ionized (and atomized). Therefore, the device comprises a second component, which is an ionization system that ionizes the atoms to form elemental ions so that they can be detected based on their mass-to-charge ratio by an MS detector component (the third component). The desorption-based sampling system and the ionization system are connected by a transport conduit. In many cases, the desorption-based sampling system is also a laser ablation-based sampling system.

[0320] Desorption sampling system

[0321] In some cases, rather than using laser ablation to create a particulate and / or vaporized plume of sample material, a bulk sample material is desorbed from a sample carrier in which the sample is located without substantially altering the sample and converting it into small sample particles and / or vaporization (see, for example, WO2016109825). Figure 8and accompanying descriptions, which are incorporated herein by reference). Herein, the term "slug" is used to refer to the desorbed material (a specific form of the sample material package discussed herein). The size of the slug can be 10 nm to 10 μm, 100 nm to 10 μm, and in some cases 1 μm to 100 μm. This process can be referred to as sample ejection. Typically, the slug represents a single cell (in which case the process can be referred to as cell ejection).

[0322] The chunks of sample material released from the sample can be a portion of the sample that has been cut into individual chunks for desorption prior to the desorption step, optionally during the process before the sample is inserted into the device. Dividing the sample into discrete chunks prior to analysis is called a structured sample. Thus, each of these individual chunks represents a discrete portion of the sample that can be desorbed, ionized, and analyzed in the device. By analyzing chunks from discrete locations, an image can be constructed in the same manner as for each location of the sample ablated by the laser ablation sampling system described above, where each chunk represents a pixel of the image.

[0323] Structured samples can be prepared by various methods. For example, a sample carrier containing morphological features configured to cut a biological sample can be used. Here, the biological sample is applied to the surface of the carrier, which results in the cutting of the morphological features and the slicing of the sample, resulting in portions of the biological material being retained at multiple discrete locations between the features, thereby providing a structured biological sample. Alternatively, the sample carrier may not contain such morphological features (in practice, a flat surface such as a microscope slide, optionally functionalized as described below). In this case, the sample can be applied to the sample carrier and the sample can be sliced to define a sample block that can be desorbed for ionization and analysis. If the sample is a tissue section, the slicing of the sample can be accomplished using a mechanical tool such as a blade or stamp. Alternatively, the material surrounding the sample section to be desorbed can be removed by laser ablation in the same or a separate sample preparation setup. In some techniques, material removal can be achieved using a setup that employs a focused electron or ion beam. A focused electron or ion beam results in extremely narrow kerfs between segmented sections (perhaps on the order of 10 nm), resulting in pixel sizes on the order of 1 μm, or in some cases, 100 nm.

[0324] The block of sample material can be released from the carrier, and each discrete portion of the sample material is sequentially introduced into the detector for analysis as a discrete event (generating pixels of an image by the techniques discussed below). Advantages of sequentially introducing discrete materials include higher sample processing rates compared to random introduction of biological samples in conventional mass spectrometry or mass spectrometry. This is because the block is preferably delivered from the sample chamber to the ionization system as a single piece of material and is therefore unable to diffuse in the gas flow (particularly in a gas flow where turbulence is present) as a plume of ablated material would.

[0325] Desorption for sampling

[0326] Sample material can be desorbed from the sample using thermal energy, mechanical energy, kinetic energy, or a combination of any of the above. This type of sampling is particularly suitable for analyzing biological samples.

[0327] In some cases, the sample material can be released from the sample by a thermal mechanism. For example, the surface of the sample carrier becomes hot enough to desorb the sample material clumps. The sample carrier can be coated with, for example, a polyethylene naphthalate (PEN) polymer or a PMMA polymer film to facilitate the clump desorption process. The heat can be provided by a radiation source, such as a laser (for example, the laser of the laser ablation sampling system discussed above). The energy applied to the surface should be sufficient to desorb the biological material, preferably without changing the sample material if it comes from a biological sample. Any suitable radiation wavelength can be used, and the radiation wavelength can depend in part on the absorption characteristics of the sample carrier. The surface or layer of the sample carrier can be coated with or include an absorbent that absorbs the laser radiation to convert it into heat. The radiation can be transferred to a surface of the carrier other than the surface on which the sample is located, or can be transferred to the surface carrying the sample, such as through the thickness of the carrier. The heated surface can be a surface layer of a multilayer structure of the sample carrier or can be an internal layer. One example of using laser radiation energy is a technique known as LIFTing (Laser Induced Forward Transfer; see, e.g., Doraiswamy et al., 2006, Applied Surface Science, 52:4743-4474; Fernández-Pradas, 2004, Thin Solid Films 453-454:27–30; Kyrkis et al., Recent Advances in Laser Processing of Materials, Perriere et al., eds., 2006, Elsivier), in which the sample carrier can include a desorption membrane layer. The desorption membrane can absorb radiation to cause release of the desorption membrane and / or the biological sample (e.g., in some cases, the sample membrane is desorbed from the sample carrier along with the biological sample, in other cases, the membrane remains attached to the sample carrier and the biological sample is desorbed from the desorption membrane).

[0328] Heating desorption can be performed on the nanosecond, picosecond, or femtosecond time scale, depending on the laser used for desorption.

[0329] The sample can be attached to the sample carrier via a cleavable photoreactive moiety. Upon irradiation of the cleavable photoreactive moiety with radiation (e.g., laser light from a laser system of a laser ablation sampling system), the photoreactive moiety can be cleaved to release the sample material. The sample carrier can comprise (i) a cleavable photoreactive moiety that couples the sample to the sample carrier, and (ii) a desorption membrane as described above. In this case, a first laser radiation pulse can be used to cause cleavage of the photoreactive moiety, and a second laser radiation pulse can be used to target the desorption membrane, thereby separating the sample from the sample carrier by lifting (or a thermal energy pulse introduced by other means can be used to heat the desorption membrane, thereby causing the sample material to separate from the sample carrier). The first pulse and the second pulse can have different wavelengths. Therefore, in some methods (e.g., involving both ablation and desorption), separation of the sample from the sample carrier may involve multiple laser pulses of different wavelengths. In some cases, cleavage and lifting of the photoreactive moiety can be accomplished by the same laser pulse.

[0330] The sample carrier can include a coating or layer of a chemically reactive substance that imparts kinetic energy to the sample, thereby releasing the sample from the surface. For example, the chemically reactive substance can release a gas, such as, for example, H2, CO2, N2, or hydrochlorofluorocarbons. Examples of such compounds include blowing agents and foaming agents, which release gases upon heating. The generation of gas can be used to transfer kinetic energy to the desorbed sample material, thereby improving the reproducibility and direction of release of the material.

[0331] The sample carrier may contain light-triggered chemical reactants that react exothermically to generate heat to desorb the sample material. The carrier coatings discussed in the paragraph above, or indeed specific chemical linkages in the carrier (which are irradiated by the laser to release the sample material clumps from the carrier) are examples of materials that can be targeted by the wavelength of the laser radiation.

[0332] In devices according to aspects of the present invention, the laser scanning systems discussed above with respect to sampling systems based on laser ablation may also be applied to devices and techniques in which some or all of the sample material is introduced for ionization and analysis by desorption. The advantage of the laser scanning system again stems from the system's ability to rapidly ablate various spots on the sample. Thus, LIFTing can be performed by firing rapid bursts of laser pulses at a sample targeted at, for example, a desorbing membrane, thereby releasing a clump of material from the sample. In doing so, a specific pattern of laser pulses can be used to effectively desorb the clump. One such example is a spiral pattern that moves from the periphery of a cell inward, such as Figure 10 Thus, in some embodiments, desorption is achieved by directing a series of laser radiation pulses onto the sample material to be desorbed in a spiral pattern, optionally delivered in bursts in a series of pulses, such as where the duration of the pulses in the burst is less than 10 -12Typically, when performing ablation, the ablation locations are resolved as separate, non-overlapping spots. However, when desorption is used as a means of introducing sample material into the device, overlapping spots can be used, for example to ensure removal of all desorbed membranes that anchor the sample to the sample carrier at a specific location. The inventors have discovered that desorbing cells using a single laser pulse with a spot size large enough to completely desorb the cells from the sample carrier often results in the breakup of the material mass. Once the sample material mass is broken into smaller pieces, the transient time of the material in the ablated mass increases because the material inevitably spreads as it travels from the chamber from which the sample is desorbed, through the delivery conduit, to the ionization system, and then to the detector. Therefore, if the sample is, for example, a cell smear, maintaining the integrity of the desorbed mass enables the fastest analysis of the ablated mass, which translates to the fastest cell analysis rate. Desorption of individual cells as discrete masses, which typically maintain their integrity until ionization, offers the opportunity to analyze single cells on a slide at rates similar to those used by CyTOF (Fluidigm, CA, USA) to analyze cells in liquid solution. However, desorption of single cells from the slide offers the additional advantage that the cells can first be analyzed visually, thus meaning that cells of interest can be selected and, for example, cells of the wrong cell type can be excluded, thereby improving the efficiency of the analysis. Furthermore, it means that the clumps of material to be desorbed can be selected so that they are actually single cells. Sometimes, when analyzing liquid samples, cells will cluster together as higher polymers, doublets, triplets, or occasionally, due to the sample introduction process, two discrete cells may be analyzed in the same event. As a result, atoms from two or more cells enter the ionization and detection system together, leading not only to inaccurate results, but also to possible damage to the equipment due to overloading of the MS detector. Therefore, single cell analysis by desorption, as provided by the use of laser scanning, without or with minimal disruption of the desorbed clumps, provides an analysis mode that is superior to that known in the art.

[0333] Typically, a sample feature / region of interest does not represent a discrete entity at a discrete location, such as a solitary cell, that is easily isolated for desorption. Instead, the cell of interest may be surrounded by other cells or materials that are not needed or desired for analysis. Therefore, attempting to desorb (e.g., lift) the feature / region of interest may desorb both the cell of interest and the surrounding material. Atoms from surrounding areas of the sample (e.g., from other cells that have been labeled) that are carried in a block of desorbed material other than the specific feature / region of interest (e.g., cell), such as labeled atoms used in elemental labeling (see discussion below), may therefore contaminate the reading of the location of interest.

[0334] Ablation and desorption (such as by lifting) techniques can be combined in a single method. For example, in order to perform precise desorption of a feature / region of interest (e.g., a cell) on a biological sample (e.g., a tissue section sample or a cell suspension dispersion) on a sample carrier, laser ablation can be used to ablate the area surrounding the cell of interest to remove other material. After the surrounding area has been removed by ablation, the feature / region of interest can be desorbed from the sample carrier and then ionized and analyzed by a mass spectrometer according to standard mass spectrometer or mass spectrometer procedures. Consistent with the above discussion, optionally after ablation has been used to remove the area surrounding the location to be desorbed, thermal, photolytic, chemical, or physical techniques can be used to desorb material from the feature / region of interest. Typically, lifting is used to separate the material block from the sample carrier (e.g., a sample carrier on which a desorption film has been coated to assist in the lifting process, as discussed above with respect to the desorption of discrete blocks of sample material).

[0335] Therefore, aspects of the present invention provide a method for analyzing a sample, the method comprising

[0336] (i) desorbing the bulk of sample material using laser radiation, wherein the laser radiation is directed onto the sample on the sample stage using a laser scanning system; and

[0337] (ii) ionizing the bulk of sample material and detecting atoms in the bulk by mass spectrometry.

[0338] The sample may be on a sample carrier, and in some cases, laser radiation is directed through the sample carrier to desorb the mass of sample material from the sample carrier.

[0339] In some embodiments, the method further comprises laser ablation of the sample prior to step (i). Sometimes, ablation of the sample produces one or more plumes of sample material that are individually ionized and the atoms in the plumes are detected by mass spectrometry. Sometimes, the method further comprises an additional step, prior to step (i), of labeling a plurality of different target molecules in the sample with one or more different labeling atom / element tags to provide a labeled sample. In some variations of the method, laser ablation is used to ablate material surrounding the feature / region of interest to clear the surrounding area before desorbing the sample material in the feature / region of interest as a block of material from the sample carrier.

