Plasma and sampling geometries for imaging mass cytometry

Abstract

To provide systems and method for imaging mass spectrometry, including imaging mass cytometry.SOLUTION: Apparatus and method for imaging mass spectrometry (IMS) that improve speed of sample acquisition, signal sensitivity, and / or signal stability may minimize the transfer time of and / or the spread of plume of a sample material removed from a sample to be transferred to components of the imaging mass spectrometer or mass cytometer that ionize and analyze the sample material.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 114,313, filed November 16, 2020, and U.S. Provisional Patent Application No. 62 / 951,556, filed December 20, 2019, the contents of both of which are incorporated herein by reference for all purposes.

[0002] Background of the Invention Imaging mass spectrometry applications, such as imaging mass cytometry, benefit from the rapid acquisition of distinct ablation plumes. For example, laser ablation ICP-MS systems can ablate spots on the order of one micron in diameter and independently detect the elemental or isotopic composition of millions of spots within a single sample. Rapid delivery of the ablation plume with minimal transient diffusion can improve sensitivity by enabling rapid analysis of many ablation spots. Transient diffusion can be increased by turbulence caused by changes in the orientation of the plume transport during formation compared to the direction of plume expansion. System components, such as optical components, can inhibit the rapid injection of the plume into the plasma source. Furthermore, altering the conventional composition of the gas stream can improve signal sensitivity and / or stability.

[0003] The present invention relates to devices and methods for laser ablation-based imaging mass spectrometry, including imaging mass cytometry. Summary of the Invention

[0004] In this invention, the inventors have devised a number of developments of existing laser ablation-based imaging mass cytometers and imaging mass spectrometers, particularly those related to improvements that minimize the transit time and / or minimize the diffusion of the plume of ablated sample material from the delivery sample to the imaging mass spectrometer or mass cytometer components that ionize and analyze the sample material.

[0005] An apparatus of the present invention, such as an imaging mass spectrometer or imaging mass cytometer, typically includes three components. The first component is a laser ablation system for generating a plume of vapor and particulate material from a sample for analysis. Before atoms in the plume of ablated sample material (including any detectable label atoms described below) can be detected by a mass spectrometer component (the MS component; the third component), the sample must be atomized and ionized. (Some ionization of the sample material may occur during ablation, but space charge effects cause charge neutralization long before the charge is detectable, thus requiring a separate ionization component.) Therefore, the apparatus includes a second component, an ionization system, that ionizes elements to form elemental ions, allowing their detection by the MS component based on their mass-to-charge ratio. A transfer conduit is provided between the laser ablation system and the ionization system, the transfer conduit being adapted to couple the laser ablation system to the ionization system, the transfer conduit having an inlet positioned within the laser ablation system and configured to capture the ablated plume as it is generated and deliver the captured, ablated plume to the ionization system. (In some cases, the ionization system is a plasma, such as an inductively coupled plasma (ICP), and the transfer conduit is the same conduit that introduces the sample directly into the ICP torch via a central injector tubing; in this case, the transfer conduit may be referred to as an injector.) Thus, during operation, a sample is delivered to the device, ablated to produce vapor / particle material, ionized by the ionization system, and the sample ions are passed into the MS component. While the MS component can detect many ions, most of these will be ions of atoms that naturally comprise the sample. For some applications, such as typical analysis of minerals in geological or archaeological applications, this may be sufficient. In imaging mass spectrometry applications such as imaging mass cytometry, the MS component can be time-of-flight (TOF) or magnetic sector MS.

[0006] Therefore, the present invention provides (i) a laser ablation system adapted to generate a plume of sample material from the sample; (ii) a plasma source adapted to receive material removed from the sample by the laser ablation system and ionize the material to form elemental ions; (iii) a mass spectrometer for receiving elemental ions from the ionization system and analyzing the elemental ions; the laser ablation system and the ionization system are coupled together by a transfer conduit adapted to convey a gas stream containing the plume of ablated sample material from the laser ablation system to the ionization system; The plasma is not oriented along the same axis as the sample stage.

[0007] The present invention also provides An apparatus is provided, comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; a plasma source; and an injector configured to deliver an ablation plume created from the sample by the laser ablation source to the plasma source, wherein at least one of the plasma source and the sample stage are oriented orthogonal to one another. For example, the plasma can be oriented more than 60 degrees, more than 70 degrees, or more than 80 degrees, such as 90 degrees, away from any axis of the sample stage (the axis of a planar sample mounted on the sample stage).

[0008] In certain embodiments, the injector is rigid and / or straight. The inner diameter of the injector can be less than 2 mm, less than 1 mm, or less than 0.5 mm. The length of the injector is less than 20 cm, less than 10 cm, less than 5 cm, or less than 3 cm. The device can be configured to direct the laser on a path that does not pass through the injector.

[0009] The device may be operable to deliver at least 500, 1000, 5000, 10000, or 50000 distinct ablation plumes to the ICP source per second. In certain embodiments, a short plasma (e.g., less than 5 mm, less than 3 mm, less than 2 mm in length) helps prevent transient diffusion, allowing ions from distinct ablation plumes to remain distinct by mass spectrometry detection.

[0010] The apparatus may further comprise a mass spectrometer (MS) coupled to the plasma source, such as a time-of-flight or magnetic sector mass spectrometer, The MS is configured to receive a vertical beam of ions.

