WO2016090356A1 - Mass cytometry imaging - Google Patents

Abstract

The inventor has improved LA-ICP-MS to facilitate its use for imaging of biological samples. Additionally, mass cytometry systems having imaging capability less than 1 micron and high sensitivity are disclosed.

Description

MASS CYTOMETRY IMAGING

CROSS-REFERENCE TO RELATED APPLICATOINS

[0001] This application claims priority to and the benefit of US Provisional Patent Application 62/097,406 filed December 29, 2014 and US Provisional Patent Application 62/088,306 filed December 5, 2014, the contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] The present disclosure relates to mass cytometry systems and the imaging of samples using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS).

BACKGROUND

[0003] Single-cell measurements and multiplexed quantitative detection of molecular targets can provide insights on the state and behavior of individual cells. Techniques which have been used for single-cell analysis include microscopy in combination with specific labelling techniques (e.g., using immunocytochemistry), single-cell imaging mass

spectrometry, surface-enhanced Raman scattering spectroscopy, and mass cytometry based on LA-ICP-MS.

[0004] Some of these techniques can readily be extended to in situ imaging of tissues (e.g., using immunohistochemistry), but others cannot. For instance, approaches such as immunofluorescence microscopy (IFM) are useful for imaging down to around 50 nanometer resolutions, but are limited in practice to measuring seven or fewer targets simultaneously. In contrast, Imaging Mass Cytometry based on LA-ICP-MS offers highly multiplexed quantitative analysis of antigen expression in single cells.

[0005] It is an object of the disclosure to provide further and improved apparatuses and techniques for imaging of samples, and in particular to adapt LA-ICP-MS for use as a subcellular resolution and/or single-cell imaging technique.

SUMMARY

[0006] In a first aspect, LA-ICP-MS systems are provided, comprising: a solid state laser; an ablation chamber comprising a stage; an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to ablate the biological sample on the stage by focusing a beam from the solid state laser to a spot size of less than 2 μιη; an inductively coupled plasma torch; and a mass spectrometer.

[0007] In a second aspect, LA-ICP-MS systems are provided, comprising: a laser; an ablation chamber comprising a stage; an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to ablate the biological sample on the stage by focusing a beam from the laser to a spot size of less than 2 μιη; an inductively coupled plasma torch; a mass spectrometer; and a camera directed on the sample stage.

[0008] In a third aspect, LA-ICP-MS systems are provided, comprising: a laser; an ablation chamber comprising a stage, wherein the stage for holding the sample is mobile within the ablation chamber; an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to ablate the biological sample on the stage by focusing a beam from the laser to a spot size of less than 2 μιη; an inductively coupled plasma torch; and a mass spectrometer.

[0009] In a fourth aspect, methods of imaging a biological sample comprising a plurality of biological cells using an LA-ICP-MS system of the present disclosure are provided.

[0010] In a fifth aspect, methods of imaging a biological sample comprising a plurality of cells are provided, the method comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating the sample with a laser to excite the one or more fluorescent labels; (iii) recording locational information of the areas of the sample which fluoresce; (iv) using the locational information of where fluorescence occurs to direct laser ablation sequentially at multiple known locations, to form a plurality of plumes; and (v) subjecting plumes to inductively coupled plasma mass spectrometry, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated.

[0011] In a sixth aspect, methods of imaging a biological sample comprising a plurality of cells and/or tissues are provided, the method comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating a known location of the sample with light to excite the one or more fluorescent labels; (iii) observing and recording whether there is fluorescence at the location; (iv) if there is fluorescence, directing laser ablation at 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 further known locations on the sample, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated.

[0012] In a further aspect, mass cytometry systems are provided, comprising a laser ablation apparatus; an injector operably coupled to the laser ablation apparatus; a scanning TOF mass spectrometer operably coupled to the injector to provide a sensitivity sufficient to detect 500 copies of affinity molecules in each pixel; wherein, the laser ablation apparatus is configured to provide a sample spatial resolution of less than 1 μιη².

[0013] In a still further aspect, methods of scanning a mass cytometry sample are provided, comprising staining a sample with a fluorescence label and one or more mass tags; irradiating a first area of the sample with a first radiation to provide a first fluorescence signal; detecting the first fluorescence signal; determining whether an intensity of the first fluorescence signal satisfies a criteria; irradiating the sample with a second radiation to cause the sample to ablate, if the criteria is satisfied; and injecting the ablated sample into a mass cytometer to measure the mass tag.

[0014] In some aspects, the imaging mass cytometer may include a laser and optics system that provides a beam spot size of 1 μιη, or less than 1 μιη, such as 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm, or less than 100 nm. Optionally, the one or more lenses of the beam optics system includes a Schwarzschild Cassegrain objective lens; and/or one or more apertures including a diaphragm or an array of substitutable apertures.

[0015] In some embodiments, the laser may be a solid state laser. The solid state laser may be Nd doped laser, such as an Nd:YAG laser; and/or may emit a beam of wavelength 213 nm. In some embodiments, the laser may emit a beam with a Gaussian profile or an approximately Gaussian profile. In some embodiments, the imaging mass cytometer may be configured to ablate the biological sample with a non-homogenized beam. Optionally, the imaging mass cytometer may be configured to ablate the biological sample with a beam having a Gaussian or an approximately Gaussian profile.

[0016] Optionally, embodiments of the imaging mass cytometry system may be adapted to ablate the sample at a rate of more than 40 Hz, such as 50 Hz or more, 100 Hz or more, 200 Hz or more, 300 Hz or more, 400 Hz or more, 500 Hz or more, or 1000 Hz or more.

[0017] In further embodiments of the present invention, system may be provided that includes a laser; an ablation chamber comprising a stage; an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to ablate the biological sample on the stage by focusing a beam from the laser to a spot size of less than 2 um; an inductively coupled plasma torch; a mass spectrometer; and a camera directed on the sample stage.

[0018] In some embodiments, the system may further include a camera directed on the sample stage. The camera may be connected to a microscope, optionally a confocal microscope. In certain embodiments the focal point of the camera or microscope is the focal point of the laser beam.

[0019] In still further embodiments, the system may include a laser or LED light source directed on the sample stage for exciting a sample on the stage of the system. The laser that is for exciting the sample in the system may be the laser that is for ablating the sample.

[0020] Some embodiments may include an optical filter and/or an array of lenses located between the camera and the sample.

[0021] In some embodiments, a system may be provided where the system includes: a laser; an ablation chamber comprising a stage, wherein the stage for holding the sample is mobile within the ablation chamber; an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to ablate the biological sample on the stage by focusing a beam from the laser to a spot size of less than 2 μιη; an inductively coupled plasma torch; and a mass spectrometer.

[0022] A stage may be provided for holding a sample to be ablated. The stage for holding the sample may be mobile within the ablation chamber. In some embodiments, the stage for holding a sample may be mobile in the x,y and z axes within the ablation chamber. Further, in some embodiments, the stage can be moved in increments of less than 10 μιη, such as less than 5 μιη, less than 4 um, less than 3 μιη, less than 2 μιη, 1 μιη, or less than 1 μιη, in the x and y axes; and/or the z axis. Optionally, the range of movement of the stage in the x and/or y axis, or horizontal plane, is at least 40 μιη, at least 50 μιη, at least 75 μιη, at least 100 μιη, at least 250 μιη, at least 500 μιη, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 30 mm, at least 50 mm, or at least 75 mm.

[0023] In some embodiments, a gap of at least 10 mm is maintained between the side wall of the ablation chamber and the edge of the sample stage. In some embodiments, the MS may be a time-of-flight MS.

[0024] In further embodiments, ablation may occur in an ablation chamber having a washout time of 50 ms or less, such as 25 ms or less or 10 ms or less.

[0025] In some aspects, a method of imaging a biological sample comprising a plurality of cells using an LA-ICP-MS system or imaging mass cytometry system is provided using system embodiments described herein.

[0026] In some aspects, a method of imaging a biological sample comprising a plurality of cells is provided that may include: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating the sample with a laser to excite the one or more fluorescent labels; (iii) recording locational information of the areas of the sample which fluoresce; (iv) using the locational information of where fluorescence occurs to direct laser ablation sequentially at multiple known locations, to form a plurality of plumes; and

(v) subjecting plumes to inductively coupled plasma mass spectrometry, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated.

[0027] In some aspects, a method of imaging a biological sample comprising a plurality of cells is provided that may include: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating a known location of the sample with light to excite the one or more fluorescent labels; (iii) observing and recording whether there is fluorescence at the location; (iv) if there is fluorescence, directing laser ablation at 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 further known locations on the sample, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated.

[0028] Optionally, the laser used for laser ablation may be (i) a laser emitting light at 213 nm, such as an Nd:YAG laser; or (ii) an excimer or exciplex laser.

[0029] The light that is used to excite the one or more fluorescent labels may be from the laser that ablates material from the biological sample.

[0030] At least 20 (e.g., at least 30, or at least 50) different labelling atoms having different atomic masses may be used. The labelling atoms may be transition metals such as lanthanides, optionally the labelling atoms may have atomic masses within the range 80-250 amu.

[0031] Methods and systems described herein may use time-of- flight detection for the mass spectrometer. The target molecules may be quantified.

[0032] Embodiments of the mass cytometry system may include a control module for performing any of the methods described herein.

[0033] In some embodiments, a mass cytometry system may be provided that includes: a laser ablation apparatus; an injector operably coupled to the laser ablation apparatus; a scanning TOF mass spectrometer operably coupled to the injector; wherein, the laser ablation apparatus is configured to provide a sample spatial resolution of less than 1 μιη and to provide a sensitivity of at least 500 copies of affinity molecules per ablation area. [0034] The system may be an imaging mass cytometry system. The laser ablation apparatus may output a laser characterized by an emission wavelength within a range of 190 nm to 300 nm. Optionally, the laser ablation apparatus comprises a laser characterized by an emission wavelength of 213 nm. In some embodiments, the laser may be configured to provide a pulse duration less than 30 ns. The laser may be configured to provide a pulse duration less than 1 nsec.

[0035] In some embodiments, system may be characterized by a sensitivity of 500 affinity molecules per ablation area. The TOF mass spectrometer may be characterized by a scan rate of at least 200 kHz.

[0036] Optionally, the laser ablation apparatus may be configured to provide an ablation area on a surface of a sample of 100 100 (10⁴) μιη² to 1000 1000 (10⁶) μιη². A scanning TOF mass spectrometer may include an orthogonal accelerator configured to provide a voltage from 700 V to 1000 V. In some embodiments, the scanning TOF mass spectrometer comprises a straight TOF section. The system may be configured to provide a sampling rate of at least 1 kHz.

[0037] A gas flow into the injector may be at least 1 slpm. The gas flow may include helium. The injector may include an injection tube. The injection tube may have a tip having diameter less than 1 mm. Optionally, the tip diameter may be about 0.85 mm.

[0038] The system may be configured to provide simultaneous measurement of from 100 to 150 mass tags in some embodiments. The system may be configured to provide simultaneous measurement of from 20 to 30 mass tags. The TOF mass spectrometer may optionally be configured to provide measurement of a mass range from 75 amu to 214 amu.

[0039] The system may be characterized by a dynamic range from 4 to 5 orders of magnitude.

[0040] The laser ablation apparatus may include a laser ablation chamber and a sample mounted within the laser ablation chamber. The sample may include a plurality of mass probes. Each of the plurality of mass probes may include an affinity probe coupled to a nanoparticle. A plurality of the mass tags may be coupled to each of the nanoparticles.

[0041] The plurality of mass tags may be at least 400.

[0042] Optionally, the laser ablation apparatus may include an ablation chamber and a plasma torch. The injector may have a first end and a second end. The second end may be inserted into the plasma torch. In some embodiments, the system may include a sampling tube. The sampling tube may extend from the ablation chamber to the first end of the injector. The sampling tube may have a length from 30 mm to 300 mm. Optionally, the sampling tube may be flexible.

[0043] In further aspects, a method of scanning a mass cytometry sample may be provided that may include: staining a sample with a fluorescence label and a mass tag;

obtaining a fluorescence image of a first region characterized by a first area of the sample; selecting a second region characterized by a second area of the sample based on the fluorescence image, wherein the second area is less than the first area; and obtaining a mass cytometry image of the second region.

[0044] In some embodiments, a method for imaging a biological sample may be provided. The method may include receiving the biological sample on a stage in an ablation chamber, the biological sample labeled with labeling atoms specific for target molecules of the biological sample; ablating at least a portion of the biological sample labeled with labeling atoms at a location with a laser emitted from a solid-state laser to eject the portion of the biological sample, the laser emitted from the solid-state laser may be focused onto the portion of the biological sample with a spot size of 2 μιη or less; receiving the ejected portion of the biological sample into an inductively coupled plasma to atomize and ionize the ejected portion of the biological sample; and analyzing the atomized and ionized portion of the biological sample to detect labeling atoms ejected from the location on the biological sample.

[0045] Optionally, the biological sample is ablated using a non-homogenized laser from the solid-state laser. In some embodiments, the biological sample may be ablated using a Gaussian or approximately Gaussian laser from the solid-state laser.