[0340] Features / regions of interest can be identified by another technique before performing laser ablation and desorption (e.g., by lifting). Including a camera (such as a charge-coupled device (CCD) image sensor camera, a CMOS camera, or an active pixel sensor-based camera) or any other light detection device described in the previous section is one way to enable these techniques for both online and offline analysis. The camera can be used to scan the sample to identify cells of particular interest or features / regions of particular interest (e.g., cells of a particular morphology). Once such a location is determined, the location can be lifted after directing the laser pulse to the area surrounding the feature / region of interest to remove other material by ablation before lifting the location (e.g., cell). This process can be automated (the system can identify, ablate, and lift the feature / region of interest) or semi-automated (the user of the system, such as a clinical pathologist, can identify the feature / region of interest, and the system will then perform ablation and lifting in an automated manner). This can significantly increase the speed with which analysis can be performed because, rather than ablating the entire sample to analyze a specific cell, cells of interest can be specifically ablated.

[0341] The camera can record images from a microscope (e.g., a confocal microscope). Identification can be made by optical microscopy, for example by examining cell morphology or cell size, or as to whether the cell is a discrete single cell (as opposed to being a member of a cluster of cells). Sometimes, the sample can be specifically labeled to identify the feature of interest (e.g., a cell). As discussed above with respect to methods of ablating visually identified features / regions of interest, typically, fluorescent markers are used to specifically stain the cells of interest (such as by using labeled antibodies or labeled nucleic acids); for the sake of brevity, this section is not repeated in full here, but those skilled in the art will immediately understand that the features of those methods can be applied to desorption-based methods and that this is within the technical teachings of this document. High-resolution optical images are advantageous in this coupling of optical techniques and lifting because the accuracy of the optical image then determines the accuracy with which the ablation laser source can be directed to ablate the area surrounding the cell of interest, which area can then be desorbed.

[0342] Aspects of the present invention also provide a method for analyzing a sample comprising a plurality of cells, the method comprising the following steps:

[0343] (i) labeling a plurality of different target molecules in a sample with one or more labeling atoms to provide a labeled sample;

[0344] (ii) illuminating the sample to identify one or more features of interest;

[0345] (iii) recording the location of one or more features of interest on the sample;

[0346] (iv) desorbing a mass of sample material from the feature of interest using the position information of the feature of interest, comprising first performing laser ablation to remove sample material surrounding the feature of interest using laser radiation before desorbing the mass of sample material from the location using laser radiation, wherein the laser radiation is directed onto the sample using a laser scanning system;

[0347] (v) ionizing the desorbed bulk sample material; and

[0348] (vi) performing mass spectrometry on the ionized sample material to detect the labeled atoms in the sample material.

[0349] Aspects of the present invention also provide variations of the above methods, for example, a method of performing mass cytometry on a plurality of cells, the method comprising the steps of:

[0350] (i) labeling a plurality of different target molecules in a sample with one or more different labeling atoms and one or more fluorescent labels to provide a labeled sample;

[0351] (ii) irradiating the sample with laser radiation to excite one or more fluorescent labels;

[0352] (iii) recording position information of one or more locations of the sample based on the fluorescence pattern;

[0353] (iv) desorbing a block of sample material from the feature of interest using positional information based on the fluorescence pattern, comprising first performing laser ablation to remove sample material surrounding the feature of interest using laser radiation before desorbing the block of sample material from the location using laser radiation, wherein the laser radiation is directed onto the sample using a laser scanning system;

[0354] (v) ionizing the desorbed bulk sample material; and

[0355] (vi) performing mass spectrometry on the ionized sample material to detect the labeled atoms in the sample material.

[0356] Sometimes, data is not recorded for ablation to clear the area surrounding the location to be desorbed (e.g., the cell of interest). Sometimes, data is recorded based on ablation of the surrounding area. Useful information that can be obtained from the surrounding area includes which target molecules, such as proteins and RNA transcripts, are present in the surrounding cells and intercellular environment. This may be of particular interest when imaging solid tissue samples, in which case direct cell-to-cell interactions are common and proteins expressed in surrounding cells may provide information about the state of the cell of interest.

[0357] Consistent with the above, here desorption of the bulk can be achieved by firing bursts of laser pulses at the sample, guided by a laser scanning system.

[0358] camera

[0359] The camera used in the desorption-based sampling system can be used in the laser ablation-based sampling system as described above, and the discussion of the camera for the laser ablation-based sampling system should be read here.

[0360] Sample Room

[0361] The sample chamber used in the desorption-based sampling system can be used as described above for the laser ablation-based sampling system. In the case of sampling a bulk sample material, one skilled in the art will appreciate that it may be necessary to increase the gas flow rate to ensure that the bulk material is entrained in the gas flow and carried into the transport conduit for delivery to the ionization system.

[0362] delivery catheter

[0363] The sample chamber used in the desorption-based sampling system can be used as described above for the laser ablation-based sampling system. In the case of sampling large blocks of sample material, one skilled in the art will appreciate that the diameter of the catheter lumen will need to be appropriately sized to accommodate any blocks without the blocks contacting the sides of the lumen (as any contact could cause the blocks to fragment and result in signal overlap—causing atoms from the block of the nth desorption event to spread into the detection window of the n+1th or subsequent blocks).

[0364] Ionization system based on desorption system

[0365] In many cases, the lifting techniques discussed above involve removing relatively large pieces of sample material (10 nm to 10 μm, from 100 nm to 10 μm, and in some cases from 1 μm to 100 μm) that have not yet been converted into particulate and gaseous material. Therefore, an ionization technique that can vaporize and atomize such relatively large amounts of material is needed.

[0366] Inductively coupled plasma torch

[0367] One such suitable ionization system is inductively coupled plasma, as already discussed above in the section beginning on page 57 with respect to laser ablation based sampling and ionization systems.

[0368] Optional components for desorption-based sampling and ionization systems

[0369] ion deflector

[0370] Ion deflectors used in desorption-based sampling systems can be used in laser ablation-based sampling systems as described above. Ion deflectors are particularly useful in such systems for protecting detectors, given that desorption-based sampling can potentially remove large, intact pieces of sample material.

[0371] 2. Quality detector system

[0372] Exemplary types of mass detector systems include quadrupole, time-of-flight (TOF), magnetic sector, high resolution, single collector or multiple collector based mass spectrometers.

[0373] The time it takes to analyze the ionized material will depend on the type of mass analyzer used to detect the ions. For example, instruments using a Faraday cup are generally too slow to analyze fast signals. Overall, the desired imaging speed, resolution, and degree of multiplexing will dictate the type of mass analyzer that should be used (and conversely, the choice of mass analyzer will dictate the speed, resolution, and multiplexing that can be achieved).

[0374] Mass spectrometers that detect ions at only one mass-to-charge ratio (m / Q, often referred to as m / z in MS) at a time (e.g., using point ion detectors) will produce poor results in imaging detection. First, the time spent switching between mass-to-charge ratios limits the speed at which multiple signals can be determined. Second, if the ion is less abundant, signals will be lost when the instrument focuses on other mass-to-charge ratios. Therefore, it is preferred to use techniques that provide for the essentially simultaneous detection of ions with different m / Q values.

[0375] Detector type

[0376] Quadrupole detector

[0377] A quadrupole mass analyzer consists of four parallel rods with a detector at one end. Alternating RF potentials and fixed DC offset potentials are applied between one pair of rods and the other, causing one pair of rods (each rod facing the other) to have an alternate potential opposite to that of the other pair. The ionized sample passes through the middle of the rods, parallel to the rods, toward the detector. The applied potential influences the ion trajectories, so that only ions with a specific mass-to-charge ratio have stable trajectories and therefore reach the detector. Ions of other mass-to-charge ratios will collide with the rods.

[0378] Sector magnetic detector

[0379] In a sector magnetic mass spectrometer, the ionized sample flows through a curved flight tube toward an ion detector. A magnetic field across the flight tube deflects the ions from their path. The amount of deflection for each ion is based on its mass-to-charge ratio, so only some ions will collide with the detector—others will be deflected from it. In field instruments with multiple collector sectors, an array of detectors is used to detect ions of different masses. In some instruments, such as the ThermoScientific Neptune Plus and Nu Plasma II, the sector magnetics are combined with electrostatic sectors to provide a dual-focusing sector magnetic instrument that can analyze ions by kinetic energy in addition to mass-to-charge ratio. Specifically, multiple detectors with Mattauch-Herzog geometry can be used (such as the SPECTRO MS, which can simultaneously record all elements from lithium to uranium in a single measurement using semiconductor direct charge detectors). These instruments can measure multiple m / Q signals essentially simultaneously. Their sensitivity can be improved by including an electron multiplier in the detector. However, array sector instruments are always applicable because, although they can be used to detect increasing signals, they are less useful when signal levels decrease, making them less suitable for situations where, in particular, label concentrations are highly variable.

[0380] Time-of-Flight (TOF) detectors

[0381] A time-of-flight mass spectrometer consists of a sample inlet, an acceleration chamber with a strong electric field applied to it, and an ion detector. Ionized sample molecules are introduced through the sample inlet and enter the acceleration chamber. Initially, each ionized sample molecule has the same kinetic energy. However, as the ionized sample molecules are accelerated through the acceleration chamber, they are separated by their respective masses, with lighter ionized sample molecules traveling faster than heavier ions. The detector then detects all ions as they arrive. The time it takes for each particle to reach the detector depends on its mass-to-charge ratio.

[0382] Therefore, the TOF detector can record multiple masses quasi-simultaneously in a single sample. In theory, TOF technology is not suitable for ICP ion sources due to its space charge characteristics, but TOF instruments can actually analyze ICP ion mists quickly and sensitively enough to achieve feasible single-cell imaging. Although TOF mass analyzers are generally not popular for atomic analysis due to the need to deal with the effects of space charge in the TOF accelerator and flight tube, tissue imaging according to the present invention can be effective by detecting only labeled atoms, so other atoms (such as those with an atomic mass below 100) can be removed. This results in a less dense ion beam, which is concentrated in the mass of (for example) the 100-250 Dalton region, which can be more efficiently manipulated and focused, thereby facilitating TOF detection and utilizing the high spectral scan rate of TOF. Therefore, rapid imaging can be achieved by combining TOF detection with the selection of uncommon labeled atoms in the sample, ideally, their mass should be higher than the mass visible in the unlabeled sample, for example, by using higher mass transition elements. Therefore, using a narrower label mass window means that TOF detection is used for effective imaging.

[0383] Suitable TOF instruments are available from Tofwerk, GBC Scientific Instruments (e.g. Optimass 9500 ICP-TOFMS) and Fluidigm of Canada (e.g. CyTOFTM and CyTOF TM 2 instruments). These CyTOF™ instruments have higher sensitivity than Tofwerk and GBC instruments and are known for mass cytometry due to their ability to rapidly and sensitively detect ions in the mass range of rare earth metals, such as lanthanides (particularly in the 100-200 m / Q range).[xi] The mass cytometers of the present application can preferentially detect ions in such a mass range. For example, the apparatus of the present application can be configured to selectively detect the presence of multiple mass tags, such as lanthanide isotopes of the mass tags.

[0384] Therefore, these are the preferred instruments for use with the present disclosure and they can be set up for imaging with instruments known in the art, such as references xii and xiii. Their mass analyzers can detect a large number of labels quasi-simultaneously at high mass spectrometry acquisition frequencies on the time scale of high-frequency laser ablation or sample desorption. They can measure the abundance of labeled atoms with a detection limit of about 100 per cell, allowing for sensitive construction of images of tissue samples. Due to these features, mass cytometry can now be used to meet the sensitivity and multiplexing needs of tissue imaging at subcellular resolution. By combining a mass cytometer with a high-resolution laser ablation sampling system and a high-speed, low-dispersion sample chamber, images of tissue samples can be constructed in a highly multiplexed manner on a practical time scale.

[0385] TOF can be coupled with a mass distribution corrector. Most ionization events produce M + Ions in which a single electron has been removed from an atom. Due to the operating mode of a TOF MS, sometimes ions of one mass (M) can bleed into (or crosstalk with) the channel of an adjacent mass (M ± 1), especially if a large number of ions of mass M enter the detector (i.e., the ion count is high, but not so high that an ion deflector located between the sample ionization system and the MS will prevent them from entering the MS, if the instrument were to include such an ion deflector). + The time when the ion arrives at the detector follows a probability distribution with a mean (each M is known), and when the mass is M + When the number of ions is high, some ions will reach the + or M+1 + The time associated with the ions. However, since each ion has a known distribution curve when it enters the TOF MS, the overlap of ions of mass M into the M±1 channels can be determined based on the peak in the mass M channel (compared to the known peak shape). This calculation is particularly applicable to TOF MS because the ion peaks detected in TOF MS are asymmetric. Therefore, the readings of the M-1, M and M+1 channels can be corrected to properly assign all detected ions to the M channel. Due to the nature of the large ion packets generated by the sampling and ionization systems, such as those disclosed herein involving laser ablation (or desorption as described below) as a technique for removing material from the sample, such corrections have particular use in correcting imaging data. Procedures and methods for improving data quality by deconvolving data from TOF MS are discussed in References xiv, xv and xvi.

[0386] Dead time corrector

[0387] As described above, signals in an MS are detected based on collisions between ions and the detector, as well as collisions between ions and electrons released from the detector surface. When a high ion count detected by the MS results in a large number of electrons being released, the MS detector may temporarily fatigue, resulting in the analog signal output from the detector being temporarily suppressed for one or more subsequent ion packets. In other words, if the ion count in the ionized sample material packet is particularly high, many electrons may be released from the detector surface and secondary multiplier during the detection of ions from the ionized sample material packet. This means that when ions from subsequent ionized sample material packets strike the detector, fewer electrons may be released until the detector surface and secondary multiplier are replenished.

[0388] Based on the characterization of the detector's behavior, this dead time phenomenon can be compensated. The first step is to analyze the ion peaks in the analog signal resulting from the detection of the nth packet of ionized sample material by the detector. The peak amplitude can be determined by the peak height, the peak area, or a combination of the peak height and the peak area.

[0389] The amplitude of the peak is then compared to see if it exceeds a predetermined threshold. If the amplitude is below this threshold, no correction is required. If the amplitude is above the threshold, the digital signal from at least one subsequent ionized sample material package will be corrected (at least the (n+1)th ionized sample material package, but possibly further ionized sample material packages, such as the (n+2), (n+3), (n+4), etc.) to compensate for temporary suppression of the analog signal in these ionized sample material packages due to detector fatigue caused by the nth ionized sample material package. The greater the amplitude of the peak of the nth ionized sample material package, the more corrections will need to be made to the peaks of subsequent ionized sample material packages, and the amplitudes of the corrections will need to be greater. References xvii, xviii, xix, xx and xxi discuss methods for correcting for such phenomena, and these methods can be applied to the data by the dead time corrector, as described herein.

[0390] Analyzer equipment based on optical emission spectroscopy detection

[0391] 1. Sampling and ionization system

[0392] a. Laser ablation-based sampling and ionization system

[0393] Laser ablation sampling systems including the laser scanning systems described above for mass-based analyzers can be used with OES detector-based systems. To detect atomic emission spectra, most preferably, the sample material removed from the sample is ionized using ICP, but any hard ionization technique capable of producing elemental ions can be used.

[0394] As will be appreciated by those skilled in the art, certain optional additional components of the above-described laser ablation-based sampling and ionization system described with respect to avoiding overloading of the mass-based detector may not be applicable to all OES detector-based systems and, if not, will not be incorporated by the skilled artisan.

[0395] b. Desorption-based sampling and ionization systems

[0396] The desorption-based sampling system including the laser scanning system described above for mass-based analyzers can be used in systems based on OES detectors. To detect atomic emission spectra, the sample material removed from the sample is most preferably ionized using ICP, but any hard ionization technique capable of producing elemental ions can be used.

[0397] As will be appreciated by those skilled in the art, certain optional additional components of the above-described desorption-based sampling and ionization system, described with respect to avoiding overloading of a mass-based detector, may not be applicable to all OES detector-based systems and, if not, would not be incorporated by the skilled artisan.

[0398] c. Laser desorption / ionization system

[0399] Laser desorption / ionization-based analyzers typically contain two components. The first is a system for generating ions from the sample for analysis. In this device, this is achieved by directing a laser beam onto the sample to generate ions; in this article, it is referred to as a laser desorption ion generation system. These ejected sample ions (including any detectable ions from the labeled atoms as described below) can be detected by a detector system (the second component), such as a mass spectrometer (the detector will be discussed in more detail below). This technology is called laser desorption / ionization mass spectrometry (LDI-MS). LDI differs from the desorption-based sampling systems discussed in more detail below because in desorption-based sampling systems, the sample material is desorbed as a charged block of neutral material, which is then ionized to form elemental ions. In contrast, here, the ions are generated directly because the laser is irradiated on the sample, so there is no need for a separate ionization system.

[0400] A laser desorption ion generation system comprises: a laser; a sample chamber for holding a sample onto which radiation from the laser is directed; and an ion optical device that can absorb ions generated from the sample and direct them to a detector for analysis. Accordingly, aspects of the present invention provide an apparatus for analyzing a sample, the apparatus comprising: a. a sample chamber for holding a sample; b. a laser adapted to desorb and ionize material from the sample to form ions; c. an ion optical device arranged to sample ions formed by the desorption ionization and direct them away from the sample toward a detector; and d. a detector that receives ions from the ion optical device and analyzes the ions, optionally further comprising a laser scanning system as described above in various aspects of the present invention. In some embodiments, the apparatus comprises a laser adapted to desorb and ionize material from the sample to form elemental ions, and wherein the detector receives elemental ions from the sampling and ionization system and detects the elemental ions. In some cases, LDI is matrix-assisted (i.e., MALDI).

[0401] During this process, some molecules reach energy levels at which they desorb from the sample and become ionized. Ions may arise directly as primary ions due to laser irradiation, or as secondary ions formed by collisions of neutrally charged species with primary ions (e.g., proton transfer, cationization, and electron capture). In some cases, as described below, ionization is aided by compounds (e.g., matrices) added to the sample during sample preparation.

[0402] laser

[0403] A variety of different lasers can be used for LDI, including the commercial lasers discussed above with respect to the lasers of the laser ablation sampling system, which are adapted to desorb ions as needed. Thus, in some embodiments, the apparatus comprises a laser adapted to desorb and ionize material from a sample, forming elemental ions, and wherein the detector receives the elemental ions from the sampling and ionization system and is adapted to detect the elemental ions. Sometimes, the apparatus comprises a laser adapted to desorb and ionize material from a sample, forming molecular ions, and wherein the detector receives the molecular ions from the sampling and ionization system and is adapted to detect the molecular ions. In other cases, the apparatus comprises a laser adapted to desorb and ionize material from a sample, forming elemental ions and molecular ions, and wherein the detector receives ions from the sampling and ionization system and is adapted to detect both elemental ions and molecular ions.

[0404] Exemplary lasers include those emitting at 193 nm, 213 nm, or 266 nm (deep ultraviolet lasers, which can cause ions to be released from a sample without the need for a matrix to promote ionization, as in MALDI). Le Pogam et al. (2016) (Scientific Reports 6, Article No. 37807) describe the desorption of ions representing lichen metabolites from a sample after laser irradiation at 355 nm.

[0405] Femtosecond lasers as described above are also advantageous in certain LDI applications.

[0406] In order to analyze samples quickly, high-frequency ablation is required, for example, greater than 200 Hz (i.e., more than 200 laser shots per second, generating more than 200 ion clouds per second). Typically, the frequency of the ion cloud generated by the laser system is at least 400 Hz, such as at least 500 Hz, at least 1 kHz, at least 10 kHz, at least 100 kHz, or at least 1 MHz. For example, the ablation frequency of the laser system is in the range of 200 Hz-1 MHz, in the range of 500 Hz-100 kHz, or in the range of 1 kHz-10 kHz.

[0407] As explained above with respect to laser ablation sampling systems, laser radiation can be directed to the sample via various optical components and focused to a spot size (i.e., the size of the laser beam when the laser radiation impinges on the sample) of less than 100 μm, such as less than 50 μm, less than 25 μm, less than 20 μm, less than 15 μm, or less than 10 μm or less than 1 μm. When used to analyze biological samples including tissue sections, the spot size of the laser beam used will depend on the size and spacing of the cells in order to analyze individual cells. For example, if single-cell analysis is to be performed, the spot size of the laser spot should be no larger than the cells if the cells are closely packed together (such as in a tissue section). This size will depend on the specific cells in the sample, but typically, the diameter of the laser spot used for LDI should be less than 4 μm, for example, in the range of 0.1-4 μm, 0.25-3 μm, or 0.4-2 μm. To analyze cells with subcellular resolution, the LDI system uses a laser spot size no larger than the cells, and more specifically, a laser beam spot size that can ablate material with subcellular resolution. Sometimes, single-cell analysis can be performed using a spot size larger than the cell size, for example, by spreading the cells on a slide and leaving space between them. Therefore, the specific spot size used should be appropriately selected depending 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 and a constant spot size is maintained throughout the ion generation process, the laser spot size should be smaller than the smallest cell.

[0408] Sometimes, the laser may include a laser scanner as discussed above with respect to laser ablation sampling (see page 8).

[0409] Sample Room

[0410] The sample chamber of an LDI system shares many features with the sample chambers of the aforementioned laser ablation- and desorption-based sampling systems. It contains a stage for supporting the sample. This stage can be a translational stage that can move in the xy or xyz axes. The sample chamber also contains an outlet through which material removed from the sample by laser radiation can be directed. The outlet is connected to a detector, enabling analysis of the sample ions.

[0411] The sample chamber can be at atmospheric pressure. Atmospheric pressure LDI (particularly MALDI) is known. Here, ions generated by auxiliary LDI are transported from the ionization chamber to a high vacuum region for analysis (e.g., MS detector) by a pneumatic flow of a gas (e.g., nitrogen) (Laiko et al., 2000, Anal. Chem., 72:652–657).

[0412] In some cases, the sample chamber is maintained under a vacuum or partial vacuum. Thus, in some cases, the sample chamber pressure is less than 50,000 Pa, less than 10,000 Pa, less than 5,000 Pa, less than 1,000 Pa, less than 500 Pa, less than 100 Pa, less than 10 Pa, less than 1 Pa, about 0.1 Pa, or less than 0.1 Pa, such as less than 0.01 Pa. For example, the partial vacuum pressure can be about 200-700 Pa, and the vacuum pressure is less than 0.2 Pa.

[0413] As will be understood by those skilled in the art, the choice of whether the sample pressure is at atmospheric pressure, under a (partial) vacuum, or not depends on the specific analysis being performed. For example, at atmospheric pressure, sample handling is easier and softer ionization can be applied. Furthermore, the presence of gas molecules may be required to enable the phenomenon of collisional cooling to occur, which may be of concern when the marker is a macromolecule, the fragmentation of which is undesirable, such as a molecular fragment containing the marker atom or a combination thereof.

[0414] Maintaining the sample chamber under vacuum prevents sample ions generated by the LDI from colliding with other particles in the chamber. In some cases, this may be preferable because collisions with gas molecules in the chamber could cause the generated sample ions to lose their charge. This charge loss would prevent the device from detecting them.

[0415] In some embodiments, the sample chamber includes one or more gas ports arranged to deliver one or more gas streams to the location of laser desorption / ionization on the sample during laser desorption / ionization, such as where one or more gas ports are in the form of nozzles. The gas ports (e.g., nozzles) are operable to deliver gas at the time of desorption and ionization to provide collisional cooling for the desorbed ions, but only at that specific time. At other times, they do not introduce gas into the chamber, thereby reducing the strain on the vacuum pump.

[0416] Ion optics

[0417] The sample ion beam is captured from the sample by an electrostatic plate located near the sample, known in the art as an extraction electrode. The extraction electrode removes sample ions desorbed by laser ablation from a localized area of the sample. This is typically achieved by placing the sample and an electrode (sample electrode) on a plate that also functions as a plate, and the extraction electrode at a large voltage potential difference. Depending on the polarity of the sample relative to the extraction electrode, positively or negatively charged secondary ions are captured by the extraction electrode.