[0046] The stage may be moveable within the ablation chamber. The stage may be moveable in increments of less than 2 μιη in the x and y axes within the ablation chamber. The method may further include translating the stage so as to move the biological sample relative to a focal point of the solid-state laser such that the focal point of the solid-state laser is moved to a second portion of the biological sample labeled with labeling atoms; and ablating the second portion of the biological sample at a second location with a laser emitted from the solid-state laser to eject the second portion of the biological sample. The laser emitted from the solid-state laser may be focused onto the second portion of the biological sample with a spot size of less than 2 μιη.

[0047] Ablation of the second portion of the biological sample may be triggered by fluorescent properties of the biological sample at the second location. The second location of the biological sample may be labeled with fluorescent tags. The solid-state laser may be configured to excite the fluorescent tags at the second location. [0048] The laser emitted from the solid-state laser may be focused onto the portion of the biological sample with a 1 μιη or less spot size, in some embodiments. The laser emitted from the solid-state laser may be focused using one or more apertures. The one or more apertures may include a diaphragm or an array of substitutable apertures. The solid-state laser may be an Nd doped laser. Optionally, the solid-state laser may emit a laser having a 213 nm wavelength.

[0049] The method may further include labeling the biological sample with labeling atoms by attaching an affinity probe to the target molecule. The affinity probe may be coupled with a nanoparticle comprising the labeling atoms.

[0050] In some aspects, a method for analyzing a biological sample may be provided, the method may include: receiving the biological sample on a stage in an ablation chamber, the biological sample labeled with labeling atoms specific for target molecules of the biological sample; ablating at least a portion of the biological sample labeled with labeling atoms at a location with a laser emitted from a laser system to eject the portion of the biological sample; and analyzing the ejected portion of the biological sample to detect labeling atoms ejected from the location on the biological sample. The laser emitted from the laser system may be focused onto the portion of the biological sample with a spot size of 2 μιη or less.

[0051] The biological sample may be ablated using a non-homogenized laser from the laser system. Optionally, the biological sample may be ablated using a Gaussian or approximately Gaussian laser from the laser system.

[0052] Reference is now made in detail to certain embodiments of compounds, compositions, and methods. The disclosed embodiments are not intended to be limiting of the claims. To the contrary, the claims are intended to cover all alternatives, modifications, and equivalents.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The drawings described below are for illustration purposes only and are not intended to limit the scope of this disclosure.

[0054] FIGURE 1 shows 1 μιη ablation spots generated using a 193 nm excimer laser, with 2 μιη spacing between spots in x and y axes.

[0055] FIGURE 2 shows 1 μιη ablation spots generated using a 213 nm Nd:YAG laser, with 2 μιη spacing between spots in x and y axes.

[0056] FIGURE 3 shows monitoring using a camera and confocal phase contrast microscopy of subcellular ablation of a cell pellet by a 213 nm Nd:YAG laser, 1 μιη spot size. [0057] FIGURE 4 A, 4B, 4C shows subcellular resolution imaging of a tissue sample using a 213 nm Nd:YAG laser-based mass cytometry imaging system. Image A is staining of pS6 protein with a ¹⁷⁰Yb labelled antibody. Image B is staining of H3 protein with a ¹⁷⁶Yb labelled antibody. Image C is the composition of images A and B. The numbering along the x and y axes is the scale in μιη.

[0058] Figure 5 shows an exemplary mass cytometry system according to some embodiments of the disclosure.

DETAILED DESCRIPTION

[0059] Mass cytometry is a popular tool for flow cytometry analysis of biological samples. In certain implementations, mass cytometry is based on affinity probing of antigens in biological cells using affinity probes having elemental tags. Tagged samples can then be analyzed by injecting material into an inductively coupled plasma (ICP) ion source where the elemental tags are atomized and ionized. The ionized cloud containing the elemental tags can be sampled into a mass spectrometer for analysis. CyTOF2 (Fluidigm Canada, Inc.) is a current commercial platform for mass cytometry. A benefit of using elemental tagging in mass cytometry is in the ability to simultaneously measure a large number of probes. For example, over 40 elemental probes can be analyzed in the ionized cloud from each sample.

[0060] Recently, the application of mass cytometry has been extended to the field of immunohistochemistry-based imaging. This method is referred to as imaging mass cytometry (IMC). In IMC, a tissue is stained with affinity probes containing elemental tags. The spatial distribution of the elemental tags across the tissue is then analyzed using mass cytometry. For example, stained tissue can be subjected to laser ablation (LA) and then sampled into an ICP source for further analysis by mass spectrometry. In IMC, the quantitative distribution of target molecules can be determined indirectly by measuring the elemental tags attached to the affinity probe.

[0061] For imaging mass cytometry applications it is desirable that a large sample area be accessed at high resolution. For example, it can be useful to scan an area of about 500 μιη x 500 μιη (250,000 μιη²) with a spatial resolution of less than 1 μιη². It is also useful that the image be obtained in a reasonable time such as less than about 3 hours. To maximize the information obtained for each sample area, it is desirable that a large number of mass tags be evaluated at high sensitivity. Several improvements to an imaging mass cytometry system can be implemented to achieve these goals.

[0062] Imaging mass cytometry systems, apparatus and methods are disclosed in PCT International Application Publication No. WO 2014/146724, PCT International Application Publication No. WO 2014/147260, PCT International Application Publication No.

2014/169394, and U.S. Application No. 62/079,448 filed on November 13, 2014, each of which is incorporated by reference in its entirety. In imaging mass cytometry, an ablation source such as a laser pulse is scanned across a sample to obtain an image of the sample. The ablation source such as a laser pulse can generate a gas plume that is then transferred to a mass spectrometer. The ablation energy can be provided by a focused laser pulse. In certain embodiments, it is desirable that the laser pulse have a duration of about 1 nsec, such as from about 0.5 nsec to 5 nsec, and the ablated area be less than 1 μιη².

[0063] ICP-MS has been used for analysis of various biological substances [1]. The inventor has now made improvements to laser ablation (LA) followed by ICP-MS so that this technique is more applicable to the analysis of biological samples. Imaging of biological samples via LA-ICP-MS has previously been reported for imaging at a cellular resolution [2,3,4]. Detailed imaging at a sub-cellular resolution has also recently been reported [5], but such fine precision ablation has been limited to the use of lasers emitting a beam with a wavelength of 193 nm. Such lasers were preferred because of the comparative ease with which their beam can be homogenized (due to the relative lack of coherency in the light emitted by such lasers), which is considered in the art to be important for laser ablation of biological materials, and because the shorter wavelength of this light means that it can be more easily demagnified to make small spot sizes. Lasers that produce light of 193 nm wavelength are typically excimer lasers.

[0064] An LA-ICP-MS system which overcomes the drawbacks of using excimer laser and is still able to achieve sub-cellular resolution is described below (e.g., a system that ablates the sample with a spot size of less than 2 μιη, which corresponds to the longest internal dimension of the beam, e.g., for a circular beam it is a beam of diameter 2 μιη, and for a square beam corresponds to the length of the diagonal between opposed corners), together with other advantageous modifications. The disclosure therefore provides a new LA- ICP-MS system and new methods of using LA-ICP-MS systems that take advantage of the developments discussed herein.

LA-ICP-MS and mass cytometry

[0065] In some non-limiting embodiments, uses laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is used to image a biological sample.

Different target molecules in the sample are labelled with different labelling atoms and LA- ICP-MS is then used across multiple cells of the biological sample (e.g., a tissue section, a monolayer of cells or individual cells, such as where a cell suspension has been dropped onto a microscope slide, as discussed in more detail below). By linking detected signals to the known positions of the laser ablations which gave rise to those signals, the localization of the labelled target molecule to specific locations on the sample is possible, and thus enables the construction of an image of the sample.

[0066] The components of an LA-ICP-MS system include a laser that emits a beam that is directed upon the labelled sample in the system. Optical components can be placed in the path of the laser beam between the laser source and the sample to modify the properties of the beam (e.g., demagnify the beam). The sample is positioned on a stage in an ablation chamber in the LA-ICP-MS system. A gas is flowed through the ablation chamber, and the flow of gas carries away the plumes of aerosolized material, including the labelling atoms, generated when the laser ablates the sample. The gas carries the material to the ICP, which ionizes the material to enable detection by an MS. The labelling atoms in the sample can be

distinguished by MS and so their detection reveals the presence or absence of multiple targets in a plume and so a determination of what targets were present at the ablated locus on the sample.

[0067] The spatial resolution of signals generated in this way depends, in at least some instances, on two main factors: (i) the spot size of the laser, as signal is integrated over the total area which is ablated; and (ii) the speed at which a plume can be analyzed, relative to the speed at which plumes are being generated, to avoid overlap of signal from consecutive plumes as mentioned above.

Lasers

[0068] Generally, the choice of wavelength and power of the laser used for ablation of the sample can follow normal usage in cellular analysis by ICP-MS. In many cases, the laser must have sufficient fluence to cause ablation to a desired depth, without substantially ablating the supporting sample holder. A laser fluence of between 2-5 J/cm² is typically suitable e.g., from 3-4 J/cm² or about 3.5 J/cm², and the laser will ideally be able to generate a pulse with this fluence at a rate of 40 Hz or greater. A single laser pulse from such a laser should be sufficient to ablate cellular material for analysis, such that the laser pulse frequency matches the frequency with which ablation plumes are generated. In at least some instances, to be a laser useful for imaging biological samples by LA-ICP-MS, the laser should produce a pulse with duration below 300 ns (preferably below 30 ns) with the wavelength in the range of 150 nm to 220 nm which can be focused to a spot size of 2 μιη or below, for example the specific spot sizes discussed below. [0069] In some LA-ICP-MS apparatuses and methods according to the disclosure, the laser used is a solid state laser. Solid state lasers have the advantage that they are compact, the laser light being emitted through the use of a crystalline material that has been doped with an element, typically a rare earth or transition metal. Of particular use with the present disclosure is a laser based on a crystal of yttrium aluminum garnet (YAG) Y₃AI₅O₁₂. An alternative crystal is yttrium lithium fluoride (YLF) L1YF₄. The crystal of a solid state laser is often doped with neodymium, ytterbium, erbium, thulium, holmium or chromium. In some embodiments, the laser is a neodymium doped laser. Sometimes, the laser is a neodymium YAG laser, which emits λ = 213 nm.

[0070] Accordingly, in some embodiments, an LA-ICP-MS system utilizes a laser which emits λ = 213 nm, adapted to ablate material with a spot size (i.e. the diameter of the crater of ablated material) of less than 2 μιη, 1 μιη, or less than 1 μιη, such as 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm, or less than 100 nm. In order to analyze cells at a subcellular resolution the disclosure uses a laser spot size, which is no larger than these cells, and more specifically uses a laser spot size which can ablate material with a subcellular resolution. Sometimes, single cell analysis can be performed using a spot size larger than the size of the cell, for example where cells are spread out on the slide, with space between the cells. Here, a larger spot size can be used and single cell characterization achieved, because the additional ablated area around the cell of interest does not comprise additional cells. The particular spot size used can therefore be selected appropriately dependent upon the size of the cells being analyzed. In biological samples, the cells will rarely all be of the same size, and so if subcellular resolution imaging is desired, the ablation spot size should be smaller than the smallest cell, if constant spot size is maintained throughout the ablation procedure. Small spot sizes can be achieved using demagnification of wider laser beams and near-field optics. A laser spot diameter of 1 μιη corresponds to a laser focus point (i.e. the diameter of the laser beam at the focal point of the beam) of 1 μιη, but the laser focus point can vary by +20% due to numerical aperture of the objective that transfers the laser beam onto the sample surface. For example, using a 25 μιη diameter laser beam, and subjecting this to 25 -fold

demagnification onto the tissue samples will give a spot size with a 1 μιη diameter. Suitable objectives for 25-fold demagnification include a reflecting objective, such as an objective of a Schwarzschild Cassegrain design (reverse Cassegrain). Refracting objectives can also be used, as can combination reflecting-refracting objectives. A single aspheric lens can also be used to achieve the required demagnification. Another means for controlling the spot size of the laser, which can be used alone or in combination with the above objectives is to pass the beam through an aperture. Different beam diameters can be achieved by passing the beam through apertures of different diameter from an array of diameters. In some instances, there is a single aperture of variable size, for example when the aperture is a diaphragm aperture. In some embodiments, the diaphragm aperture is an iris diaphragm. Variation of the spot size can also be achieved through dithering of the optics. The one or more lenses and one or more apertures are positioned between the laser and the sample stage.

[0071] In certain embodiments, it can be desirable that a large area be analyzed such as 2x2 mm, 5><5 mm, or 10^x 10 mm or other suitable area, ablation spot can have a square shape such as 5x5 μιη², 10x 10 μιη², 15x 15 μιη² or rectangular shape or hexagonal shape or a rhomboid shape or a circular shape. An example of rectangular shape could be 1 μιη by 20 μιη. The challenge with ablating large surface areas is in providing sufficient energy over the desired ablation area, which can be greater than about 10 μΧ By using a wide rectangular line one can cover a length of a cell in one dimension while maintaining the energy level at a reasonable level.