[0418] In some embodiments, the charge across the electrodes is constant during laser desorption / ionization. Sometimes, the charge changes after desorption / ionization, such as with delayed extraction, where the accelerating voltage is applied after a short delay following desorption / ionization caused by the laser pulse. This technique can compensate for the time-of-flight spread of ion energies, where ions with higher kinetic energy travel from the sample to the detector faster than ions with lower kinetic energy. This velocity difference can reduce detector resolution because not all ions move at the same speed. Therefore, by delaying the application of the voltage between the sample and extraction electrodes, ions with lower kinetic energy remain closer to the sample electrode when the accelerating voltage is applied, and thus begin accelerating at a greater potential than ions farther from the target electrode. With an appropriate delay, slower ions are accelerated sufficiently to capture ions with higher kinetic energy after laser desorption / ionization at a distance from the pulsed acceleration system. Ions of the same mass-to-charge ratio then drift simultaneously through the flight tube to the detector. Thus, in some embodiments, the sample electrode and extraction electrode are controllable to apply a charge to the electrodes at a set time after the laser is shorted, thereby causing desorption / ionization of the sample.

[0419] The sample ions are then transferred to the detector via one or more other electrostatic lenses (referred to in the art as transfer lenses). The transfer lens focuses the sample ion beam into 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 other ion manipulation components between the electrodes and the detector, such as one or more apertures, mass filters, or a set of deflection plates. The electrodes, transfer lenses, and any other components together constitute the ion optical device. Components for producing suitable ion optical arrangements can be obtained from commercial suppliers, such as Agilent, Waters, Bruker, and can be appropriately positioned by those skilled in the art to deliver ions to the detector, as described below.

[0420] In addition to the detectors discussed below, because LDI can be performed resulting in soft ionization (e.g., ionization that does not disrupt bonds in the molecules being analyzed), in some cases the detector can be a tandem MS, in which ions are selected from the sample by a first m / z separation before the selected ions are broken into fragments and subjected to a second m / z separation (the fragments are then detected).

[0421] Using LDI method

[0422] Aspects of the present invention also provide methods for analyzing biological samples using LDI. In this analysis, cells are labeled with markers, and these markers are then detected in ions produced after the sample is subjected to LDI. Thus, aspects of the present invention provide a method for mass cytometry of a sample comprising a plurality of cells, the method comprising: a. labeling a plurality of different target molecules in the sample with one or more different markers to provide a labeled sample; b. performing laser desorption / ionization of the sample, wherein the laser desorption / ionization is performed at a plurality of locations to form a plurality of separate ion clouds; and c. performing mass spectrometry analysis on each of the ion clouds, whereby detection of the markers in the plume can construct an image of the sample, optionally wherein the plurality of locations are a plurality of known locations.

[0423] In some embodiments, one or more labels comprise a label atom. In this case, the effect of the label is as described below, wherein a member of a specific binding pair (e.g., an antibody bound to a protein antigen, or a nucleic acid bound to RNA in a sample) is attached to an elemental tag (e.g., a lanthanide or actinide) comprising one or more label atoms. The elemental tag may comprise only a single type of label atom (e.g., one or more atoms of a single isotope of a particular element), or may comprise a variety of different label atoms (e.g., different elements / isotopes), such that a large number of different labels generated as a specific combination of elements / isotopes are used as labels. In some cases, the label atom is detected as an elemental ion. In some embodiments, the label atom is emitted from the sample within a molecular ion. Therefore, instead of detecting the label atom in the mass channel, the presence of the label material in the sample will be detected in the mass channel of the molecular ion (i.e., by subtracting the label atom from the mass of the molecule, the mass channel will simply be transformed, relative to a single label atom). However, in some embodiments, the molecule containing the label atom can vary between different label atoms. In that case, the ions containing the molecular residue and the label atom will be subjected to a fragmentation method to produce a more consistent mass peak for each reagent, such as by the application of tandem MS. The goal of all these changes and modifications to the main LDI imaging mass cytometry protocol was to maximize the number of available mass channels while reducing overlap between them.

[0424] In certain embodiments, dyeing reagent can be designed to promote the release and ionization of mass label material and single element ion or molecular ion containing single copy label atom.Dyeing reagent can also be designed to promote the release and ionization of single element ion or molecular ion of mass label material and label atom (or its combination, as described above) containing multiple copies.As another alternative, the quality of dyeing reagent itself can be utilized to create a detection channel for mass cytometry.In this case, rare earth isotopes will not be used in dyeing, and the quality of dyeing reagent is changed by changing the chemical property of dyeing reagent to produce many mass channels.This change can be completed with carbon, oxygen, nitrogen, sulphur, phosphorus, hydrogen and similar isotopes, without the need for rare earth isotopes.

[0425] In some embodiments, the sample is also treated with a laser radiation absorber composition that acts to enhance the sample's absorption of the laser light when irradiated, thereby increasing energy transfer to excite the labeled atoms (and thereby promoting the generation of elemental or molecular ions, or a combination thereof, containing the labeled atoms).

[0426] Numbering Examples Related to LDI

[0427] 1. A device for analyzing a sample, comprising: a. a sample chamber for holding the sample; b. a laser adapted to desorb and ionize material from the sample to form ions; c. an ion optical device arranged to sample ions formed by the desorption ionization and to direct them toward a detector away from the sample; and d. a detector to receive ions from the ion optical device and analyze the ions, optionally wherein the device includes a laser scanning system of aspects of the present invention.

[0428] 2. The apparatus of embodiment 1, wherein the apparatus comprises a laser adapted to desorb and ionize material from the sample to form elemental ions, and wherein the detector receives the elemental ions from the sampling and ionization system and is adapted to analyze the elemental ions.

[0429] 3. The apparatus according to any preceding embodiment, wherein the apparatus comprises a laser adapted to desorb and ionize material from the sample to form molecular ions, and wherein the detector receives the molecular ions from the sampling and ionization system and is adapted to detect the molecular ions.

[0430] 4. The apparatus of any preceding embodiment, wherein the apparatus comprises a laser adapted to desorb and ionize material from the sample to form elemental ions and molecular ions, and wherein the detector receives ions from the sampling and ionization system and is adapted to detect both elemental ions and molecular ions.

[0431] 5. The apparatus according to any preceding embodiment, wherein the laser is a deep UV laser, such as a laser emitting 193 nm, 213 nm or 266 nm radiation.

[0432] 6. The apparatus according to any preceding embodiment, wherein the laser is a femtosecond laser.

[0433] 7. The device according to any preceding embodiment, wherein desorption ionization occurs in the sample chamber under vacuum, partial vacuum or atmospheric pressure.

[0434] 8. An apparatus according to any preceding embodiment, wherein the sample chamber comprises one or more gas ports arranged so as to enable one or more gas pulses to be delivered to the location of laser desorption ionization on the sample during laser desorption ionization, such as wherein the one or more gas ports are in the form of nozzles.

[0435] 9. The apparatus of embodiment 8, wherein the one or more gas ports are arranged to enable one or more gas pulses to collisionally cool ions generated from the sample by the laser radiation from the laser.

[0436] 10. A method for performing mass cytometry on a sample comprising a plurality of cells, comprising: a. labeling one or more different target molecules in the sample with one or more mass tags to provide a labeled sample; b. performing laser desorption ionization on the sample, wherein the laser desorption ionization is performed at a plurality of known positions to form a plurality of ion clouds; and c. performing mass spectrometry on the ion cloud to detect ions from the one or more mass tags in the cloud, allowing an image of the sample to be constructed.

[0437] 11. The method of embodiment 10, wherein the plurality of ion clouds is a plurality of individual ion clouds, each individual ion cloud formed by laser desorption ionization at a known position, and wherein subjecting the ion clouds to mass spectrometry includes subjecting the individual ion clouds to mass spectrometry.

[0438] 12. The method of embodiment 10 or 11, wherein each different target is bound by a different specific binding pair member (SBP), and each different SBP is linked to a mass tag, such that each target is labeled with a specific mass tag.

[0439] 13. The method of any one of embodiments 10 to 12, further comprising treating the sample with an ionization promoter composition before step a, or between steps a and b.

[0440] 14. The method of embodiment 13, wherein the ionization promoter composition promotes ionization of the labeled atom and / or ionization of molecular ions containing the labeled atom.

[0441] 15. The method of any one of embodiments 10 to 14, further comprising treating the sample with a laser radiation absorber composition before step a, or between steps a and b.

[0442] 2. Photodetector

[0443] Example types of photodetectors include photomultiplier tubes and charge coupled devices (CCDs).Photodetectors can be used to image a sample and / or identify features / regions of interest prior to imaging by an elemental mass spectrometer.

[0444] A photomultiplier tube consists of a vacuum chamber containing a photocathode, several dynodes, and an anode. Due to the photoelectric effect, photons incident on the photocathode cause it to emit electrons. Due to a process called secondary emission, the electrons are multiplied by the dynodes to produce a multiplied electron current. This multiplied electron current is then detected by the anode, providing a means of detecting electromagnetic radiation incident on the photocathode. Photomultiplier tubes are available, for example, from ThorLabs.

[0445] A CCD consists of a silicon chip containing an array of light-sensitive pixels. During exposure, each pixel generates an electric charge proportional to the intensity of the light incident on it. After exposure, control circuitry generates a series of charge transfers to produce a series of voltages. These voltages can then be analyzed to generate an image. Suitable CCDs are available, for example, from Cell Biosciences.

[0446] Build the image

[0447] The above apparatus can provide signals for multiple atoms in ionized sample material packets removed from a sample. Detection of an atom in the sample material packet reveals its presence at the ablation location, either because the atom is naturally present in the sample or because the atom has been localized at that location by a labeling reagent. By generating a series of ionized sample material packets from known spatial locations on the sample surface, the detector signals can reveal the positions of the atoms on the sample, and these signals can therefore be used to construct an image of the sample. By labeling multiple targets with distinguishable labels, the positions of the labeled atoms can be correlated with the positions of homologous targets, and thus the method can construct complex images with a level of multiplexing far exceeding that achievable using traditional techniques (such as fluorescence microscopy).

[0448] Assembling the signals into an image will use a computer and can be accomplished using known techniques and software packages. For example, the GRAPHIS software package from Kylebank Software can be used, or other software packages such as TERAPLOT can be used. Imaging using MS data from techniques such as MALDI-MSI is known in the art, for example, reference xxii discloses an "MSiReader" interface for viewing and analyzing MS imaging files on a Matlab platform, and reference xxiii discloses two software tools for rapid data exploration and visualization of 2D and 3D MSI data sets over full spatial and spectral resolution, such as the "Datacube Explorer" program.

[0449] Images obtained using the methods disclosed herein can be further analyzed, for example, in the same manner as IHC results. For example, images can be used to depict cell subpopulations in a sample and can provide information useful for clinical diagnosis. Similarly, SPADE analysis can be used to extract a cellular hierarchy from the high-dimensional cytometry data provided by the disclosed methods [xxiv]. In some aspects, cell types (e.g., identified by SPADE analysis) can be colored to allow for simultaneous visualization of multiple cell types (at least some of which are characterized by a combination of markers).

[0450] Alternatively or additionally, serial sections can be imaged and stacked to provide a 3D image of the sample by imaging mass cytometry. A large number of labeled atoms can be integrated across features or regions of interest (ROIs) in 2 or 3 dimensions, such as across cells, cell clusters, micrometastases, tumors, or tissue subregions. In certain aspects, laser scanning can be performed to quickly analyze such features or ROIs on one or more tissue sections. Such integration of signals can simplify analysis and / or improve sensitivity.

[0451] Multiple imaging modes

[0452] Multiple imaging modalities can be used to image one or more tissue slices. In some cases, slices from the same tissue can be imaged separately using different modalities and then co-registered (e.g., mapped to the same coordinate system, stacked, overlaid, and / or combined to identify higher-level features).

[0453] Aspects of the present invention include a method for co-registering images, the method comprising: obtaining a first image from a first tissue section of a tissue sample using an imaging modality other than imaging mass cytometry, obtaining a second image from a second tissue section of the tissue sample using imaging mass cytometry, and co-registering the first and second images. In certain aspects, the first image, or both the first and second images, may be provided by a third party.

[0454] In some cases, the imaging mass cytometer can be equipped with other modes of imaging, including but not limited to optical microscopy, such as bright field, fluorescence and / or nonlinear microscopy. For example, the imaging mass cytometer can stack optical devices for laser ablation and optical microscopy. Histochemical stains can be imaged by optical microscopy to identify regions of interest (ROIs) for analysis by imaging mass cytometry. Alternatively or additionally, as described herein, an optical microscope can be used to co-register images obtained from a first tissue slice by imaging mass cytometry with images obtained from a second tissue slice (e.g., serial slices) by another mode (e.g., by another system). When a high-speed (e.g., femtosecond) laser is used, nonlinear microscopy can be performed at one or more harmonics to image the structural aspects of the sample. When an antibody is labeled simultaneously with a marker atom and a fluorophore label, analyzing the distribution of the fluorophore label may be non-destructive to the sample, and then the marker atom can be subjected to IMC analysis. In some aspects, the fluorophore label can be a fluorescent barcode that is cut (e.g., photo-cut) from the region of interest and analyzed after aspiration.

[0455] In some cases, an additional imaging mode can be an electron microscope, such as a scanning electron microscope or a transmission electron microscope. Typically, an electron microscope comprises an electron gun (e.g., with a tungsten cathode), an electrostatic / electromagnetic lens, and an aperture (which controls the beam to direct it onto a sample in a sample chamber). The sample is kept under vacuum so that gas molecules do not obstruct or diffract the electrons on their way from the electron gun to the sample. In a transmission electron microscope (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.

[0456] 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 prepared using an ultramicrotome are 100 nm or thinner. Biological tissue samples are chemically fixed, dehydrated, and embedded in a polymer resin to stabilize them sufficiently for ultrathin sectioning. Sections of biological specimens, organic polymers, and similar materials may need to be stained with heavy atom labels to achieve the desired image contrast, because unstained biological specimens, in their natural unstained state, rarely interact strongly with electrons to deflect them for the image to be recorded by electron microscopy.

[0457] As described 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 (e.g., transmission electron microscopy), which can then be used to refine the resolution of the image data obtained by IMS or IMC to a resolution beyond that achievable using laser radiation ablation (due to the much shorter wavelength of electrons than photons). In some cases, both electron microscopy and elemental analysis by IMC or IMS are performed on a sample in a single instrument (because IMC / IMS are destructive processes, electron microscopy is performed before IMC / IMS).

[0458] One or more tissue sections can be analyzed by imaging mass cytometry and one or more other imaging modalities and co-registered based on a reference (such as a coordinate system) present on the slide holding the tissue sections. Alternatively or additionally, co-registration can be performed by aligning features (e.g., structures or patterns) present on two sections from the same tissue. Features can be identified by the same or different imaging modalities. Even when identified by the same imaging modality, features or their x, y coordinates can be used to co-register different imaging modalities.

[0459] In some aspects, other imaging modes are MALDI mass spectrometry imaging. The sample preparation of the tissue slice for MALDI imaging may be incompatible with the preparation for imaging mass cytometry. Thus, the MALDI imaging of the first slice can be co-registered with the imaging mass cytometry of the second slice (e.g., serial slices) from the same tissue. Laser desorption ionization in MALDI imaging can provide molecular ions detected by mass spectrometry. The MALDI image of the sample can identify the distribution of analytes (e.g., drugs, such as cancer drugs, potential cancer drugs or their metabolites) in tissue slices or subregions comprising tumors and / or healthy tissues. When the analyte is a drug, it can be applied to a subject (e.g., a human patient or an animal model), from which tissue samples are collected for analysis, as described herein. Other identical analytes can be isotopically labeled, such as using non-naturally abundant isotopes (e.g., isotopes of H, C or N) to carry out isotopic labeling, and then applied to tissue together with matrix to identify and expect the peak in the mass spectrum associated with the original analyte. Alternatively or in addition to imaging the distribution of analytes, MALDI images can also provide the distribution of endogenous biomolecules (or their molecular ions). MALDI images can be co-registered with IMC images using shared or similar histochemical stains (such as cresol violet, Ponceau S, bromophenol blue, ruthenium red, trichrome stain, osmium tetroxide, etc.). In some aspects, labeled atoms of a sample analyzed by MALDI imaging can survive the process, allowing IMC analysis. However, MALDI sample preparation may complicate sample preparation for IMC imaging, in which case MALDI and IMC images can be obtained from different tissue sections.

[0460] Co-registration of MALDI images with mass spectrometry images can provide greater understanding of the tissue portions that retain the drug and / or the drug's effect on the tissue. For example, metal-containing histochemical stains, active agents, and / or cell state indicators can identify whether the drug targets connective tissue (e.g., stroma, extracellular matrix, or macromolecules (such as collagen or glycoproteins, fibrin (such as actin), keratin, tubulin), cells or cell subregions (e.g., cell membrane, cytoplasm, and / or nucleus), proliferating cells, live or dead cells, hypoxic cells or regions, necrotic regions, tumor cells, or cells with tumor characteristics (e.g., a combination of surface markers and / or cell state marker characteristics of a tumor), and / or at least one of healthy tissue. In some cases, the effect of a drug can be inferred by combining drug distribution (e.g., identified by MALDI imaging) and tissue status at or around the drug (e.g., identified by imaging mass cytometry). For example, the number, location, cell type, and distribution of tumor cells or tumor-infiltrating immune cells can be determined. Cell activity surface markers, intracellular signal transduction markers, cell type markers can be used to identify the effects of drugs and / or identify other drug targets (such as receptors that are upregulated or downregulated in tumor cells or tumor-infiltrating immune cells in response to drugs). Tumor-infiltrating immune cells may include one or more of dendritic cells, lymphocytes (such as B cells, T cells and / or NK cells) or immune cell subsets (such as CD4+, CD8+ and / or CD4+CD25+T cells). In some cases, imaging mass cytometry can identify multiple immune cell types in the tumor microenvironment and can further identify cell states (e.g., intracellular signaling and / or expression of receptors involved in activation or inhibition of immune responses). The drug distribution area of MALDI imaging can identify ROIs for imaging cytometry analysis and / or co-register with mass cytometry images.

[0461] In certain aspects, co-registration of the IMC image with the non-IMC image provides the distribution of multiple (e.g., at least 5, 10, 20, or 30) different targets (e.g., or their associated labeled atoms) at cellular or subcellular resolution. The IMC image can be obtained by LA-ICP-MS and optionally by using a femtosecond laser and / or laser scanning system as described herein.

[0462] Co-registration can include mapping (e.g., aligning) two images (obtained by different imaging modalities) to each other (e.g., a shared coordinate system). The two co-registered images (or aspects of each image) can be superimposed or combined to reveal higher-level features, such as the common expression of two objects detected by the two different imaging modalities. In some aspects, co-registration may be performed only in the region of interest.

[0463] sample

[0464] Certain aspects of the present disclosure provide methods for imaging biological samples. Such samples can contain a plurality of cells, and imaging mass cytometry (IMC) can be performed on the cells to provide images of these cells in the sample. Generally, aspects of the present invention can be used to analyze tissue samples currently studied by immunohistochemistry (IHC) techniques, but using labeled atoms suitable for detection by mass spectrometry (MS) or optical emission spectroscopy (OES).

[0465] In some aspects, sample can comprise multiple sections (for example, continuous tissue sections). In some aspects, tissue sections can be frozen (for example, frozen) and / or embedded with wax (for example paraffin) before sectioning. Any sectioning method known to those skilled in the art can be used, although most sectioning methods relate to using a sharp blade applied at a certain angle to cut tissue samples, and then the resulting tissue sections are mounted on a solid support (such as a slide). Sections from the same tissue (for example, continuous sections) can be imaged by imaging mass cytometry and / or different modes, and as described herein, are co-registered with each other. When the penetration of dye and / or imaging mode only allow the top layer of the tissue section to be analyzed, tissue sectioning may relate to preparing two continuous sections that are dyed and / or imaged on one side facing each other. For example, a section can flip so that it presents a face adjacent to another section. When identifying ROI based on the first section, and / or when co-registering images from two sections, the image obtained from a section may flip. Alternatively or additionally, serial sections can be aligned with the benchmarks on the corresponding slides (or on the same slides) so that their approximate positions relative to each other are retained or represented before sectioning. Any suitable tissue sample can be used for the methods described herein. For example, tissue can include one or more tissues or any other tissues from epithelium, muscle, nerve, skin, intestine, pancreas, kidney, brain, liver, blood (e.g., blood smear), bone marrow, cheek brush, cervical brush. The biological sample can be an immortal cell line or a primary cell obtained from a living organism. For diagnosis, prognosis, or experimental (e.g., drug development) purposes, the tissue can be from a tumor. In certain embodiments, the sample can be from a known tissue, but whether the sample comprises tumor cells may be unknown. Imaging can reveal the presence of a target that indicates the presence of a tumor, thereby facilitating diagnosis. Tissue from a tumor can include immune cells also characterized by the present method, and an understanding of tumor biology can be provided. The tissue sample can include formalin-fixed, paraffin-embedded (FFPE) tissue. Tissue can be obtained from any living multicellular organism, such as a mammal, an animal research model (eg, a model of a particular disease, such as an immunodeficient rodent bearing a human tumor xenograft), or a human patient.

[0466] The tissue sample can be, for example, a slice having a thickness in the range of 2-10 μm, such as between 4-6 μm. The technology for preparing such slices is well known in the field of IHC, for example using a microtome, including a dehydration step, fixation, embedding, permeabilization, sectioning, etc. Thus, the tissue can be chemically fixed and then sections can be prepared in the desired plane. Cryosectioning or laser capture microdissection can also be used to prepare tissue samples. The sample can be permeabilized, for example to allow the uptake of reagents for labeling intracellular targets (see above).

[0467] The size of the tissue sample to be analyzed will be similar to that of current IHC methods, although the maximum size will be determined by the laser ablation device, specifically the size of the sample that can fit into its chamber. A typical maximum size is 5 mm × 5 mm, but smaller samples (e.g., 1 mm × 1 mm) are also useful (these dimensions refer to the size of the slice, not its thickness).

[0468] In addition to being useful for imaging tissue samples, the present disclosure can also be used to image cell samples, such as monolayer adherent cells or cells immobilized on a solid surface (as in conventional immunocytochemistry). These embodiments are particularly useful for analyzing adherent cells that are not readily soluble for cell suspension mass cytometry. Thus, the present disclosure can be used to enhance not only current immunohistochemical analyses, but also immunocytochemistry.

[0469] Serial sectioning and resampling

[0470] In certain aspects, serial sections of tissue can be analyzed by imaging mass cytometry. Serial sections can be stained identically or for different markers. For example, a first serial section can be stained for a protein marker (or primarily a protein marker), while a second serial section can be stained for an RNA marker (or primarily an RNA marker). This capability is particularly useful when sample preparation for one set of markers (such as antigens used to retrieve protein markers) may damage or impair the ability to detect another set of markers (such as RNA markers).

[0471] Multiple consecutive sections can be stained with different sets of SBPs containing the same or overlapping mass tags. Alternatively, consecutive sections can be stained with the same or overlapping sets of SBPs containing the same or overlapping mass tags. Markers present on features shared across consecutive sections can be integrated or otherwise combined for analysis. For example, identical markers detected across subsequent sections in a feature such as a cell (e.g., bound by the same SBP) can be summed to determine expression in that feature. This can provide greater sensitivity and may be particularly useful for detecting and / or determining the abundance of low-expressed markers. Features such as cells may be larger than a single section or may be divided into multiple sections. Various methods can be used to slice thin sections down to the micron level. Dehydration of the sections during sample preparation, combined with the depth of laser ablation, can remove a significant portion of the section thickness to be ablated. If the thickness of the section is significantly greater than the depth of laser ablation, resampling at a location can yield more material from the feature to be analyzed. Lasers with short, intense pulses (e.g., fs lasers) can more cleanly sample the sample (e.g., with minimal heat outside the ablation site), thus enabling better resampling. As described above, resampling and / or analysis of multiple serial slices can allow for higher sensitivity. Additionally, resampling and / or analysis of multiple serial slices can allow for reconstruction of 3D mass cytometry images.