[0072] Previously, it was considered that lasers emitting 213 nm light (e.g., Nd:YAG) would be of no use in LA-ICP-MS for ablation of biological materials at a cellular or subcellular resolution. This is because the prevailing thinking in the art was that the light from such a laser had properties which prevented it being focused with sufficient spot size and sufficient energy to ablate the necessarily small spots from the biological sample in the manner described for an excimer laser in reference 5. In particular, it was believed that the diffraction limit and coherence of light from an Nd:YAG laser prevented the necessary small, high energy, spot size from being achieved. The inventor has demonstrated that, in spite of this prevalent thinking in the art, a sufficiently small spot size for sub-cellular resolution ablation of biological samples can be achieved using a 213 nm Nd:YAG laser. The spot size of a Nd:YAG laser in comparison to the type of laser used previously is presented in the figures (Nd:YAG Figure 2 vs excimer laser in Figure 1). Evidence of subcellular resolution ablation of by a 213 nm Nd:YAG laser is presented in Figure 3.

[0073] A development leading to the disclosure was the discovery that the use of homogenized laser light was not necessary for ablation of biological samples at subcellular resolution. Accordingly, in some embodiments, the laser light is not homogenized and in some embodiments the system does not include a beam homogenizer. Previously, homogenized light was considered to be necessary, and 213 nm light from a Nd:YAG was considered to be too coherent to homogenize, meaning that the art was prejudiced against the use of such a laser for ablation of samples with high resolution. High resolution imaging can be accomplished using a non-homogenized (Gaussian) beam from the Nd:YAG laser.

Accordingly, sometimes, the disclosure uses a non-homogenized laser (e.g., Nd:YAG) with λ = 213 nm, including in the embodiments discussed above relating to specific spot sizes when the laser ablates the sample. A Gaussian beam is one in which the energy profile of the beam would follow a normal distribution (i.e. bell shaped curve) if along a line drawn through the beam. In some laser beams the distribution may be approximately Gaussian. In an

approximately Gaussian beam, the laser energy along a line drawn through the center of the beam does not precisely follow a Gaussian distribution, but a Gaussian distribution can be fitted to the intensity profile. In other words, a beam with a Gaussian or approximately Gaussian distribution is one which is characterized by a higher energy at the center of the beam, with the energy of the beam dropping towards the edge of the beam. Such a beam profile results from the laser operating on the fundamental transverse mode, or "TEMoo mode" of the laser's optical resonator. This may be contrasted to a homogenized beam, in which the beam is manipulated such that the energy of the beam is more consistent across it.

[0074] For rapid analysis of a tissue sample a high frequency of ablation is needed in at least some instances, for example more than 20 Hz (i.e. more than 20 ablations per second, giving more than 20 plumes per second). In some embodiments the frequency of ablation by the laser is within the range 40-200 Hz, within the range 40-150 Hz, or within the range 75-150 Hz. An ablation frequency of more than 40 Hz allows imaging of typical tissue samples to be achieved in a reasonable time. The frequency with which laser pulses can be directed at a spot on the sample (assuming full ablation of the material at that spot) and still be individually resolved determines how quickly the pixels of the image can be obtained. Accordingly, if the duration of laser pulse required to ablate the material at a point means that only less than 5 pulses can be directed at a sample per second, the time taken to study a 100 μιη x 100 μιη area with ablation at a spot size of 1 μιη would be over two days. With a rate of 40 Hz, this would be around 6-7 hours, with further reductions in the analysis time for further increases in the frequency of pulses. At these frequencies the instrumentation must be able to analyze the ablated material rapidly enough to avoid substantial signal overlap between consecutive ablations, if it is desired to resolve each ablated plume individually. In at least some instances, it is preferred that the overlap between signals originating from consecutive plumes is <10% in intensity, more preferably <5%, and ideally <2%. The time required for analysis of a plume will depend on the washout time of the ablation chamber (see ablation chamber section below), the transit time of the plume aerosol to and through the ICP, and the time taken to analyze the ionized material. Each laser pulse can be correlated to a pixel on the image of the sample that is subsequently built up, as discussed in more detail below.

[0075] For completeness, the standard lasers for LA-ICP-MS at sub-cellular resolution, as known in the art (e.g., [5]), are excimer or exciplex lasers. Suitable results can be obtained using an argon fluoride laser (λ = 193 nm). Pulse durations of 10-15 ns with these lasers can achieve adequate ablation. Such lasers may be used in other LA-ICP-MS systems described below.

[0076] Overall, the laser pulse frequency and strength are selected in combination with the response characteristics of the MS detector to permit distinct detection of individual laser ablation plumes. In combination with using a small laser spot and an ablation chamber having a short washout time, rapid and high resolution imaging is now feasible.

Spatial resolution

[0077] In certain applications, it is desirable that the spatial dimensions of the sample that is ablated for introduction into the injector be minimized. The ability to sample small spatial dimensions can increase the amount of information about the distribution of a probe across or within a sample.

[0078] In certain embodiments, a laser ablation system can be configured to provide a spatial resolution less than 2 μιη, less than 1 μιη, or in certain embodiments, less than 0.5 μιη.

[0079] The spatial resolution of a laser ablation system can depend on a number of factors including, for example and in some instances, the optical characteristics of laser radiation, the temporal characteristics of the laser radiation, the absorption properties of the sample being irradiated, heat flow within and/or in the vicinity of the irradiated sample, and the gas flow dynamics of the laser ablation system.

[0080] High spatial resolution laser ablation depends in part and in some instances on the ability to focus the laser radiation incident on the sample to a small spot size. Achieving submicron laser resolution can be achieved by selection of suitable wavelengths and optics. Lasers operating at different wavelengths and having different pulse characteristics are commercially available and can be adapted for sample laser ablation.

[0081] Excimer lasers can be used to produce tightly focused beams of sample

irradiation. Examples of suitable excimer lasers include ArF lasers operating at 193 nm and KrF lasers operating at 248 nm. Excimer lasers can operate at pulse repetition rates from about 1 Hz to about 100 Hz, such as about 40 Hz, and are characterized by a pulse width from about 3 ns to about 30 ns. The output of excimer lasers can be optically manipulated to provide submicron beams. Optical manipulation includes homogenizing the excimer beam profile using a mask to define the ablation area and reimaging the masked area onto the target using an objective.

[0082] Pulsed solid state lasers can also provide useful UV radiation for laser ablation. For example, the fifth harmonic of a Nd:YAG laser operating at 1064 nm provides radiation at 213 nm. The output of a solid state laser can also be optically manipulated to provide sub- micron spatial resolution. Solid state laser pulse widths can range from about 1 ps to about 20 ns. Other UV sources and wavelengths can be used including, for example, diode pumped solid state lasers operating at 266 nm (fourth harmonic of Nd-YAG laser). Apparatus and methods for providing high sub-micron resolution have generally been developed in the semiconductor industry, laser material processing industry (micro-drilling, cutting, etc.) and similar methods and apparatus can be employed to provide sub-micron ablation areas.

[0083] The laser radiation wavelength used for sample ablation is in general not limited to the specified wavelengths. To achieve small spot sizes, depending on the radiation wavelength and the optical characteristics of the laser radiation, a suitable optical system can be designed to provide an ablation area of 1 μιη or less.

[0084] For integration with a mass cytometry system, it is desirable that the laser source is small, easy to use, and ancillary operating systems are minimized. In this regard, solid state lasers have advantages over excimer lasers in that they can be small and do not require the gas handling infrastructure required for excimer lasers.

[0085] High spatial resolution can also be achieved using femtosecond (fs) lasers.

Femtosecond laser can provide pulse durations from 5 fs to 500 fs. Short pulse durations can provide a better transfer of energy into ablation resulting in smaller particles entering the plume, and less thermal damage to the remaining sample which can in turn increase the sensitivity of the instrument. Femtosecond lasers often feature higher repetition rates that could be utilized by the mass cytometry instrument. Ablation efficiency or quality of ablation cut may be increased by applying multiple pulses to an ablation area. As a result of the short pulse duration, ablation using femtosecond pulses can also minimize heat spread within the sample. Generally, femtosecond laser ablation is governed by non-linear processes and not by linear absorption of the incident radiation by the sample. Femtosecond lasers also are available over a wide wavelength range from about 190 nm to about 3000 nm, for example, from 800 nm to 1030 nm.

Laser ablation focal point

[0086] To maximize the efficiency of a laser to ablate material from a sample, the sample should be at a suitable position with regard to the laser's focal point, for example at the focal point, as the focal point is where the laser beam will have the smallest diameter and so most concentrated energy. This can be achieved in a number of ways. A first way is that the sample can be moved in the axis of the laser light directed upon it (i.e. up and down the path of the laser light / towards and away from the laser source) to the desired point at which the light is of sufficient intensity to effect the desired ablation. Alternatively, or additionally, lenses can be used to alter the focal length of the laser light and so its effective ability to ablate material at the location of the sample, for example by demagnification. The one or more lenses are positioned between the laser and the sample stage. A third way, which can be used alone or in combination with either or both of the two preceding ways, is to alter the position of the laser.

[0087] To assist the user of the system in placing the sample at the most suitable location for ablation of material from it, a camera can be directed at the stage holding the sample. Accordingly, the disclosure provides a LA-ICP-MS system that in some instances includes a camera directed on the sample stage. The image detected by the camera can be focused to the same point at which the laser is focused (see Figure 3, which is an image from a camera showing ablation of a sample by a laser focused to the same point as the camera). This can be accomplished by using the same objective lens for both laser ablation and optical imaging. By bringing the focal point of two into accordance, the user can be sure that laser ablation will be most effective when the optical image is in focus. Accordingly, in one embodiment, the only movement of the sample relative to the focal point of the laser (and camera) is by movement of the sample (i.e. the absolute position of the laser and any lenses is fixed). The precision with which the stage supporting the sample can be moved will determine how precisely the sample is positioned at the focus of the laser (and camera). Accordingly, in some embodiments the stage is configured to move the sample in the Z-axis in increments of less than 10 μιη, less than 5 μιη, less than 4 μιη, less than 3 um, less than 2 μιη, 1 μιη, or less than 1 μιη, such as less than 500 nm, less than 200 nm, or less than 100 nm. Precise stage movements will be in increments of about 1 μιη, such as 1 μιη ± 0.1 μιη. The range of movement in the Z-axis can be 100 μιη or more, for example 200 μιη or more, 300 μιη or more, 400 μιη or more or 500 μιη or more, such as 1 mm. This movement can be effected by use of piezo activators, as available from Physik Instrumente, Cedrat-technologies, Thorlabs and other suppliers.

Additional applications of a camera

[0088] In addition to identifying the most effective positioning of the sample for laser ablation, the inclusion of a camera (such as a charged coupled device image sensor based (CCD) camera or an active pixel sensor based camera), or any other light detecting means in an LA-ICP-MS system enables various further analyses and techniques. A CCD is a means for detecting light and converting it into digital information that can be used to build up an image. In a CCD image sensor, there are a series of capacitors that detect light, and each capacitor represents a pixel on the determined image. These capacitors allow the conversion of incoming photons into electrical charges. The CCD is then used to read out these charges, and the recorded charges can be converted into an image. An active-pixel sensor (APS) is an image sensor consisting of an integrated circuit containing an array of pixel sensors, each pixel containing a photodetector and an active amplifier, e.g., a CMOS sensor.

[0089] A camera can be incorporated into any of the other LA-ICP-MS systems discussed herein. The camera can be used to scan the sample to identify cells of particular interest or regions of particular interest (for example cells of a particular morphology). Once such cells have been identified, then laser pulses can be directed at these particular cells to ablate material for analysis, for example in an automated (where the system both identifies and ablates the area(s) of interest) or semi-automated process (where the user of the system, for example a clinical pathologist, identifies the area(s) of interest, which the system then ablates in an automated fashion). This enables a significant increase in the speed at which analyses can be conducted, because instead of needing to ablate the entire sample to analyze particular cells, the cells of interest can be specifically ablated. This leads to efficiencies in methods of analyzing biological samples in terms of the time taken to perform the ablation, but in particular in the time taken to interpret the data from the ablation, in terms of constructing images from it. Constructing images from the data is one of the more time-consuming parts of the imaging procedure, and therefore by minimizing the data collected to the data from relevant parts of the sample, the overall speed of analysis is increased.

[0090] In one embodiment, the camera records the image from a confocal microscope. Confocal microscopy is a form of optical microscopy that offers a number of advantages, including the ability to reduce interference from background information (light) away from the focal plane. This happens by elimination of out-of-focus light or glare. Confocal microscopy can be used to assess unstained samples for the morphology of the cells. Often, the sample is specifically labelled with fluorescent markers (such as by labelled antibodies or by labelled nucleic acids). These fluorescent makers 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 an appropriate wavelength of light, these regions of the sample are specifically identifiable. In some embodiments, LA-ICP-MS systems described herein therefore can comprise a laser for exciting fluorophores in the labels used to label the sample. Alternatively, an LED light source can be used for exciting the fluorophores. Non-confocal (e.g., wide field) fluorescent microscopy can also be used to identify certain regions of the biological sample, but with lower resolution than confocal microscopy.