[0472] In some aspects, identification of features can be performed during optical interrogation and the laser can be scanned along the optically identified feature of interest. Alternatively, features can be identified from a pixel-by-pixel mass cytometry image, such as an array of pixels on the micrometer scale (e.g., 0.5 to 2 micrometers in diameter). Pixels associated with a feature can be identified during the analysis phase and signals from markers in that feature can be integrated. Laser scanning along the feature, grouping pixels (obtained by stage translation and / or laser scanning) into features, resampling at a location and / or integrating features across consecutive sections can be arbitrarily combined to improve the sensitivity of markers associated with the feature. When laser scanning is applied, significant time savings can be achieved, which becomes even more valuable when analyzing consecutive sections.

[0473] IMC offers inherent advantages over immunohistochemistry or immunofluorescence microscopy because there is little or no overlap in signals from metal labels, enabling simultaneous imaging of more than 40 proteins (and / or other markers) from a single tissue section. In some cases, IMC can be less sensitive than other methods. For example, based on antibodies labeled with 100 atoms and the typical transmittance of ICP-TOF-MS, the detection limit of conventional IMC might be 400 copies of the antibody per 1-micron diameter laser spot (pixel). Features such as cells may be larger than 10, 20, 50, or 100 square microns. In conventional IMC, tissue 3-10 microns thick (i.e., 5-7 microns thick) is typically dried to a thickness of 1 micron or less, which is the approximate limit for complete ablation for typical laser energies used in IMC (assuming 1 microjoule at the laser head). If there are not many cells, some initial sections may be thicker. As a result, tissue sections often contain cell fragments rather than intact cells. It is important to note that varying laser speed, wavelength, and energy may alter these assumptions. In some cases, fast (eg, fs) lasers may allow resampling and "drilling" into thicker tissue sections.

[0474] Interrogating features of cells such as those of the cell through IMC may result in reduced detection of low-abundance markers, which may be evenly distributed (e.g., throughout the cytoplasm) and whose abundance in a portion of the cell may be lower than in the entire cell. In addition, certain markers may be underexpressed in specific portions of the cell, as some markers may be present in specific cellular compartments. For example, in a particular tissue section, a cell nucleus (e.g., detectable by an iridium nucleic acid intercalator) may be completely present, completely absent, or present in a portion thereof. As a result, it may be completely detected with a good signal-to-noise ratio, partially detected, or not detectable at all / not present at all; similarly, protein markers may be detected, partially detected, or not detectable at all, depending on their presence in the cellular compartment / section. Even for markers above the detection threshold, higher sensitivity may improve or allow for qualitative or quantitative assessment of the abundance of the marker.

[0475] As described herein, a method or system can measure the main markers present in cells in high abundance, measured in continuous tissue sections. The main marker signals can then be used to identify objects / segment cell-like objects representing specific cells in each cross section, or to form typical cell phenotypes present in each tissue section. The marker signature or cell phenotype can then be linked to the XY coordinates of each identified object. Objects with similar main marker characteristics / phenotypes and close XY coordinates are then linked to each other as fragments of the same cell that was sliced during the sectioning process. Once the objects in the continuous sections are identified as representing the same cell, the signals of all markers between the continuous tissue sections are integrated (e.g., summed), thereby effectively generating a "volume integral" of the marker signals. Since the sum of the marker signals may be proportional to the number of summed slices, and the background signal will be proportional to the square root of the number of summed slices, this improves the signal-to-noise ratio.

[0476] Furthermore, where a particular cell compartment (or a marker in a compartment) is not present in one tissue slice, it may be present in the previous or next slice of the same tissue block. Thus, the detection of some markers can be increased many folds, or even achieved. A variety of methods for identifying that primary marker signatures belong to the same cell can be used, including methods known in the art of image segmentation (e.g., watershed methods). While the above examples are provided for cells, the method can be used for any feature described herein. Features having similar properties (such as shape and / or marker expression) and / or having similar XY coordinates with a similar set of surrounding features can be identified as belonging to the same cell feature (e.g., cell) after such segmentation.

[0477] Sample carrier

[0478] In certain embodiments, the sample can be fixed on a solid support (i.e., a sample carrier) to position it for imaging mass spectrometry. The solid support can be optically transparent, for example made of glass or plastic. In the case where the sample carrier is optically transparent, it enables ablation of sample material through the support, such as Figure 5 Sometimes, the sample carrier will include features that serve as reference points for use with the devices and methods described herein, for example to allow calculation of the relative position of features / regions of interest to be ablated or desorbed and analyzed. The reference points may be optically resolvable or may be resolvable by mass analysis.

[0479] Target element

[0480] In imaging mass spectrometry, it may be necessary to pay attention to the distribution of one or more target elements (i.e., elements or element isotopes). In some aspects, the target element is a labeled atom as described herein. The labeled atom can be added to the sample alone or covalently bound to or within the bioactive molecule. In certain embodiments, the labeled atom (e.g., metal tag) can be conjugated to a member of a specific binding pair (SBP), such as an antibody (combined with its cognate antigen), an aptamer or an oligonucleotide hybridized with a DNA or RNA target, as described in more detail below. The labeled atom can be connected to the SBP by any method known in the art. In some aspects, the labeled atom is a metal element, such as a lanthanide or transition element or another metal tag, as described herein. The mass of the metal element can be greater than 60 amu, greater than 80 amu, greater than 100 amu or greater than 120 amu. The mass spectrometer described herein can consume elemental ions lower than the mass of the metal element so that a large amount of lighter elements will not produce space charge effects and / or overload the mass detector.

[0481] Labeling of tissue samples

[0482] The present disclosure produces an image of a sample that has been labeled with a marker atom, for example, a plurality of different marker atoms, where the marker atoms are detected by an apparatus capable of sampling a specific, preferably subcellular region of the sample (thus, the marker atoms represent elemental labels). Reference to a plurality of different atoms refers to labeling the sample with more than one atomic species. A mass detector can be used to distinguish between these atomic species (e.g., they have different m / Q ratios) such that the presence of two different marker atoms in the plume will produce two different MS signals. A spectrometer can also be used to distinguish between atomic species (e.g., different atoms have different emission spectra) such that the presence of two different marker atoms in the plume will result in two different emission spectral signals.

[0483] Reagents with mass labels

[0484] As used herein, a mass-tagged reagent comprises a number of components. The first is the SBP. The second is the mass tag. The mass tag and the SBP are connected by a linker, formed at least in part by the conjugation of the mass tag and the SBP. The connection between the SBP and the mass tag may also comprise a spacer. The mass tag and the SBP can be conjugated together through a range of reaction chemicals. Exemplary conjugation reaction chemicals include thiolmaleimides, NHS esters and amines, or click chemistry reactants (preferably Cu(I)-free chemicals), such as strained alkynes and azides, strained alkynes and nitrones, and strained alkenes and tetrazines.

[0485] Quality Label

[0486] The mass tags (also referred to as element tags) used in the present invention can take a variety of forms. Typically, the tags contain at least one tag atom. Tag atoms are discussed below.

[0487] Thus, a mass tag can, in its simplest form, comprise a metal-chelating moiety, which is a metal-chelating group in which the metal label atom is coordinated in a ligand. In some cases, it is sufficient for each mass tag to detect only a single metal atom. However, in other cases, it may be desirable for each mass tag to contain more than one label atom. This can be achieved in a variety of ways, as described below.

[0488] The first method of producing a mass tag that can comprise more than one labeled atom is to use a polymer that comprises a metal chelate ligand connected to more than one subunit of a metal. The number of metal chelate groups that can be combined with at least one metal atom in the polymer can be between about 1 and 10,000, such as 5-100, 10-250, 250-5000, 500-2500 or 500-1000. At least one metal atom can be combined with at least one metal chelate group. The degree of polymerization of the polymer can be between about 1 and 10,000, such as 5-100, 10-250, 250-5000, 500-2500 or 500-1000. Therefore, the mass tag based on the polymer can comprise about 1 to 10,000, such as 5-100, 10-250, 250-5000, 500-2500 or 500-1000 labeled atoms.

[0489] The polymer may be selected from the group consisting of linear polymers, copolymers, branched polymers, graft copolymers, block polymers, star polymers, and hyperbranched polymers. The backbone of the polymer may be derived from substituted polyacrylamides, polymethacrylates, or polymethacrylamides, and may be a substituted derivative of a homopolymer or copolymer of acrylamide, methacrylamide, acrylate, methacrylate, acrylic acid, or methacrylic acid. The polymer may be synthesized by the group consisting of reversible addition fragmentation polymerization (RAFT), atom transfer radical polymerization (ATRP), and anionic polymerization. The step of providing the polymer may comprise synthesizing the polymer from a compound selected from the group consisting of N-alkyl acrylamides, N,N-dialkyl acrylamides, N-aryl acrylamides, N-alkyl methacrylamides, N,N-dialkyl methacrylamides, N-aryl methacrylamides, methacrylates, acrylates, and functional equivalents thereof.

[0490] The polymer can be water-soluble. This part is not limited by chemical content. However, if the skeleton has a relatively reproducible size (such as length, number of tag atoms, reproducible dendrimer characteristics, etc.), the analysis can be simplified. The requirements for stability, solubility and non-toxicity are also taken into account. Therefore, functional water-soluble polymers are prepared and characterized by a synthetic strategy that places many functional groups and different reactive groups (linking groups) along the main chain. The reactive groups can be used to connect the polymer to a molecule (such as SBP) through a linker and an optional spacer. The size of the polymer can be controlled by controlling the polymerization reaction. Generally, the size of the polymer will be selected so that the gyroradiation of the polymer is as small as possible, such as between 2 and 11 nanometers. The length of the IgG antibody (an exemplary SBP) is about 10 nanometers. Therefore, relative to the size of the SBP, an overly large polymer tag may spatially interfere with the binding of the SBP to its target.

[0491] The metal chelating group capable of binding at least one metal atom may comprise at least four acetic acid groups. For example, the metal chelating group may be a diethylenetriamine pentaacetate (DTPA) group or a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group. Alternative groups include ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0492] The metal chelating group may be linked to the polymer via an ester or via an amide.Examples of suitable metal chelating polymers include X8 and DM3 polymers available from Fluidigm, Canada.

[0493] The polymer can be water-soluble. Due to their hydrolytic stability, N-alkyl acrylamide, N-alkyl methacrylamide and methacrylate or functional equivalents can be used. A degree of polymerization (DP) of about 1 to 1000 (1 to 2000 main chain atoms) covers most target polymers. Larger polymers have the same functionality within the scope of the present invention and are feasible, as those skilled in the art will appreciate. Typically, the degree of polymerization will be between 1 and 10,000, such as 5-100, 10-250, 250-5000, 500-2500 or 500-1000. The polymer can be synthesized by a route that results in relatively narrow polydispersity. The polymer can be synthesized by atom transfer radical polymerization (ATRP) or reversible addition fragmentation (RAFT) polymerization, and its Mw (weight average molecular weight) / Mn (number average molecular weight) value should be within the range of 1.1 to 1.2. An alternative strategy involving anionic polymerization, in which polymers having Mw / Mn of about 1.02 to 1.05 can be obtained. Both methods can control the end groups by selecting initiators or terminators. This allows the synthesis of polymers that can be connected to joints. A strategy can be adopted to prepare polymers containing functional side groups in the repeating unit, to which the transition metal unit of the ligand (e.g., Ln unit) can be attached in a subsequent step. This embodiment has several advantages. It avoids the complexity that may arise from the polymerization of ligand-containing monomers.

[0494] To minimize the charge repulsion between side groups, (M 3+ ) should impart a net charge of -1 on the chelate.

[0495] Polymers useful in various aspects of the present invention include:

[0496] Random copolymers poly(DMA-co-NAS): Relógio et al. (2004) (Polymer, 45, 8639-49) reported the RAFT synthesis of random copolymers of N-acryloyloxysuccinimide (NAS) and N,N-dimethylacrylamide (DMA) in a 75 / 25 molar ratio with high conversion, excellent molar mass control (ranging from 5000 to 130,000), and Mw / Mn ≈ 1.1. Reactive NHS esters were reacted with metal chelating groups bearing reactive amino groups to produce metal chelating copolymers synthesized via RAFT polymerization.

[0497] - Poly(NMAS):N MAS can be polymerized by ATRP to obtain polymers with average molar masses of 12 to 40 kDa and Mw / Mn of about 1.1 (see, for example, Godwin et al., 2001; Angew. Chem. Int. Ed, 40:594-97).

[0498] - Poly(MAA): Polymethacrylic acid (PMAA) can be prepared by anionic polymerization of its tert-butyl or trimethylsilyl (TMS) esters.

[0499] Poly(DMAEMA): Poly(dimethylaminoethyl methacrylate) (PDMAEMA) can be prepared by ATRP (see Wang et al., 2004, J. Am. Chem. Soc, 126, 7784-85). This is a well-known polymer that can be easily prepared with average Mn values between 2 and 35 kDa and an Mw / Mn of approximately 1.2. This polymer can also be synthesized by anionic polymerization with a narrow particle size distribution.

[0500] - Polyacrylamide or polymethacrylamide.

[0501] The metal chelating group can be attached to the polymer by methods known to those skilled in the art, for example, the side group can be attached via an ester or via an amide. For example, for a methyl acrylate-based polymer, the metal chelating group can be attached to the polymer backbone by first reacting the polymer with ethylenediamine in methanol and then reacting DTPA anhydride in a carbonate buffer under alkaline conditions.

[0502] The second approach is to produce nanoparticles that can be used as mass tags. The first approach to producing such mass tags is to use nanoscale particles of metals that have been coated in polymers. Here, the metal is isolated by the polymer and isolated from the environment, and when the polymer shell can be made to react, for example, by being incorporated into a functional group in the polymer shell, the metal does not react. The functional group can react with a linker component (optionally containing a spacer) to attach a click chemistry reagent, thus allowing this type of mass tag to be inserted into the synthesis strategy discussed above in a simple, modular manner.

[0503] Incorporation and grafting are the two main mechanisms for producing polymer brushes around nanoparticles. In the incorporation method, the polymer is synthesized separately, so the synthesis is not limited by the need to maintain colloidal stability of the nanoparticles. Reversible addition-fragmentation chain transfer (RAFT) synthesis is particularly advantageous due to the wide variety of monomers and the ease of functionalization. Chain transfer agents (CTAs) can be easily used as the functional group themselves, functionalized CTAs can be used, or the polymer chains can be post-functionalized. The polymer is attached to the nanoparticles using chemical reactions or physical adsorption. A disadvantage of the incorporation method is the generally low grafting density due to steric repulsion of the coiled polymer chains during attachment to the particle surface. All incorporation methods have the disadvantage that rigorous post-treatment is required to remove excess free ligands from the functionalized nanocomposite particles. This is usually achieved by selective precipitation and centrifugation. In the grafting method, molecules (such as initiators for atom transfer radical polymerization (ATRP) or CTAs for (RAFT) polymerization) are fixed to the particle surface. A disadvantage of this method is the development of new initiator coupling reactions. Furthermore, in contrast to the intercalation method, the particles must be colloidally stable under the polymerization conditions.

[0504] Another approach to mass tagging is through the use of doped beads. Chelated lanthanide (or other metal) ions can be used in miniemulsion polymerization to produce polymer particles with chelated lanthanide ions embedded in the polymer. As known to those skilled in the art, the chelating group is selected so that the solubility of the metal chelate in water is negligible, while the solubility in the monomers used for miniemulsion polymerization is reasonable. Typical monomers that can be used, as known to those skilled in the art, are styrene, methylstyrene, and various acrylates and methacrylates. To improve mechanical strength, the glass transition temperature (Tg) of the metal-tagged particles is above room temperature. In some cases, core-shell particles are used, in which the metal-containing particles prepared by miniemulsion polymerization serve as seed particles for seeded emulsion polymerization to control the nature of the surface functionality. By selecting appropriate monomers for this second stage polymerization, surface functionality can be introduced. In addition, acrylate (and possibly methacrylate) polymers are preferred over polystyrene particles because the ester groups can bind or stabilize unsatisfactory ligand sites on the lanthanide complex. An exemplary method for preparing such doped beads is: (a) combining a complex containing at least one marker atom in a solvent mixture containing at least one organic monomer (such as styrene and / or methyl methacrylate, in one embodiment) in which the complex containing at least one marker atom is soluble, and at least one different solvent in which the organic monomer and the complex containing at least one marker atom are not readily soluble, (b) emulsifying the mixture of step (a) for a sufficient time to provide a uniform emulsion; (c) initiating a polymerization reaction and continuing the reaction until a substantial portion of the monomer is converted to polymer; and (d) incubating the product of step (c) for a sufficient time to obtain a latex suspension of polymer particles having the complex containing at least one marker atom incorporated into or on the particles, wherein the complex containing at least one marker atom is selected such that different mass signals are obtained from the at least one marker atom when the polymer mass signature is examined. By using two or more complexes containing different marker atoms, doped beads comprising two or more different marker atoms can be prepared. In addition, the ratio of the complexes containing different marker atoms is controlled to allow the preparation of doped beads having different ratios of marker atoms. By using multiple label atoms, and in varying ratios, the number of distinctly identifiable mass labels is increased. In core-shell beads, this can be achieved by incorporating a complex containing a first label atom into the core and a complex containing a second label atom into the shell.

[0505] Another approach is to create polymers that include a labeling atom in the polymer backbone rather than as a coordinating metal ligand. For example, Carerra and Seferos (Macromolecules 2015, 48, 297-308) disclose methods for incorporating tellurium into the polymer backbone. Other polymers that incorporate atoms that can serve as labeling atoms include tin, antimony, and bismuth. Such molecules are particularly discussed in Priegert et al., 2016 (Chem. Soc. Rev., 45, 922-953).

[0506] Thus, a mass tag can comprise at least two components: a labeling atom and a polymer that chelates, contains, or incorporates the labeling atom. Furthermore, the mass tag also comprises a linker group (when not bound to an SBP) that, upon chemical reaction between the two components, forms part of the chemical bond between the mass tag and the SBP in a click chemistry reaction, as discussed above.

[0507] Polydopamine coating can be used as another way that SBP is connected to doped beads or nanoparticles. Given the functional range in polydopamine, SBP can be conjugated to a mass tag, which is formed by the beads or particles coated with PDA by the reaction of, for example, amino or sulfhydryl groups on SBP (such as antibodies). Alternatively, the functional groups on PDA can be reacted with reagents such as bifunctional linkers, which introduce further functional groups to react with SBP in turn. In some cases, the linker can contain a spacer, as described below. These spacers increase the distance between the mass tag and the SBP, thereby minimizing the steric hindrance of the SBP. Therefore, various aspects of the present invention include SBP with a mass tag, a mass tag comprising SBP and containing polydopamine, wherein polydopamine comprises at least a portion of the connection between the SBP and the mass tag. Nanoparticles and beads (particularly nanoparticles and beads coated with polydopamine) can be used for signal enhancement to detect low-abundance targets because they can have thousands of metal atoms and can have multiple copies of the same affinity reagent. The affinity reagent can be a second antibody, which can further enhance the signal.

[0508] Label atoms

[0509] Labeled atoms that can be used with the present disclosure include any substance that is detectable by MS or OES and that is substantially absent from an unlabeled tissue sample. Thus, for example, 12 C atoms are not suitable as labeling atoms due to their natural abundance. 11C can be used for MS because it is an artificial isotope that occurs naturally. Usually, the marker atom is a metal. However, in a preferred embodiment, the marker atom is a transition metal, such as a rare earth metal (15 kinds of lanthanides, plus scandium and yttrium). These 17 elements (which can be distinguished by OES and MS) provide many isotopes that are easy to distinguish (by MS). Various of these elements can be obtained in the form of enriched isotopes, for example samarium has 6 kinds of stable isotopes and neodymium has 7 kinds of stable isotopes, all of which can be obtained in enriched form. 15 kinds of lanthanides can provide at least 37 kinds of isotopes with non-redundant unique masses. Examples of elements suitable for use as labeling atoms include lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc) and yttrium (Y). In addition to rare earth metals, other metal atoms are also suitable for detection, such as gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), bismuth (Bi), etc. Radioactive isotopes are not preferred because they are less convenient to handle and are unstable. For example, among the lanthanides, Pm is not a preferred labeling atom.

[0510] To facilitate time-of-flight (TOF) analysis (as discussed herein), it is helpful to use label atoms with atomic masses in the range of 80-250, for example, in the range of 80-210 or in the range of 100-200. This range includes all lanthanides, but excludes Sc and Y. The range of 100-200 allows, by using different label atoms, theoretically 101 clusters of analysis while taking advantage of the high spectral scan rate of TOF MS. As described above, by selecting label atoms with masses that are in a window above the masses visible in unlabeled samples (e.g., in the range of 100-200), TOF detection can be used to provide rapid imaging at biologically significant levels.

[0511] In some embodiments, the mass label of the present invention can be connected to a single SBP member.Depending on the mass label used (and the number of label atoms of each mass label) and the mass label number that each SBP is connected, various number of label atoms can be connected to a single SBP member.When more label atoms are connected to any SBP member, higher sensitivity can be...

Claims

1. An apparatus for analyzing a biological sample, the apparatus comprising: (i) a sampling and ionization system comprising a sampling system and an ionization system, the sampling system comprising a laser source, a laser scanning system, and a sample stage, and the ionization system being adapted to receive material removed from the sample by the laser system and to ionize the material to form elemental ions; (ii) a detector that receives the elemental ions from the sampling and ionization system and detects the elemental ions; in, The laser scanning system includes a positioner capable of imparting a first relative movement to the laser beam emitted by the laser source relative to the sample stage; wherein the laser scanning system further includes a second positioner capable of imparting a second relative movement to the laser beam emitted by the laser source relative to the sample stage, wherein the first relative movement and the second relative movement are not parallel.

2. The apparatus of claim 1 , the second positioner of the laser scanning system further capable of rastering the laser beam relative to the sample stage.

3. The device according to claim 1, wherein The laser scanning system has a response time 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 1 ns.

4. The apparatus according to claim 1, wherein The positioner and / or the second positioner is: (i) a mirror-based positioner, wherein the mirror includes a galvanometer mirror, a MEMS mirror, a polygon scanner, a piezoelectric device mirror and / or (ii) a solid-state positioner, wherein the solid-state positioner includes an acousto-optic device (AOD) or an electro-optic device (EOD).

5. The device according to claim 4, wherein The laser scanning system comprises: (i) the positioner of an EOD in which two sets of electrodes have been orthogonally connected to a refractive medium; or (ii) the positioner and the second positioner are in the form of two orthogonally arranged AODs; or (iii) the positioner and the second positioner are in the form of a galvanometer mirror pair.

6. The device according to claim 4 or 5, wherein: The laser scanning system comprises: (i) the positioner being the galvanometer mirror and the second positioner being the AOD; (ii) the positioner for the galvanometer mirror and the second positioner for the EOD; (iii) said positioner and said second positioner being in the form of a galvanometer mirror pair and further comprising an AOD; or (iv) The positioner and the second positioner are in the form of a galvanometer mirror pair and further include an EOD.

7. The apparatus according to claim 4, wherein The AOD refractive medium is formed of a material selected from the group consisting of tellurium dioxide, fused silica, lithium niobate, arsenic trisulfide, tellurite glass, lead silicate, Ge 55 As 12 S 33 , monovalent mercury chloride and divalent lead bromide.

8. The apparatus according to claim 4, wherein The EOD refractive medium is formed of a material selected from the following: KTN (KTa x Nb 1-x O3), LiTaO3, LiNbO3, BaTiO3, SrTiO3, SBN (Sr 1-x Ba x Nb2O6)、BSKNN(Ba 2-x Sr x K 1- y Na y Nb5O 15 ) and PBN(Pb 1-x Ba x Nb2O6).