[0091] When a laser is used to excite fluorophores for fluorescence microscopy, in some embodiments this laser is the same laser that generates the laser light used to ablate material from the biological sample, but used at a power that is not sufficient to cause ablation of material from the sample. In some embodiments, the fluorophores are excited by the wavelength of light that the laser then ablates the sample with. In others, a different wavelength may be used, for example by exploiting different harmonics of the laser to obtain light of different wavelengths.

[0092] As an example technique combining fluorescence and LA-ICP-MS, it is possible to label the nuclei of cells in the biological sample with an antibody or nucleic acid conjugated to a fluorescent moiety. Accordingly, by exciting the fluorescent label and then observing and recording the positions of the fluorescence using a camera, it is possible to direct the ablating laser specifically to the nuclei, or to areas not including nuclear material. The division of the sample into nuclei and cytoplasmic regions will find particular application in field of cytochemistry. By using an image sensor (such as a CCD detector or an active pixel sensor, e.g., a CMOS sensor), it is possible to entirely automate the process of identifying regions of interest and then ablating them, by using a control module (such as a computer or a programmed chip) which correlates the location of the fluorescence with the x,y coordinates of the sample and then directs the ablation laser to that location. As part of this process, in some embodiments, the first image taken by the image sensor has a low objective lens magnification (low numerical aperture), which permits a large area of the sample to be surveyed. Following this, a switch to an objective with a higher magnification can be used to home in on the particular features of interest that have been determined to fluoresce by higher magnification optical imaging. These features recorded to fluoresce may then be ablated by a laser. Using a lower numerical aperture lens first has the further advantage that the depth of field is increased, thus meaning features buried within the sample may be detected with greater sensitivity than screening with a higher numerical aperture lens from the outset.

[0093] In methods and systems in which fluorescent imaging is used before LA-ICP-MS imaging, the emission path of fluorescent light from the sample to the camera may include one or more lenses and/or one or more optical filters. By including an optical filter adapted to pass a selected spectral bandwidth from one or more of the fluorescent labels, the system is adapted to handle chromatic aberrations associated with emissions from the fluorescent labels. Chromatic aberrations are the result of the failure of lenses to focus light of different wavelengths to the same focal point. Accordingly, by including an optical filter, the background in the optical system is reduced, and the resulting optical image is of higher resolution. A further way to minimize the amount of emitted light of undesired wavelengths that reaches the camera is to exploit chromatic aberration of lenses deliberately by using a series of lenses designed for the transmission and focus of light at the wavelength transmitted by the optical filter, akin to the system explained in reference 6.

[0094] A higher resolution optical image is advantageous in this coupling of optical techniques and LA-ICP-MS imaging, because the accuracy of the optical image then determines the precision with which the ablating laser can be directed to ablate the sample.

[0095] Accordingly, the disclosure provides for, in some embodiments, a method of imaging a biological sample comprising a plurality of cells, the method comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating the sample with light to excite the one or more fluorescent labels; (iii) recording locational information of the areas of the sample which fluoresce; (iv) using the locational information of where fluorescence occurs to direct laser ablation sequentially at multiple known locations, to form a plurality of plumes; and (v) subjecting plumes to inductively coupled plasma mass spectrometry, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated. In some embodiments, the image of the sample from the laser ablation data may be overlaid on the image of the fluorescent data.

[0096] In a further mode of operation combining both fluorescence analysis and LA-ICP- MS, instead of analyzing the entire slide for fluorescence before targeting laser ablation to those locations, it is possible to fire a pulse from the laser at a spot on the sample (at low energy so as only to excite the fluorescent moieties in the sample rather than ablate the sample) and if a fluorescent emission of expected wavelength is detected, then the sample at the spot can be ablated by firing the laser at that spot at full energy, and the resulting plume analyzed by ICP-MS as explained above. This has the advantage that the rastering mode of analysis is maintained, but the speed is increased, because it is possible to pulse and test for fluorescence and obtaining results immediately rather than the time taken to analyze and interpret data from the ICP-MS, again enabling only the loci of importance to be targeted for ICP-MS analysis. Accordingly, the disclosure provides a method of imaging a biological sample comprising a plurality of cells, the method comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating a known location of the sample with light to excite the one or more fluorescent labels; (iii) observing and recording whether there is fluorescence at the location; (iv) if there is fluorescence, directing laser ablation at 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 further known locations on the sample, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated.

[0097] The disclosure also provides an LA-ICP-MS system comprising a control module programmed to perform the methods set out in the preceding two paragraphs.

Ablation chamber

[0098] One component of the LA-ICP-MS system is the ablation chamber, which is the component in which the sample is placed when it is subjected to laser ablation. The ablation chamber comprises a stage, which holds the sample (typically the sample is on a microscope slide, e.g., a tissue section, a monolayer of cells or individual cells, such as where a cell suspension has been dropped onto the microscope slide, and the slide is placed on the stage). When ablated, the material in the sample forms plumes, and the flow of gas passed through the ablation chamber from an inlet to an outlet carries away the plumes of aerosolized material, including any labelling atoms that were at the ablated location. The gas carries the material to the ICP, which ionizes the material to enable detection by an MS. The labelling atoms in the sample can be distinguished by MS and so their detection reveals the presence or absence of multiple targets in a plume and so a determination of what targets were present at the ablated locus on the sample. Accordingly, the ablation chamber plays a dual role in hosting the solid sample that is analyzed, but also in being the starting point of the transfer of aerosolized material to the ICP and MS. This means that the gas flow through the chamber can affect how spread out the ablated plume of material becomes as it passes through the system. A measure of how spread out the ablated plume becomes is the washout time of the cell. This figure is a measure of how long it takes material ablated from the sample to be carried out of the ablation chamber by the gas flowing through it. When a chamber has a shorter washout time it means that more plumes can be generated and carried from the chamber per unit of time. For example, in a system with a washout time of 25 ms, it will be possible to transfer 40 plumes resulting from ablation of a sample from the ablation chamber per second, permitting an ablation frequency of 40 Hz in such an ablation chamber.

[0099] One feature of an ablation chamber, which is of particular use where specific portions in various discrete areas of sample are ablated, is a wide range of movement in which the sample can be moved in the x and y (i.e. horizontal) axes in relation to the laser (where the laser beam is directed onto the sample in the z axis), with the x and y axes being perpendicular to one another. The inventor has discovered that more reliable and accurate relative positions are achieved by moving the stage within the ablation chamber and keeping the laser's position fixed in the LA-ICP-MS apparatus. The greater the range of movement, the more distant the discrete ablated areas can be from one another. The sample is moved in relation to the laser by moving the stage on which the sample is placed. Accordingly, in one embodiment, the sample stage has a range of movement within the ablation chamber of at least 10 mm in the x and y axes, such as 20 mm in the x and y axes, 30 mm in the x and y axes, 40 mm in the x and y axes, 50 mm in the x and y axes, such as 75 mm in the x and y axes. In one embodiment, the range of movement is such that it permits the entire surface of a standard 25 mm by 75 mm microscope slide to be analyzed within the chamber. Of course, to enable subcellular ablation to be achieved, in addition to a wide range of movement, the movement should be precise. Accordingly, in some embodiments the stage is configured to move the sample in the x and y axes in increments of less than 10 μιη, such as less than 5 μιη, less than 4 μιη, less than 3 μιη, less than 2 μιη, 1 μιη, or less than 1 μιη, such as less than 500 nm. Precise stage movements can be in increments of about 1 μιη, such as 1 μιη±0.1 μιη. Commercially available microscope stages can be used, for example as available from

Thorlabs, Prior Scientific, and Applied Scientific Instrumentation. Alternatively, the motorized stage can be built from components, based on positioners providing the desired range of movement and suitably fine precision movement, such as the SLC-24 positioners from Smaract.

[0100] Naturally, when a sample stage in an ablation chamber has a wide range of movement, the ablation chamber must be sized appropriately to accommodate the movements of the stage. Sizing of the ablation chamber is therefore dependent on size of the sample to be involved, which in turn determines the size of the mobile sample stage. Exemplary sizes of ablation chamber have an internal chamber of 10 cm x 10 cm, 15 cm x 15 cm or 20 cm x 20 cm. The depth of the chamber may be 3 cm, 4 cm or 5 cm. The skilled person will be able to select appropriate dimensions following the teaching herein. The inventor considers, however, that the internal dimensions of the ablation chamber for analyzing biological samples by LA-ICP-MS must be bigger than the range of movement of the sample stage, for example in some embodiments by as much as at least 5 mm bigger, such as at least 10 mm bigger. This is because if the walls of the chamber are too close to the edge of the stage, the flow of the carrier gas passing through the chamber which takes the ablated plumes of material from the away sample and into the ICP-MS can become turbulent. Turbulent flow disturbs the ablated plumes, and so instead of remaining as a tight cloud of ablated material, the peak of material begins to spread out as it is ablated and carried away to the ICP-MS part of the apparatus. A broader peak of the ablated material has negative effects on the data produced by the LA-ICP-MS because it leads to interference due to peak overlap, and so ultimately, less spatially resolved data, unless the rate of ablation is slowed down to such a rate that it is no longer experimentally of interest.

[0101] An ablation chamber with a short washout time (e.g., 100 ms or less) is advantageous for use with the apparatus and methods of the disclosure. A cell with a long washout time will either limit the speed at which an image can be generated or will lead to overlap between signals originating from consecutive sample spots (e.g., reference 7, which had signal duration of over 10 seconds). Therefore, in at least some embodiments, aerosol washout time is a key limiting factor for achieving high resolution without increasing total scan time. Ablation chambers with washout times of <100 ms are known in the art. For example, reference 8 discloses an ablation chamber with a washout time below 100 ms. An ablation chamber was disclosed in reference 9 (see also reference 10) which has a washout time of 30 ms or less, thereby permitting a high ablation frequency (e.g., above 20 Hz) and thus rapid analysis. Another such ablation chamber is disclosed in reference 11. The ablation chamber in reference 11 comprises a sample capture cell configured to be arranged operably proximate to the target, the sample capture cell including: a capture cavity having an opening formed in a surface of the capture cell, wherein the capture cavity is configured to receive, through the opening, target material ejected or generated from the laser ablation site and a guide wall exposed within the capture cavity and configured to direct a flow of the carrier gas within the capture cavity from an inlet to an outlet such that at least a portion of the target material received within the capture cavity is transferrable into the outlet as a sample. The volume of the capture cavity in the ablation chamber of reference 11 is less than 1 cm³ and can be below 0.005 cm³.

[0102] Sometimes the ablation chamber has a washout time of 25 ms or less, such as 20 ms or 10 ms or less. Example System

[0103] Figure 5 shows an exemplary system 100 according to some embodiments of the disclosure. In some embodiments of the disclosure, system 100 is an imaging mass cytometry system. System 100 may include a UV laser 102 configured to direct laser energy along a beam path 104 toward a sample 106. The sample 106 may be positioned within a flow cell 107 and may be on a moveable stage 108 (e.g., an XYZ stage 108). System 100 may further include a light source 109 (e.g., LED or the like) configured to direct

illumination light toward the sample 106 along a beam path 110. A CCD camera 112 may be provided to receive light returned from the sample 106 for imaging of the sample 106.

[0104] In some embodiments, a raster shutter 114 may be disposed along the first beam path 104 between the laser 102 and the sample 106. The rastering shutter 114 may provide energy stability to the system 100 by allowing continuous operation of the laser 102 while turning off delivery to the sample 106 during movement of the XYZ stage 108. The rastering shutter 114 may also acts as a safety feature which is activated when a safety interlock has been triggered in the system 100. In the safety interlock mode, a shutter can be tripped by the signals that covers have been opened. This provides a second level of protection for laser safety concerns. The shutter in this system may not be employed at a primary safety shutter.

[0105] In some embodiments, a laser attenuator 116 may be disposed along the first beam path 104 between the rastering shutter 114 and the sample 106. The laser attenuator 116 may provide operators the ability to vary the energy of laser beam for accurate definition of ablation conditions for a given sample 106. There are several possible technologies to provide optical attenuation. In some embodiments, an attenuator may be employed that is based on polarization rotation and a polarizer filtering of the laser light. But alternative technologies based on neutral density filter or based on variable refraction when two glass plates are rotated may be utilized.

[0106] The beam shaping optics 118 may be disposed along the first beam path 104 between the laser attenuator 116 and the sample 106. The beam shaping optics 118 may be configured to shape the laser beam to produce a focused spot that is directed to the sample 106. Beam shaping optics 118 may include any of the optical components described herein to produce the focused spot that is directed to the sample 106. For example, beam shaping optics 118 may include one or more objectives and/or apertures as described above for focusing the laser 102. Lenses can be used to focus and defocus the beam. UVFS material is commonly used for these wavelengths. [0107] A first beam splitter 120 may be positioned along the first beam path 104 between the beam shaping optics 118 and the sample 106. The first beam splitter 120 may also be disposed along the second beam path 110. The first beam splitter 120 may be configured to direct light received along the first beam path 104 and light received along the second beam path 110 along a third path 122 toward the sample 106. The beam splitter 120 may be a dichroic mirror. In other embodiments, other beam splitter solutions such as half-mirror may be employed.