9. The device according to claim 1 further comprises: at least one dispersion compensator between the locator and / or the second locator and the sample, the dispersion compensator being suitable for compensating for any dispersion caused by the locator when the locator is an AOD and / or when the second locator is an AOD.

10. The apparatus according to claim 1, wherein The sample stage is movable at least in an x-axis, and wherein the positioner is adapted to introduce a deflection at least in a y-axis into a path of the laser beam onto the sample stage.

11. The apparatus according to claim 10, wherein: (i) the positioner is further adapted to introduce a deflection in the x-axis into the path of the laser beam onto the sample stage; or (ii) the apparatus comprises a second positioner adapted to introduce a deflection in an x-axis into a path of the laser beam onto the sample stage; The positioner of the laser scanning system is controlled by a control module, which also controls the movement of the sample stage.

12. The apparatus according to claim 1, wherein The laser source is a picosecond laser or a femtosecond laser.

13. The apparatus of claim 1 , wherein: (i) an ablation rate of 200 Hz or more; and / or (ii) a laser repetition rate of at least 1 kHz, wherein the sampling system further comprises a pulse picker, wherein the pulse picker is controlled by a control module that also controls movement of the sample stage and / or the positioner of the laser scanning system.

14. The apparatus according to claim 1, wherein The laser source is adapted to produce a spot size having a diameter of or less than 10 μm, less than 5 μm, less than 2 μm, 1 μm, or less than 1 μm.

15. The device of claim 1, further comprising a camera.

16. The apparatus according to claim 1, wherein The ionization system is ICP.

17. The apparatus according to claim 1, wherein The detector is a TOF mass spectrometer.

18. The apparatus according to claim 1, wherein The apparatus is configured to selectively detect the presence of a plurality of mass tags, wherein the mass tags comprise lanthanide isotopes.

19. A method for analyzing a sample, the method comprising: (i) performing laser ablation of a sample on a sample stage, wherein laser radiation is directed onto the sample using a laser scanning system, and wherein ablation is performed at a plurality of locations to form a plurality of plumes; and (ii) ionizing and mass spectrometrically analyzing the plume to detect atoms in the plume to allow construction of an image of the sample, wherein the plurality of locations is a plurality of known locations; in, The method also includes controlling a positioner in the laser scanning system to impart a first relative movement to the laser beam emitted by the laser relative to the sample stage; and a second positioner capable of imparting a second relative movement to the laser beam emitted by the laser relative to the sample stage, wherein the first relative movement and the second relative movement are non-parallel.

20. A method for performing mass cytometry on a sample comprising a plurality of cells, the method comprising: (i) labeling a plurality of different target molecules in the sample with one or more different labeling atoms to provide a labeled sample; (ii) performing laser ablation of the sample on a sample stage, wherein laser radiation is directed onto the sample using a laser scanning system, and wherein ablation is performed at a plurality of locations to form a plurality of plumes; and (iii) ionizing and mass spectrometrically analyzing the plume to detect atoms in the plume to allow construction of an image of the sample, wherein the plurality of locations is a plurality of known locations; in, The method also includes controlling a positioner in the laser scanning system to impart a first relative movement of a laser beam emitted by a laser relative to the sample stage; and controlling a second positioner in the laser scanning system, the second positioner being capable of imparting a second relative movement of the laser beam relative to the sample stage, wherein the first relative movement and the second relative movement are non-parallel.

21. The method according to claim 20, wherein: a. subjecting one or more of the plumes to said ionization and mass spectrometry analysis; and / or b. Generating one or more plumes from known locations.

22. The method according to claim 20, wherein The plumes from adjacent known locations, where ablation is performed at one or more features of interest in the sample, and the plumes from the adjacent known locations all originate from the one or more features of interest, are analyzed as a single event.

23. The method according to claim 22, wherein Adjacent spots are less than 10 times the diameter of the spot size of the laser radiation used to ablate the sample.

24. The method according to claim 20, wherein The method includes controlling the positioner in the laser scanning system to impart a second relative movement to the laser beam relative to the sample stage, wherein the first relative movement and the second relative movement are non-parallel.

25. The method of claim 20 , comprising: moving the sample in a first direction by controlling movement of the sample stage; and introducing a relative movement in a second direction in the laser radiation beam relative to the sample by controlling the positioner of the laser scanning system, wherein the first direction and the second direction are not parallel, and wherein the area scanned is larger than the area that could be scanned without moving the sample stage.

26. The method of claim 20, comprising: moving the sample in the X-axis by controlling movement of the sample stage; and introducing a relative movement in the Y-axis in the laser radiation beam compared to the sample by controlling the positioner of the laser scanning system.

27. The method according to claim 26, wherein The laser scanning system also introduces a relative movement in the X-axis in the laser radiation compared to the sample, thereby maintaining a regular grid pattern of ablation spots on the sample.

28. The method of claim 20, comprising performing 3D imaging of the sample, wherein at least a portion of the sample is ablated to a first depth using laser ablation, and then a portion of the sample is ablated to a second depth by ablating the sample exposed to the first depth.

29. The method according to claim 28, wherein The focal length is controlled to effect a change in ablation depth, and / or wherein the sample stage moves the sample in a Z-axis to effect a change in sample depth.

30. The method according to claim 20, wherein The positioner and / or the second positioner is: (i) a mirror-based positioner, wherein the mirror includes a galvanometer mirror, a MEMS mirror, a polygon scanner, a piezoelectric device mirror and / or (ii) a solid-state positioner, wherein the solid-state positioner includes an acousto-optic device (AOD) or an electro-optic device (EOD).

31. The method of claim 20, comprising controlling a laser that produces laser radiation and the positioner of the laser scanning system to produce bursts of laser radiation pulses directed to locations on the sample, wherein a plume produced by the bursts of laser radiation pulses is ionized and detected as a continuous event.

32. The method according to claim 31, wherein The burst of laser radiation comprises at least three laser pulses, wherein a duration between each laser pulse is shorter than 1 ms.

33. The method according to claim 32, wherein The burst of laser radiation comprises at least 10, at least 20, at least 50, or at least 100 laser pulses.

34. The method according to any one of claims 31 to 33, wherein The positioner is: (i) an EOD in which two sets of electrodes have been orthogonally connected to a refractive medium; or (ii) two orthogonally arranged AODs, wherein the method further comprises controlling the intensity of the laser radiation beam via the AODs.

35. The method of claim 20, wherein: The method comprises the steps of identifying one or more features of interest on the sample, recording positional information of the one or more features of interest on the sample, and ablating the sample, wherein the positional information of the one or more features of interest is used to direct the laser radiation onto the sample using the laser scanning system to form one or more plumes.

36. The method according to claim 35, wherein The plumes from the feature of interest are analyzed as continuous events.

37. The method according to claim 35 or 36, wherein Features are identified by examining an optical image of the sample, wherein the sample has been labeled with a fluorescent marker and the sample is illuminated under conditions such that the fluorescent marker fluoresces.

38. The method according to claim 19 or 20, wherein Performing laser ablation includes rastering laser radiation, and the ablation is performed at a plurality of locations of one of the plurality of cells.

39. A method for analyzing a sample, the method comprising using the apparatus for analyzing a biological sample according to claim 1 to perform the following steps: (i) desorbing the mass of sample material using laser radiation, wherein the laser radiation is directed onto the sample on the sample stage using a laser scanning system; and (ii) ionizing a bulk of said sample material and detecting atoms in said bulk by mass spectrometry; Wherein laser ablation is used to ablate material around the feature of interest to clear the surrounding area before desorbing the sample material at the feature of interest from the sample carrier as a block of material.

40. A method for performing mass cytometry on a sample comprising a plurality of cells, the method comprising performing the following steps using the apparatus for analyzing a biological sample according to claim 1: (i) labeling a plurality of different target molecules in a sample with one or more different labeling atoms to provide a labeled sample; (ii) desorbing the mass of sample material with laser radiation, wherein the laser radiation is directed onto the sample on the sample stage using a laser scanning system; as well as (iii) ionizing a bulk of said sample material and detecting atoms in said bulk by mass spectrometry; Wherein laser ablation is used to ablate material around the feature of interest to clear the surrounding area before desorbing the sample material at the feature of interest from the sample carrier as a block of material.

41. The method of claim 40, wherein desorption is achieved by directing a series of pulses of the laser radiation onto the sample material to be desorbed, wherein: a. a series of pulses of laser radiation in the form of a spiral onto the sample material, wherein the series of pulses is delivered as a burst; and / or b. The series of pulses are within known positions on the sample.

42. The method according to claim 41, wherein The burst of laser radiation comprises at least three laser pulses, wherein a duration between each laser pulse is shorter than 1 ms.

43. The method according to claim 42, wherein The burst of laser radiation comprises at least 10, at least 20, at least 50, or at least 100 laser pulses.

44. The method of claim 40, wherein The method includes controlling a positioner in the laser scanning system to impart a first relative movement to a laser beam emitted by a laser with respect to the sample stage.

45. The method of claim 44, wherein the method comprises controlling the positioner in the laser scanning system to impart a second relative movement to the laser beam relative to the sample stage, wherein the first relative movement and the second relative movement are non-parallel.

46. The method of claim 44, wherein The method includes controlling a second positioner in the laser scanning system to impart a second relative movement to the laser beam relative to the sample stage, wherein the first relative movement and the second relative movement are non-parallel.

47. The method of claim 46, wherein The positioner and / or the second positioner are: (i) a mirror-based positioner, the mirror comprising a galvanometer mirror, a MEMS mirror, a polygon scanner, a piezoelectric device mirror and / or (ii) a solid-state positioner, the solid-state positioner comprising an acousto-optic device (AOD) or an electro-optic device (EOD), wherein the laser scanning system comprises: (a) the positioner being the galvanometer mirror and the second positioner being the AOD; (b) the positioner for the galvanometer mirror and the second positioner for the EOD; (c) said positioner and said second positioner being in the form of a galvanometer mirror pair and further comprising an AOD; or (d) The positioner and the second positioner are in the form of a galvanometer mirror pair and further include an EOD.

48. The method of claim 40, wherein The method comprises the steps of identifying one or more features of interest on the sample, recording positional information of the one or more features of interest on the sample, and desorbing sample material from the sample, wherein the laser radiation is directed onto the sample using the laser scanning system, and the positional information of the one or more features of interest is used to desorb a block of material from the one or more features of interest.

49. The method according to claim 48, wherein Features are identified by examining an optical image of the sample, wherein the sample has been labeled with a fluorescent marker and the sample is illuminated under conditions such that the fluorescent marker fluoresces.

50. The method of claim 40, wherein The sample is on a sample carrier comprising a desorption membrane layer between the sample and the sample carrier, and the laser radiation is directed onto the desorption membrane to desorb sample material.

51. The method of claim 40, further comprising the method of any one of claims 19 to 33 and 35 to 36.

52. A laser scanning system for use in any one of methods 19 to 51.

53. A method for co-registering images, the method comprising performing the following steps using the apparatus for analyzing a biological sample according to claim 1: a) acquiring a first image from a first tissue section of the tissue sample by an imaging modality other than imaging mass cytometry; b) obtaining a second image of a second tissue section of the tissue sample by imaging mass cytometry; c) co-registering the first image and the second image, in, In addition to imaging mass cytometry, the imaging modality is non-linear microscopy.

54. A method of imaging mass cytometry, the method comprising using the apparatus for analyzing a biological sample according to claim 1 to perform the following steps: identifying features in the sample using optical microscopy; scanning radiation across the feature to produce a plume of material; delivering the plume of material to a mass analyzer; Ionize the material by ICP, in, The feature is a cell.

55. The method of claim 54, wherein the sample comprises mass-tagged SBP.

56. The method of claim 54, further comprising analyzing more than 100 single cells per second.

57. The method of claim 54, wherein: The radiation is laser radiation.

58. The method of claim 54, wherein The mass analyzer comprises a TOF detector.

59. An apparatus for performing the method of claim 54.

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