[0108] A microscope objective 124 may be positioned along the third path 122 between the first beam splitter 120 and the sample 106. The microscope objective 124 may focus illumination light from the light source 109 and imaging light from laser 102 onto sample 106. Accordingly, in some embodiments, the system 100 may have a common focusing beam path for illumination and imaging.

[0109] Additionally, a beam splitter 126 may be positioned along the beam path 110 for directing light reflected from the sample 106 toward the CCD camera 112.

[0110] A tube lens 128 may be positioned between beam splitter 120 and beam splitter 126. The tube lens 128 may allow for focusing of the specimen optical image onto the CCD camera 112. In some embodiments, it may be desirable for the IMC scheme to employ infinite conjugation setup using tube lens 128. The infinite conjugation setup may reduce some types of aberrations and may also improve the quality of the image on the CCD camera 112. The beam splitter 126 may be a half-mirror. Though, other beam splitters can be employed there.

[0111] The UV laser source 102 may be configured to transmit the laser for ablating and/or fluorescing the sample 106. The laser 102 may operate at a wavelength of 213 nm in some embodiments. While illustrated and described as a solid state laser operating at a wavelength of 213 nm, it should be understood that laser source 102 may be any of the laser systems described herein in other embodiments of the present disclosure. For example, in other embodiments, femtosecond lasers, excimer lasers, or the like may be substituted for UV laser 102.

[0112] Similarly, while system 100 is illustrated and described as including a CCD camera 1 12, it should be understood that any of the imaging sensors described herein may be used in other embodiments of the present disclosure, such as a CMOS sensor or the like.

[0113] Irradiation of various spot sizes can be accomplished by using a mechanically controlled aperture or an array of interchangeable apertures and/or an objective (e.g., along beam path 104) with proper magnification to establish the spot size or alternatively multiple laser shots can be scanned across the ablation area corresponding to one pixel by rapidly dithering the optics.

ICP-MS

Sensitivity

[0114] There is a tradeoff between sensitivity and the number of channels or mass tags detected. In some instrumentation higher sensitivity can be realized when sampling a few mass tags. However, to maximize the information obtained it is desirable to measure the largest number of mass tags possible within a reasonable acquisition time. This is particularly important in imaging mass cytometry applications where the information is spatially distributed and it is desirable to sample as large a sample area as possible in the least time.

[0115] Sensitivity can be increased in a number of ways including, for example, increasing the mass tag concentration, using high voltages in the orthogonal accelerator of the TOF mass spectrometer, including helium in the gas flow, using a short pulse to ablate the sample, using a small bore injector tube, increasing the proximity of the sample ablation chamber to the ICP torch. These parameters can also increase the practical pixel acquisition rate of the imaging mass cytometer by reducing the spread of laser ablation plumes in time as recorded by the data acquisition system.

[0116] It is believed that the addition of helium in the gas flow reduces the spread of the ablation plume. In certain embodiments, the gas flow into the injector is at least 1 slpm.

[0117] Sensitivity can be increased by increasing the number of entities of a particular mass tag in an ablation area.

[0118] As described, for example, in U.S. Application Publication No. 2014/0120550 and U.S. Application Publication No. 2010/0144056, each of which is incorporated by reference in its entirety, a sample can be labelled with one or more elemental tags. In certain embodiments, a metal isotope can be conjugated to a polymer or to a nanoparticle. The polymer or nanoparticle containing the metal isotopes can be coupled to an affinity moiety. An affinity moiety refers to a chemical moiety capable of binding to or attaching to a specific molecular and/or chemical target. An affinity moiety is part of a molecular tag. When an affinity moiety is released or cleaved from a molecular tag, the affinity moiety is referred to as an affinity molecule. Antibodies refer to immunoglobulin glycoprotein molecules.

Antibodies can be found in serum of animals. Antibodies may be made in mammals such as rabbits, mice, rats, goats, etc., and chicken. Procedures for immunization and elicitation of a high antibody production response in an animal are well known to those skilled in the art. Antibodies may also be made in cell cultures, for example by recombinant DNA methods. Antibodies may be used, for example, as whole molecules, half molecules known as Fab' and Fab² fragments, or as monovalent antibodies (combining a light chain and a modified heavy chain).

[0119] In certain embodiments, a mass probe comprises an affinity moiety coupled to a nanoparticle, wherein the nanoparticle has a plurality of mass tags bound to the surface. In certain embodiments, from 10 to 10,000 elemental mass tags can be bound to the surface of the nanoparticle. In certain embodiments, it is desirable the system have the ability to access 500 copies of mass probes in an ablation area, each having, for example, from 10 to 500 mass tags,. In certain embodiments, a nanoparticle contains element tags within the particle or as part of the particle, with the number of element tags being, for example, from about 100 to about 10,000.

[0120] The spread of the ablation plume before the ICP ion source can lead to a reduction in sensitivity. To decrease the spread of the ablation plume, helium can be added to the gas flow. In general, longer plumes result in a longer accumulation time and a higher amount of background noise. Accordingly, in certain embodiments the gas flow can comprise helium. The gas flow can be a mixture of helium and argon. In certain embodiments, nitrogen can be introduced into the gas flow such that the gas flow is a mixture of nitrogen, helium, and argon; or a mixture of nitrogen and argon.

[0121] Short pulse durations can decrease the size of the ablation plume and lead to increased sensitivity. In certain embodiments, a laser pulse has a duration from about 0.5 nsec to about 10 nsec, from about 0.5 nsec to about 5 nsec, and in certain embodiments, from about 0.5 nsec to about 2 nsec. In certain embodiments, laser ablation can be accomplished using a picosecond laser having a pulse duration from about 1 ps to about 1 ,000 ps, and in certain embodiments, using a femtosecond laser characterized by a pulse duration from about 5 fs to about 1,000 fs.

[0122] In certain embodiments, imaging mass cytometry apparatus provided by the present disclosure are configured to provide simultaneous measurement of from 100 to 150 mass tags, for example, from 20 to 30 mass tags. In certain embodiments, the relevant mass range is from 75 amu to 214 amu.

[0123] In certain embodiments, the sampling tube between the laser ablation chamber and the mass spectrometer is shortened in order to minimize the spread of the ablation plume. For example, in certain embodiments the length of the sampling tube is from about 30 mm to about 300 mm. [0124] The transit time of a plume aerosol to and through the ICP is easily controlled simply by positioning the ablation chamber near to the ICP and by ensuring a sufficient gas flow to transport the aerosol at an appropriate speed directly to the ICP. Transport using argon and helium as described in reference 9 provides good results.

Mass Analyzer

[0125] The time taken to analyze the ionized material will depend on the type of mass analyzer which is used for detection of ions. For example, instruments which use Faraday cups are generally too slow for analyzing rapid signals. Overall, the desired imaging speed, resolution and degree of multiplexing will dictate the type(s) of mass analyzer which should be used (or, conversely, the choice of mass analyzer will determine the speed, resolution and multiplexing which can be achieved).

[0126] Mass spectrometry instruments that detect ions at only one mass-to-charge ratio (m/Q, commonly referred to as m/z in MS) at a time, for example using a point ion detector, will give poor results in single cell imaging using multiple labels. Firstly, the time taken to switch between mass-to-charge ratios limits the speed at which multiple signals can be determined, and secondly, if ions are at low abundance then signals can be missed when the instrument is focused on other mass-to-charge ratios. Thus, although the instrument used in references 2 and 3 (Agilent 4500) is sensitive, its quadrupole-based detector is not well suited to imaging with multiple labels because, by design, ions of different mass-to-charge ratios pass through sequentially and so data acquisition for multiple labels is slow. Similarly, the instruments used in references 4 and 9 (Thermo Fisher ElementXR and Element2) analyze only one m/Q at a time and have a large settling time for magnet jumps when measuring multiple m/Q values over a range exceeding the range of an electrostatic field jump.

[0127] Thus it is preferred to use a technique which offers substantially simultaneous detection of ions having different m/Q values. For instance, instead of using a point ion detector, it is possible to use an array detector (e.g., see Chapter 29 of ref. 12). Multi-collector sector field ICP-MS instruments can be used (e.g., the Thermo Scientific Neptune Plus, Nu Plasma II, and Nu Plasma 1700 systems), and in particular those having a Mattauch-Herzog geometry (e.g., the SPECTRO MS, which can simultaneously record all elements from lithium to uranium in a single measurement using a semiconductor direct charge detector). These instruments can measure multiple m/Q signals substantially simultaneously. Their sensitivity can be increased by including electron multipliers in the detectors. Array sector instruments are not ideal, however, because, although they are useful for detecting increasing signals, they are less useful when signal levels are decreasing, and so they are not well suited in situations where labels are present at highly variable concentrations.

[0128] The most preferred MS for at least some embodiments is based on time-of-flight (TOF) detection, which can quasi-simultaneously register multiple masses in a single sample. In theory TOF techniques are not ideally suited to ICP ion sources because of their space charge characteristics, but the inventor has shown that TOF instruments can analyze an ICP ion aerosol rapidly enough and sensitively enough to permit feasible single-cell imaging. Whereas TOF mass analyzers are normally unpopular for atomic analysis because of the compromises required to deal with the effects of space charge in the TOF accelerator and flight tube, tissue imaging using the disclosure can be effective by detecting only the labelling atoms, and so other atoms (e.g., those having an atomic mass below 100) can be removed. This results in a less dense ion beam, enriched in the masses in (for example) the 100-250 Dalton region, which can be manipulated and focused more efficiently, thereby facilitating TOF detection and taking advantage of the high spectral scan rate of TOF. Thus rapid imaging can be achieved by combining TOF detection with choosing labelling atoms that are uncommon in the sample and ideally having masses above the masses seen in an unlabeled sample e.g., by using the higher mass transition elements. Using a narrower window of label masses thus means that TOF detection to be used for efficient imaging.

[0129] Suitable TOF instruments are available from Tofwerk, GBC Scientific Equipment (e.g., the Optimass 9500 ICP-TOFMS), and Fluidigm Canada (e.g., the CyTOF™ and CyTOF™2 instruments). These CyTOF™ instruments have greater sensitivity than the Tofwerk and GBC instruments and are known for use in mass cytometry because they can rapidly and sensitively detect ions in the mass range of rare earth metals (particularly in the m/Q range of 100-200) [13]. Thus these are preferred instruments for use with the disclosure in certain embodiments, and they can be used for imaging with the instrument settings already known in the art e.g., references 14 & 15. Their mass analyzers can detect a large number of markers quasi-simultaneously at a high mass-spectrum acquisition frequency on the timescale of high-frequency laser ablation [4]. They can measure the abundance of labelling atoms with a detection limit of about 100 per cell, permitting sensitive construction of an image of the tissue sample. Because of these features, mass cytometry can now be used to meet the sensitivity and multiplexing needs for tissue imaging at subcellular resolution. By combining the mass cytometry instrument with a high-resolution laser ablation system and a rapid-transit low-dispersion ablation chamber it has been possible to permit construction of an image of the tissue sample with high multiplexing on a practical timescale. Further details on mass cytometry can be found in references 1 and 16.

Scanning area

[0130] The value of imaging is in determining a spatial distribution of a probe or probes across a surface of a sample and/or within a sample. Information can be maximized by having a high spatial resolution and by being able to obtain information over a large surface area. In turn, for practical application, it is desirable that the surface area be sampled in a reasonable time. For example, in certain applications it is desirable that an area of

1000 1000 μιη², an area of 500x500 μιη², an area of 250x250 μιη² or an area of 100x 100 μιη² be sampled within about 3 hours.

[0131] The scan rate depends at least in part on factors such as the laser repetition rate, ablation spot size, the design of the ablation chamber, the carrier gas, the proximity of the ablation chamber to the torch, the duration of the laser pulse, the design and dimensions of the injector, and the gas flow rate.

Injector

[0132] Mass cytometer systems include an injector. One end of an injector is configured to receive a gas sample from an ablation chamber and the other end of the injector is coupled to a plasma torch. The plasma torch decomposes, atomizes and ionizes the sample. An injector is also characterized by a bore diameter extending through the length of the injector. Injectors having a small tip diameter at the input from the ablation chamber such as less than about 2 mm are generally avoided in the prior art due to the tendency to clog. However, injector tip diameters less than 1 mm such as, for example, 0.85 mm or 0.5 mm can be successfully used. The use of smaller injector tip diameters, which minimizes the spread of the response for individual cells introduced into the plasma facilitates the ability to increase the sampling rate. In certain embodiments, the injector tip diameter refers to the diameter of the bore at the entrance to the injector.

[0133] In certain embodiments, the injector bore diameter and the injector tip diameter can be configured to reduce spreading of the ablation plume. In certain of such

embodiments, an injector bore diameter can be from about 1 mm to about 4 mm, such as about 1 mm, about 2 mm, about 3 mm, or about 4 mm, and the tip diameter can be from about 0.5 mm to about 2 mm, such as about 0.5 mm, about 1 mm, about 1.5 mm, or about 2 mm.

[0134] In certain embodiment, the injector bore has a diameter that is about the same as the injector tip diameter. For example, in certain embodiments, an injector can be characterized by an end-to-end diameter of less than 2 mm, less than 1.5 mm, less than 1 mm, or less than 0.5 mm.

[0135] In other embodiments, the bore of injector diameter refers to the diameter at the entrance to the injector and along the main part of the injector. For analysis of laser ablation plumes, narrow bore diameters may reduce spreading of the plume.

[0136] Another approach that can be used independently or in combination with other methods, is to preselect certain areas of interest for subsequent mass cytometry analysis. For example, such methods include dual labeling strategies. For example, a sample can be stained with an element tag and a fluorescence probe. During sample analysis, the fluorescence probe is first interrogated with a laser pulse and the fluorescence detected. If a threshold fluorescence signal is detected, then a second laser pulse can be used to ablate the sample coincident with the fluorescence probe into the plasma. Using a dual probe method comprising interrogation followed by ablation only regions of interest across a sample can be ablated and the mass tag quantified. In embodiments in which dual probe methods or used, the size of the ablated area can be adjusted depending on the fluorescence signal detected.

[0137] In certain embodiments, a fluorescence map of a sample can be obtained. The fluorescence map can be obtained using a separate apparatus or using an apparatus configured to both provide fluorescence imaging and laser ablation. Based on the

fluorescence map selection of areas for laser ablation analysis can determined. This method has the advantage that fluorescence images can be obtained over a large area. Regions of interest can then be selected for in-depth analysis using imaging mass cytometry. This dual analysis method facilitates the ability to pre-select areas of interest for imaging mass cytometry thereby maximizing throughput. For example, fluorescence images of an area of 1 mm² can be obtained within a second and larger areas can be recorded in a few minutes. In contrast, imaging mass cytometry images of a 1 mm² area may take several hours to obtain. Quantitation

[0138] Quantification of affinity probe amount can be facilitated by detecting an internal standard with the mass signal. This can be accomplished by incorporating an internal standard into the plume. This can be accomplished in several ways. For example, an internal standard can be applied to a sample prior to analysis. During sample ablation, the mass standard will be incorporated into a plume and analyzed with the mass tags in the sample. In certain embodiments, an internal standard can be a particle such as a polystyrene particle with some amount of mass tag elements embedded into the particle. For instance, a polystyrene particle can have one or more mass tags and/or other mass standard attached to the surface. An internal standard can be applied to a sample using any suitable method, such as by spin coating on top of the sample or by forming a bottom layer on the slide prior to the deposition of the tissue sample.

[0139] In certain embodiments, and in particular for thin samples, a mass standard can be applied to a substrate or be the substrate itself. In other embodiments, a mass standard can be introduced into the gas flow before ionization.

Constructing an image

[0140] LA-ICP-MS can provide signals for multiple labelling atoms in plumes. Detection of a label in a plume reveals the presence of its cognate target at the position of oblation. By generating a series of plumes at known spatial locations on the sample's surface the MS signals reveal the location of the labels on the sample, and so the signals can be used to construct an image of the sample. By labelling multiple targets with distinguishable labels it is possible to associate the location of labelling atoms with the location of cognate targets, so embodiments of the disclosure can build complex images, reaching levels of multiplexing which far exceed those achievable using existing techniques. The inventor has shown that images generated by the methods of the disclosure can reproduce the staining patterns and the proportion of cells expressing a given marker as determined by IFM, thereby confirming the disclosure's suitability for imaging.

[0141] Assembly of signals into an image will use a computer and can be achieved using known techniques and software packages. For instance, reference 3 used the GRAPHIS package from Kylebank Software, but other packages such as TERAPLOT can also be used. Imaging using MS data from techniques such as MALDI-MSI is known in the art e.g.

reference 17 discloses the 'MSiReader' interface to view and analyze MS imaging files on a Matlab platform, and reference 18 discloses two software instruments for rapid data exploration and visualization of both 2D and 3D MSI data sets in full spatial and spectral resolution e.g., the 'Datacube Explorer' program.

[0142] Images obtained using methods of the disclosure can be further analyzed e.g., in the same way that IHC results are analyzed. For instance, the images can be used for delineating cell sub-populations within 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 which methods of the disclosure provide [19].

Labelling of the tissue sample

[0143] The disclosure produces images of samples which have been labelled with labelling atoms, for example a plurality of different labelling atoms, wherein the labelling atoms are detected in laser-ablated plumes by ICP-MS. The reference to a plurality of different atoms means that more than one atomic species is used to label the sample. These atomic species can be distinguished using ICP-MS (e.g., they have different m/Q ratios), such that the presence of two different labelling atoms within a plume gives rise to two different MS signals.

[0144] The disclosure is suitable for the simultaneous detection of many more than two different labelling atoms, permitting multiplex label detection e.g., at least 3, 4, 5, 10, 20, 30, 32, 40, 50 or even 100 different labelling atoms. Labelling atoms can also be used in a combinatorial manner to even further increase the number of distinguishable labels.

Reference 5 demonstrates the use of 32 different labelling atoms in an imaging method, but LA-ICP-MS is intrinsically suitable for parallel detection of higher numbers of different atoms e.g., even over 100 different atomic species [13]. By labelling different targets with different labelling atoms it is possible to determine the cellular location of multiple targets in a single image.

[0145] Labelling atoms that can be used with the disclosure include any species that are detectable by LA-ICP-MS and that are substantially absent from the unlabeled tissue sample. Thus, for instance, ¹²C atoms would be unsuitable as labelling atoms because they are naturally abundant, whereas ^UC could in theory be used because it is an artificial isotope which does not occur naturally. In preferred embodiments, however, the labelling atoms are transition metals, such as the rare earth metals (the 15 lanthanides, plus scandium and yttrium). These 17 elements provide many different isotopes which can be easily

distinguished by ICP-MS. A wide variety of these elements are available in the form of enriched isotopes e.g., samarium has 6 stable isotopes, and neodymium has 7 stable isotopes, all of which are available in enriched form. The 15 lanthanide elements provide at least 37 isotopes that have non-redundantly unique masses. Examples of elements that are suitable for use as labelling 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). For example, the disclosure can use any of the isotopes of the lanthanides as listed in the tables of the supplementary information to reference 5. In addition to rare earth metals, other metal atoms are suitable for detection by ICP-MS e.g., gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), bismuth (Bi), etc. The use of radioactive isotopes is not preferred as they are less convenient to handle and are unstable e.g., Pm is not a preferred labelling atom among the lanthanides. [0146] In order to facilitate TOF analysis (see above) it is helpful to use labelling atoms with an atomic mass within the range 80-250 amu, e.g., within the range 80-210 amu, or within the range 100-200 amu. This range includes all of the lanthanides, but excludes Sc and Y. The range of 100-200 amu permits a theoretical 101-plex analysis by using different labelling atoms, while permitting the disclosure to take advantage of the high spectral scan rate of TOF MS. As mentioned above, by choosing labelling atoms whose masses lie in a window above those seen in an unlabeled sample (e.g., within the range of 100-200), TOF detection can be used to provide rapid imaging at biologically significant levels.

[0147] Labelling the tissue sample generally requires that the labelling atoms are attached to one member of a specific binding pair (sbp). This labelled sbp is contacted with a tissue sample such that it can interact with the other member of the sbp (the target sbp member) if it is present, thereby localizing the labelling atom to a specific location in the sample. The method of the disclosure then detects the presence of the labelling atom at this specific location and translates this information into an image in which the target sbp member is present at that location. Rare earth metals and other labelling atoms can be conjugated to sbp members by known techniques e.g., reference 20 describes the attachment of lanthanide atoms to oligonucleotide probes for ICP-MS detection, reference 21 describes the use of ruthenium to label oligonucleotides, and Fluidigm Canada sells the MaxPar™ metal labelling kits which can be used to conjugate over 30 different labelling atoms to proteins (including antibodies).

[0148] Various numbers of labelling atoms can be attached to a single sbp member, and greater sensitivity can be achieved when more labelling atoms are attached to any sbp member. For example greater than 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 labelling atoms can be attached to a sbp member. For example, monodisperse polymers containing multiple monomer units may be used, each containing a chelator such as DTPA. DTP A, for example, binds 3+ lanthanide ions with a dissociation constant of around 10^"6 M [1]. These polymers can terminate in a thiol-reactive group (e.g., maleimide) which can be used for attaching to a sbp member. For example the thiol-reactive group may bind to the Fc region of an antibody. Other functional groups can also be used for conjugation of these polymers e.g., amine- reactive groups such as N-hydroxy succinimide esters, or groups reactive against carboxyls or against an antibody's glycosylation. Any number of polymers may bind to each sbp member. Specific examples of polymers that may be used include straight-chain (' 'X8") polymers or third-generation dendritic ("DN3") polymers, both available as MaxPar™ reagents. Use of metal nanoparticles can also be used to increase the number of atoms in a label. [0149] As mentioned above, labelling atoms are attached to a sbp member, and this labelled sbp member is contacted with the tissue sample where it can find the target sbp member (if present), thereby forming a labelled sbp. The labelled sbp member can comprise any chemical structure that is suitable for attaching to a labelling atom and then for imaging according to the disclosure.

[0150] In general terms, methods of the disclosure can be based on any sbp which is already known for use in determining the location of target molecules in tissue samples (e.g., as used in IHC or fluorescence in situ hybridization, FISH), but the sbp member which is contacted with the sample will carry a labelling atom which is detectable by ICP-MS. Thus the disclosure can readily be implemented by using available IHC and FISH reagents, merely by modifying the labels which have previously been used e.g., to modify a FISH probe to carry a label which can be detected by ICP-MS.

[0151] The sbp may comprise any of the following: a nucleic acid duplex; an

antibody/antigen complex; a receptor/ligand pair; or an aptamer/target pair. Thus a labelling atom can be attached to a nucleic acid probe which is then contacted with a tissue sample so that the probe can hybridize to complementary nucleic acid(s) therein e.g., to form a

DNA/DNA duplex, a DNA/R A duplex, or a R A/RNA duplex. Similarly, a labelling atom can be attached to an antibody, which is then contacted with a tissue sample so that it can bind to its antigen. A labelling atom can be attached to a ligand, which is then contacted with a tissue sample so that it can bind to its receptor. A labelling atom can be attached to an aptamer ligand which is then contacted with a tissue sample so that it can bind to its target. Thus labelled sbp members can be used to detect a variety of targets in a sample, including DNA sequences, R A sequences, proteins, sugars, lipids, or metabolites.

[0152] In a typical embodiment of the disclosure the labelled sbp member is an antibody. Labelling of the antibody can be achieved through conjugation of one or more labelling atom binding molecules to the antibody, for example using the MaxPar™ conjugation kit as described above. Antibodies which recognize cellular proteins that are useful for imaging are already widely available for IHC usage, and by using labelling atoms instead of current labelling techniques (e.g., fluorescence) these known antibodies can be readily adapted for use in methods of the disclosure, but with the benefit of increasing multiplexing capability. Antibodies used with the disclosure can recognize targets on the cell surface or targets within a cell. Antibodies can recognize a variety of targets e.g., they can specifically recognize individual proteins, or can recognize multiple related proteins which share common epitopes, or can recognize specific post-translational modifications on proteins (e.g., to distinguish between tyrosine and phospho-tyrosine on a protein of interest, to distinguish between lysine and acetyl-lysine, to detect ubiquitination, etc.). After binding to its target, labelling atom(s) conjugated to an antibody can be detected to reveal the location of that target in a sample.

[0153] The labelled sbp member will usually interact directly with a target sbp member in the sample. In some embodiments, however, it is possible for the labelled sbp member to interact with a target sbp member indirectly e.g., a primary antibody may bind to the target sbp member, and a labelled secondary antibody can then bind to the primary antibody, in the manner of a sandwich assay. Usually, however, the disclosure relies on direct interactions, as this can be achieved more easily and permits higher multiplexing. In both cases, however, a sample is contacted with a sbp member, which can bind to a target sbp member in the sample, and at a later stage label attached to the target sbp member is detected.

[0154] One feature of the disclosure is its ability to detect multiple (e.g., 10 or more, and even up to 100 or more) different target sbp members in a sample e.g., to detect multiple different proteins and/or multiple different nucleic acid sequences. To permit differential detection of these target sbp members their respective sbp members should carry different labelling atoms such that their signals can be distinguished by ICP-MS. For instance, where ten different proteins are being detected, ten different antibodies (each specific for a different target protein) can be used, each of which carries a unique label, such that signals from the different antibodies can be distinguished. In some embodiments, it is desirable to use multiple different antibodies against a single target e.g., which recognize different epitopes on the same protein. Thus a method may use more antibodies than targets due to redundancy of this type. In general, however, the disclosure will use a plurality of different labelling atoms to detect a plurality of different targets.

[0155] If more than one labelled antibody is used with the disclosure, it is preferable that the antibodies should have similar affinities for their respective antigens, as this helps to ensure that the relationship between the quantity of labelling atoms detected by LA-ICP-MS and the abundance of the target antigen in the tissue sample will be more consistent across different sbps (particularly at high scanning frequencies).

[0156] If a target sbp member is located intracellularly, it will typically be necessary to permeabilize cell membranes before or during contacting of the sample with the labels. For example when the target is a DNA sequence but the labelled sbp member cannot penetrate the membranes of live cells, the cells of the tissue sample can be fixed and permeabilised. The labelled sbp member can then enter the cell and form a sbp with the target sbp member. In this respect, known protocols for use with IHC and FISH can be utilized. [0157] Usually, a method of the disclosure will detect at least one intracellular target and at least one cell surface target. In some embodiments, however, the disclosure can be used to detect a plurality of cell surface targets while ignoring intracellular targets. Overall, the choice of targets will be determined by the information, which is desired from the method, as the disclosure will provide an image of the locations of the chosen targets in the sample. Samples

[0158] The disclosure provides a method of imaging a sample. The sample comprises a plurality of cells, a plurality of these cells can be subjected to LA-ICP-MS in order to provide an image of these cells in the sample. In general, the disclosure can be used to analyze tissue samples which are now studied by IHC techniques, but with the use of labels which are suitable for detection by LA-ICP-MS.

[0159] Any suitable tissue sample can be used in the methods described herein. For example, the tissue can be epithelium tissue, muscle tissue, nerve tissue, etc., and

combinations thereof. For diagnostic or prognostic purposes the tissue can be from a tumor. In some embodiments, a sample may be from a known tissue, but it might be unknown whether the sample contains tumor cells. Imaging can reveal the presence of targets which indicate the presence of a tumor, thus facilitating diagnosis. The tissue sample may comprise breast cancer tissue, for example human breast cancer tissue or human mammary epithelial cells (HMLE). The tissue sample may comprise formalin-fixed, paraffin-embedded (FFPE) tissue. The tissues can be obtained from any living multicellular organism, but will usually be human.

[0160] The tissue sample will usually be a section e.g., having a thickness within the range of 2-10 μιη, such as between 4-6 μιη. Techniques for preparing such sections are well known from the field of IHC e.g., using microtomes, including dehydration steps, including embedding, etc. Thus a tissue may be chemically fixed and then sections can be prepared in the desired plane. Cryosectioning or laser capture microdissection can also be used for preparing tissue samples. Samples may be permeabilised e.g., to permit of reagents for labelling of intracellular targets (see above).

[0161] The size of a tissue sample to be analyzed will be similar to current IHC methods, although the maximum size will be dictated by the laser ablation apparatus, and in particular by the size of sample which can fit into its ablation chamber. A size of up to 5 mm x 5 mm is typical, but smaller samples (e.g., 1 mm x 1 mm) are also useful (these dimensions refer to the size of the section, not its thickness). [0162] In addition to being useful for imaging tissue samples, the disclosure can instead be used for imaging of cellular samples such as monolayers of adherent cells or of cells which are immobilized on a solid surface (as in conventional immunocytochemistry). These embodiments are particularly useful for the analysis of adherent cells that cannot be easily solubilized for cell-suspension mass cytometry. Thus, as well as being useful for enhancing current immunohistochemical analysis, the disclosure can be used to enhance

immunocytochemistry.

[0163] After being prepared, the sample will be placed into a laser ablation chamber and then subjected to analysis according to the disclosure.

Single cell analysis

[0164] Methods of the disclosure include laser ablation of multiple cells in a sample, and thus plumes from multiple cells are analyzed and their contents are mapped to specific locations in the sample to provide an image. In most cases a user of the method will need to localize the signals to specific cells within the sample, rather than to the sample as a whole. To achieve this, the boundaries of cells (e.g., the plasma membrane, or in some cases the cell wall) in the sample can be demarcated.

[0165] Demarcation of cellular boundaries can be achieved in various ways. For instance, a sample can be studied using conventional techniques, which can demarcate cellular boundaries, such as microscopy as discussed above. When performing these methods, therefore, an LA-ICP-MS system comprising a camera as discussed above is particularly useful. An image of this sample can then be prepared using a method of the disclosure, and this image can be superimposed on the earlier results, thereby permitting the ICP-MS signals to be localized to specific cells. Indeed, as discussed above, in some cases the laser ablation may be directed only to a subset of cells in the sample as determined to be of interest by the use of microscopy-based techniques.

[0166] To avoid the need to use multiple techniques, however, it is possible to demarcate cellular boundaries as part of the imaging method of the disclosure. Such boundary demarcation strategies are familiar from IHC and immunocytochemistry, and these approaches can be adapted by using labels, which can be detected by ICP-MS. For instance, the method can involve labelling of target molecule(s), which are known to be located at cellular boundaries, and signal from these labels can then be used for boundary demarcation. Suitable target molecules include abundant or universal markers of cell boundaries, such as members of adhesion complexes (e.g., β-catenin or E-cadherin). Some embodiments can label more than one membrane protein in order to enhance demarcation. [0167] In addition to demarcating cell boundaries by including suitable labels, it is also possible to demarcate specific organelles in this way. For instance, antigens such as histones (e.g., H3) can be used to identify the nucleus, and it is also possible to label mitochondrial- specific antigens, cytoskeleton-specific antigens, Golgi-specific antigens, ribosome-specific antigens, etc., thereby permitting cellular ultrastructure to be analyzed by methods of the disclosure.

[0168] Signals which demarcate the boundary of a cell (or an organelle) can be assessed by eye, or can be analyzed by computer using image processing. Such techniques are known in the art for other imaging techniques e.g., reference 22 describes a segmentation scheme that uses spatial filtering to determine cell boundaries from fluorescence images, reference 23 discloses an algorithm which determines boundaries from brightfield microscopy images, reference 24 discloses the CellSeT method to extract cell geometry from confocal microscope images, and reference 25 discloses the CellSegm MATLAB toolbox for fluorescence microscope images. A method which is useful with the disclosure uses watershed

transformation and Gaussian blurring. These image processing techniques can be used on their own, or they can be used and then checked by eye.

[0169] Once cellular boundaries have been demarcated it is possible to allocate signal from specific target molecules to individual cells. It can also be possible to quantify the amount of a target analyte(s) in an individual cell e.g., by calibrating the methods against quantitative standards. Methods of the disclosure are highly quantitative. In comparison with known methods they do not suffer from sample autofluorescence, there are minimal or no matrix effects as compared to those common in MALDI and SIMS imaging, there is no need for an amplification step such as is often applied in IHC, the tissue can be completely sampled, and the method has a wide dynamic range of ~10⁵.

General

[0170] The term "comprising" encompasses "including" as well as "consisting" e.g., a composition "comprising" X may consist exclusively of X or may include something additional e.g., X + Y.

[0171] The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

[0172] The word "substantially" does not exclude "completely" e.g., a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the disclosure.

Other [0173] Combination of analog and digital detection works well for mass cytometry.

[0174] Finally, it should be noted that there are alternative ways of implementing the embodiments disclosed herein. Accordingly, the present embodiments are to be considered as illustrative and not restrictive. Furthermore, the claims are not to be limited to the details given herein, and are entitled their full scope and equivalents thereof.

EXAMPLES

Example 1

Ablation system using a 213 nm Nd:YAG laser compared to a 193 nm excimer laser

[0175] To make an ablation system for testing, a 213 nm Nd:YAG laser (either FQSS- 213-Q4 from CryLas or Compiler 213 from Passat Ltd) was focused to a spot size of 1 μιη. In detail, a 400 μιη diameter aperture (Edmund optics) or a variable iris (Thorlabs) was used for spatial filtering. The filtered beam was diverged by a negative lens (Thorlabs) with 10 mm focal length and then converged by a bi-convex lens (Thorlabs) with 20 mm focal length. This formed a focal point for the beam. The focal point was then reimaged by a microscope objective, which was a x25 Schwarzschild Cassegrain objective lens (Model 5003-190 from Davin Optronics).

[0176] The laser was directed into an ablation chamber (made at Fluidigm Canada, Inc., approx. dimensions 15 cm x 15 cm x 5 cm) with a mobile sample stage. The sample stage was made at Fluidigm Canada, Inc., using commercially available SLC-24 positioners from Smaract for horizontal movement, and a P-603 piezo actuator from Physik Instrumente for vertical movement. This chamber used the design set out in reference 9. The system also included a camera focused to the same point as the ablation laser using the same objective lens as the ablation laser. The sample was illuminated for optical imaging with the camera by an LED light source. The ablation chamber was connected to a CyTOF2 mass cytometer (Fluidigm Canada, Inc.) for ionization and detection of material ablated in the chamber.

[0177] A second system was made that used a 193 mm argon fluoride excimer laser. The beam was homogenized using a homogenizer (Microlens Array 10 mm x 10 mm, 300 μιη Pitch, 0.5° Divergence; Part 64-478; Edmund Optics or an EDC engineered diffuser from RPC Photonics). The beam was focused to a large spot size on a scale of 1 mm by a lens with focal distance of 100 mm (Thorlabs). It was then masked by an aperture with 25 micrometer diameter (Thorlabs). The remaining light was refocused onto the target by the same reflecting objective. A 1 micrometer ablation spot at the target was achieved using a x25 Schwarzschild Cassegrain objective lens (Model 5003-190 from Davin Optronics). [0178] The two systems were programmed to ablate the sample with 2 μιη spacing between the ablated spots in both horizontal dimensions and a test substrate was inserted into the systems to record the spots produced by each system. The results of this experiment are shown in Figures 1 and 2 for the Nd:YAG and excimer systems, respectively. The figures show comparable laser spots produced by the two systems.

Example 2

Ablation of biological samples by a 213 nm Nd:YAG laser

[0179] The Nd:YAG system described in Example 1 was tested for ablation of biological materials. An exemplary image from this testing is presented in Figure 3, which shows the ablation of a cell pellet by the system. Here, ablation was performed with a 1 μιη spot size, moving the laser in 1 μιη steps. Figure 3 shows complete ablation of the biological material in the areas targeted by the laser.

Example 3

Subcellular resolution mass cytometry imaging using

a system based on a 213 nm Nd:YAG laser

[0180] The Nd:YAG system described in Example 1 was tested for its ability to perform laser ablation imaging, as discussed recently in reference 5. Imaging was performed using two antibodies on a tissue sample. A first antibody binding to the pS6 (a component of the ribosomal 40S subunit) was labelled with an isotope of Ytterbium with mass 170 (¹⁷⁰Yb), as shown in Figure 4A. This image therefore shows staining of the cytoplasm. A second antibody binding to the H3 (a histone protein) was labelled with an isotope of Ytterbium with mass 176 (¹⁷⁶Yb), as shown in Figure 4B. This antibody therefore stains the nucleus. The A and B images are overlaid in Figure 4C.

[0181] It is appreciated that the previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure.

Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0182] Different arrangements of the components depicted in the drawings or described above, as well as components and steps not shown or described are possible. Similarly, some features and sub-combinations are useful and may be employed without reference to other features and sub-combinations. Embodiments of the invention have been described for illustrative and not restrictive purposes, and alternative embodiments will become apparent to readers of this patent. Accordingly, the present invention is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications may be made without departing from the scope of the claims below.

[0183] List of Reference, each of which are incorporated herein by reference in their entirety:

I] Tanner et al. Cancer Immunol Immunother (2013) 62:955-965

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4] Giesen et al. (2011) Anal. Chem. 83:8177-83.

5] Giesen et al. (2014) Nature Methods. 11 All -All.

6] International Application Publication No. WO 2005/121864

7] Kindness et al. (2003) Clin Chem 49: 1916-23.

8] Gurevich and Hergenroder (2007) J. Anal. At. Spectrom., 22: 1043-1050.

9] Wang et al. (2013) Anal. Chem. 85:10107-16.

10] International Application Publication No. WO 2014/146724.

I I] International Application Publication No. WO 2014/127034.

12] Herbert and Johnstone, Mass Spectrometry Basics, CRC Press 2002.

13] Bandura et a/. (2009) Anal. Chem., 81 :6813-22.

14] Bendall et al. (2011) Science 332, 687-696.

15] Bodenmiller et al. (2012) Nat. Biotechnol. 30:858-867.

16] U.S. Patent No. 7479630.

17] Robichaud et al. (2013) J Am Soc Mass Spectrom 24(5):718-21.

18] Klinkert et al. (2014) IntJMass Spectrom http://dx.doi.Org/10.1016/j.ijms.2013.12.012.

19] Qiu et al. (2011) Nat. Biotechnol. 29:886-91.

20] Briickner et al. (2013) Anal. Chem. 86:585-91.

21] Gao and Yu (2007) Biosensor Bio electronics 22:933-40.

22] Arce et al. (2013) Scientific Reports 3, article 2266.

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24] Pound et al. (2012) The Plant Cell 24: 1353-61.

25] Hodneland et al. (2013) Source Code for Biology and Medicine 8: 16.

Claims

CLAIMS What is claimed is:

1. An imaging mass cytometry system for imaging a biological sample, the system comprising:

a solid state laser for producing a beam for ablating the biological sample; an ablation chamber comprising a stage for supporting the biological sample; an arrangement of one or more lenses and/or one or more apertures located between the solid state laser and the stage in the ablation chamber, and adapted to focus the beam from the solid state laser to a spot size of less than 2 um for ablating the biological sample;

an inductively coupled plasma torch coupled with the ablation chamber and configured to receive ablated portions of the biological sample; and

a mass spectrometer coupled with the inductively coupled plasma torch.

2. The imaging mass cytometry system of claim 1, wherein the spot size is 1 μιη, or less than 1 μιη, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 100 nm, or less than 100 nm.

3. The imaging mass cytometry system of claim 1 or claim 2, wherein:

(i) the one or more lenses includes a Schwarzschild Cassegrain objective lens; and/or

(ii) the one or more apertures includes a diaphragm or an array of substitutable apertures.

4. The imaging mass cytometry system of any preceding claim wherein the solid state laser:

(i) is an Nd doped laser, such as an Nd:YAG laser; and/or

(ii) emits a beam of wavelength 213 nm.

5. The imaging mass cytometry system of any preceding claim wherein the solid state laser emits a beam with a Gaussian profile or an approximately Gaussian profile.

6. The imaging mass cytometry system of claim 5, wherein the sample is ablated with a non-homogenized beam.

7. The imaging mass cytometry system of any preceding claim wherein the system is adapted to ablate the sample at a rate of more than 40 Hz, such as 50 Hz or more, 100 Hz or more, 200 Hz or more, 300 Hz or more, 400 Hz or more, 500 Hz or more, or 1000 Hz or more.

8. The imaging mass cytometry system of any one of claims 1-7, further comprising a camera directed on the sample stage.

9. A imaging mass cytometry system for imaging a biological sample, the system comprising:

a laser;

an ablation chamber comprising a stage;

an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to ablate the biological sample on the stage by focusing a beam from the laser to a spot size of less than 2 μιη;

an inductively coupled plasma torch;

a mass spectrometer; and

a camera directed on the sample stage.

10. The imaging mass cytometry system of 9, wherein the camera is connected to a microscope, optionally a confocal microscope.

11. The imaging mass cytometry system of any one of claims 9-10, wherein the focal point of the camera or microscope is the focal point of the laser beam.

12. The imaging mass cytometry system of any one of claims 9-11, which comprises an laser or LED light source directed on the sample stage for exciting a sample on the stage of the imaging mass cytometry system.

13. The imaging mass cytometry system of claim 12, wherein the laser that is for exciting the sample in the imaging mass cytometry system is the laser that is for ablating the sample.

14. The imaging mass cytometry system of any one of claims 9-13, which further comprises an optical filter and/or an array of lenses located between the camera and the sample.

15. The imaging mass cytometry system of any one of claims 1-14, comprising a stage for holding a sample to be ablated, in which the stage for holding the sample is mobile within the ablation chamber.

16. An imaging mass cytometry system comprising:

a laser;

an ablation chamber comprising a stage, wherein the stage for holding the sample is mobile within the ablation chamber;

an arrangement of one or more lenses and/or one or more apertures located between the laser and the stage in the ablation chamber, and adapted to focus a beam from the laser to a spot size of less than 2 μιη to ablate the biological sample on the stage;

an inductively coupled plasma torch; and

a mass spectrometer.

17. The imaging mass cytometry system of claim 16, in which the stage for holding a sample is mobile in the x, y, and z axes within the ablation chamber.

18. The imaging mass cytometry system of claim 17, in which the stage can be moved in increments of less than 10 μιη, such as less than 5 μιη, less than 4 μιη, less than 3 μιη, less than 2 μιη, 1 μιη, or less than 1 μιη, in the:

(i) x and y axes; and/or

(ii) z axis.

19. The imaging mass cytometry system of claim 17 or 18 in which the range of movement in the x and/or y axis, or horizontal plane, is at least 40 μιη, at least 50 μιη, at least 75 μηι, at least 100 um, at least 250 μιη, at least 500 μιη, at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 10 mm, at least 20 mm, at least 30 mm, at least 50 mm, or at least 75 mm.

20. The imaging mass cytometry system of any preceding claim, in which a gap of at least 10 mm is maintained between the side wall of the ablation chamber and the edge of the sample stage.

21. The imaging mass cytometry system of any preceding claim, wherein the MS is a time-of-flight MS.

22. The imaging mass cytometry system of any preceding claim, wherein laser ablation occurs in an ablation chamber having a washout time of 50 ms or less, such as 25 ms or less or 10 ms or less.

23. A method of imaging a biological sample comprising a plurality of cells using the imaging mass cytometry system of any of the preceding claims.

24. A method of imaging a biological sample comprising a plurality of cells, the method comprising steps of:

(i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample;

(ii) illuminating a known location of the sample with light to excite the one or more fluorescent labels;

(iii) observing and recording whether there is fluorescence at the location;

(iv) if there is fluorescence, directing laser ablation at 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 further known locations on the sample, whereby detection of labelling atoms in the plumes permits construction of an image of the sample of the areas which have been ablated;

wherein the laser used for laser ablation is:

(i) a laser emitting light at 213 nm, such as an Nd:YAG laser; or

(ii) an excimer or exciplex laser.

25. The method of any one of claims 24, wherein the light that is used to excite the one or more fluorescent labels is from the laser that ablates material from the biological sample.

26. The method of any one of claims 24-25, wherein at least 20 different labelling atoms having different atomic masses are used.

27. The method of any one of claims 24-26, wherein the labelling atoms are transition metals such as lanthanides, optionally wherein the labelling atoms have atomic masses within the range 80-250 amu.

28. The method of any one of claims 24-27, wherein the mass spectrometry step uses time-of-fiight detection.

29. The method of any one of claims 24-28, wherein target molecules are quantified.

30. An imaging mass cytometry system of any one of claims 1-23, further comprising a control module for performing the method of any one of claims 24-29.

31. A mass cytometry system, comprising:

a laser ablation apparatus;

an injector operably coupled to the laser ablation apparatus;

a scanning TOF mass spectrometer operably coupled to the injector with a sensitivity of at least 500 copies of affinity molecules per ablation area; wherein,

the laser ablation apparatus is configured to provide a sample spatial resolution of less than 1 μιη.

32. The mass cytometry system of claim 31 , wherein the system is an imaging mass cytometry system.

33. The mass cytometry system of claim 31 , wherein the laser ablation apparatus comprises a laser characterized by an emission wavelength within a range of 190 nm to 300 nm.

34. The mass cytometry system of claim 31 , wherein the laser ablation apparatus comprises a laser characterized by an emission wavelength of 213 nm.

35. The mass cytometry system of claim 31 , wherein the laser is configured to provide a pulse duration less than 30 ns.

36. The mass cytometry system of claim 31 , wherein the laser is configured to provide a pulse duration less than 1 nsec.

37. The mass cytometry system of claim 31 , wherein the system is characterized by a sensitivity of 500 affinity molecules per ablation area.

38. The mass cytometry system of claim 31 , wherein the TOF mass spectrometer is characterized by a scan rate of at least 200 kHz.

39. The mass cytometry system of claim 31 , wherein the laser ablation apparatus is configured to provide an ablation area on a surface of a sample of 100 x 100 (10⁴) μιη² to 1000x 1000 (10⁶) μιη².

40. The mass cytometry system of claim 31 , wherein the scanning TOF mass spectrometer comprising an orthogonal accelerator configured to provide a voltage from 700 V to 1000 V.

41. The mass cytometry system of claim 31 , wherein the scanning TOF mass spectrometer comprises a straight TOF section.

42. The mass cytometry system of claim 31 , wherein the system is configured to provide a sampling rate of at least 1 kHz.

43. The mass cytometry system of claim 31 , wherein a gas flow into the injector is at least 1 slpm.

44. The mass cytometry system of claim 31 , wherein the gas flow comprises helium.

45. The mass cytometry system of claim 31 , wherein the injector comprises an injection tube, wherein the injection tube comprises a tip having diameter less than 1 mm.

46. The mass cytometry system of claim 45, wherein the tip diameter is about 0.85 mm.

47. The mass cytometry system of claim 31 , wherein the system is configured to provide simultaneous measurement of from 100 to 150 mass tags.

48. The mass cytometry system of claim 31 , wherein the system is configured to provide simultaneous measurement of from 20 to 30 mass tags.

49. The mass cytometry system of claim 31 , wherein the TOF mass spectrometer is configured to provide measurement of a mass range from 75 amu to 214 amu.

50. The mass cytometry system of claim 31 , wherein the system is characterized by a dynamic range from 4 to 5 orders of magnitude.

51. The mass cytometry system of claim 31 , wherein the laser ablation apparatus comprises a laser ablation chamber and a sample mounted within the laser ablation chamber.

52. The mass cytometry system of claim 51 , wherein the sample comprises a plurality of mass probes.

53. The mass cytometry system of claim 52, wherein each of the plurality of mass probes comprises an affinity probe coupled to a nanoparticle, wherein a plurality of the mass tags is coupled to each of the nanoparticles.

54. The mass cytometry system of claim 53, wherein the plurality of mass tags is at least 400.

55. The mass cytometry system of claim 31 , wherein,

the laser ablation apparatus comprises an ablation chamber and a plasma torch;

the injector having a first end and a second end, wherein the second end is inserted into the plasma torch; and

the system comprises a sampling tube, wherein,

the sampling tube extends from the ablation chamber to the first end of the injector; and

the sampling tube is characterized by a length from 30 mm to 300 mm.

56. The mass cytometry system of claim 55, wherein the sampling tube is flexible.

57. A method for imaging a biological sample, the method comprising:

receiving the biological sample on a stage in an ablation chamber, the biological sample labeled with labeling atoms specific for target molecules of the biological sample; ablating at least a portion of the biological sample labeled with labeling atoms at a location with a laser emitted from a solid-state laser to eject the portion of the biological sample, the laser emitted from the solid-state laser being focused onto the portion of the biological sample with a spot size of 2 μιη or less;

receiving the ejected portion of the biological sample into an inductively coupled plasma to atomize and ionize the ejected portion of the biological sample; and

analyzing the atomized and ionized portion of the biological sample to detect labeling atoms ejected from the location on the biological sample.

58. The method of claim 57, wherein the biological sample is ablated using a non- homogenized laser from the solid-state laser.

59. The method of claim 57, wherein the biological sample is ablated using a Gaussian or approximately Gaussian laser from the solid-state laser.

60. The method of claim 57, wherein the stage is moveable within the ablation chamber.

61. The method of claim 60, wherein the stage is moveable in increments of less than 2 μιη in the x and y axes within the ablation chamber.

62. The method of claim 60, further comprising translating the stage so as to move the biological sample relative to a focal point of the solid-state laser such that the focal point of the solid-state laser is moved to a second portion of the biological sample labeled with labeling atoms; and ablating the second portion of the biological sample at a second location with a laser emitted from the solid-state laser to eject the second portion of the biological sample, the laser emitted from the solid-state laser being focused onto the second portion of the biological sample with a spot size of less than 2 μιη.

63. The method of claim 62, wherein ablation of the second portion of the biological sample is triggered by fluorescent properties of the biological sample at the second location.

64. The method of claim 63, wherein the second location of the biological sample is labeled with fluorescent tags.

65. The method of claim 64, wherein the solid-state laser is configured to excite the fluorescent tags at the second location.

66. The method of claim 57, wherein the laser emitted from the solid-state laser is focused onto the portion of the biological sample with a 1 μιη or less spot size.

67. The method of claim 57, wherein the laser emitted from the solid-state laser is focused using one or more apertures.

68. The method of claim 66, wherein the one or more apertures includes a diaphragm or an array of substitutable apertures.

69. The method of claim 57, wherein the solid-state laser comprises an Nd doped laser.

70. The method of claim 57, wherein the solid-state laser emits a laser having a 213 nm wavelength.

71. The method of claim 57, further comprising labeling the biological sample with labeling atoms by attaching an affinity probe to the target molecule, the affinity probe being coupled with a nanoparticle comprising the labeling atoms.

72. A method for analyzing a biological sample, the method comprising:

receiving the biological sample on a stage in an ablation chamber, the biological sample labeled with labeling atoms specific for target molecules of the biological sample; ablating at least a portion of the biological sample labeled with labeling atoms at a location with a laser emitted from a laser system to eject the portion of the biological sample, the laser emitted from the laser system being focused onto the portion of the biological sample with a spot size of 2 μιη or less; and

analyzing the ejected portion of the biological sample to detect labeling atoms ejected from the location on the biological sample.

73. The method of claim 71, wherein the biological sample is ablated using a non- homogenized laser from the laser system.

74. The method of claim 72, wherein the biological sample is ablated using a Gaussian or approximately Gaussian laser from the laser system.