[0011] In certain embodiments, the sample stage may be vertical and operable to move in a vertical position (e.g., adjusted by a motor capable of countering gravity while still providing steps on the scale of the diameter of the ablation spot).

[0012] In certain embodiments, the plasma source may be vertically oriented. The plasma source may be vacuum sealed (e.g., excluding the injector inlet to the plasma source).

[0013] The plasma source may be an inductively coupled plasma torch (ICP source). For example, the apparatus may be an LA-ICP-MS system.

[0014] The method can include analyzing a sample by LA-ICP-MS using an apparatus described herein. The sample can be a biological sample and can include a labeled atom (such as a labeled atom of SBP attached to an analyte of the biological sample). The method can further include labeling the sample with the labeled atom before analyzing the sample by LA-ICP-MS.

[0015] Aspects of the subject application also include apparatus and methods for introducing hydrogen-containing molecules into an ICP torch in an LA-ICP-MS, e.g., for signal enhancement. The hydrogen-containing molecule can be a gas, such as hydrogen gas, ammonia, or methane, as described further below. Alternatively or additionally, a hydrogen-containing molecule, such as water or an alcohol (e.g., ethanol), can be introduced into the gas stream as a vapor, as described further below. [Brief explanation of the drawings]

[0016] Those skilled in the art will understand that the drawings described below are for illustrative purposes only and are not intended to limit the scope of applicant's described subject matter in any way.

[0017] [Figure 1] FIG. 1 is a diagram of a laser ablation system showing sampling of the laser ablated plume through an aperture configured to convey the plume to an injector. [Figure 2] Figure 2 is a diagram of the horizontal plasma sampling configuration. [Figure 3] FIG. 3 is a diagram of the sampling configuration with a vertical plasma. [Figure 4] FIG. 4 is a schematic diagram of a typical LA-ICP-MS system in the art. [Figure 5] FIG. 5 is a graph of the sensitivity (average counts of Lu ions per shot) as a function of the humidity (in microbars) of the argon carrier gas. [Figure 6] FIG. 6 is an illustration of a humidification system suitable for LA-ICP-MS. [Figure 7] FIG. 7 is a graph of sensitivity (Lu ion counts per shot) over a long sample transfer (60,000 seconds) while maintaining the humidity of the argon carrier gas. [Figure 8] FIG. 8 is a graph of sensitivity (average counts of Lu ions per shot) as a function of hydrogen gas flow (L / min). [Figure 9]Figure 9 compares the improvement in image signal in imaging mass cytometry by humidification (left) or by using a premixed gas source of 3% hydrogen / helium for the trapping gas (right). DETAILED DESCRIPTION OF THE INVENTION

[0018] The indefinite articles "a" or "an," as used in conjunction with the present description in reference to various elements, are to be understood to encompass "one or more" or "at least one," unless the context clearly dictates otherwise.

[0019] The term "comprising" encompasses "including" in addition to "consisting only of," e.g., a composition comprising X may consist solely of X or may include additional items such as X plus Y.

[0020] The term "about" in relation to a numerical value x is used optionally and means, for example, x±10%.

[0021] The term "substantially" does not exclude "completely", for example, a composition that is "substantially free" of Y may be completely free of Y. If desired, the term "substantially" may be omitted from the definition of the present invention.

[0022] The term "invention" does not refer to all embodiments of the invention, but rather to particular aspects, examples, or embodiments of the invention.

[0023] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be understood by those skilled in the relevant art that, once familiar with the present disclosure, various changes in form and detail are possible without departing from the scope of the invention as defined by the appended claims. Accordingly, the present invention is not limited to the precise components or details of the methods or structures described above. Except to the extent necessary or inherent in the process itself, no particular order is intended or implied in the steps or stages of the methods or processes described in the present disclosure, including the drawings. In many cases, the order of process steps may be changed without altering the purpose, effect, or impact of the described methods. All publications and patent documents cited herein are incorporated by reference as if each such publication or patent document was specifically and individually indicated to be incorporated by reference herein. Citation of publications and patent documents (patents, published patent applications, and unpublished patent applications) is not intended as an admission that any such document is relevant prior art, nor does it constitute any admission as to the contents or publication date of the same.

[0024] The present invention relates to imaging mass spectrometry, including laser ablation combined with inductively coupled plasma mass spectrometry (LA-ICP-MS), which has been described for the measurement of endogenous elements in biological materials and, more recently, for imaging by detection of element-tagged antibodies. See, for example, U.S. Patent Publication No. 2012 / 0061561 to Antonov, A. and Bandura, D., published in 2012, which are incorporated herein by reference; Seuma et al., "Combination of immunohistochemistry and laser ablation ICP mass spectrometry for imaging of cancer biomarkers," Proteomics 8:3775-3784, published in 2008; "Imaging and spatial distribution of β-amyloid peptide and metal ions in Alzheimer's plaques by laser ablation-inductively coupled plasma-mass spectrometry," Analytical Biochemistry 346.2:225-233, published in 2005; and "Journal of Analytical Atomic Becker et al., "Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in elemental imaging of biological tissues and in proteomics," published in "Spectrometry" 22.7:736-744.", Binet et al., "Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry," Analytical Biochemistry 318:30-38, 2003; Quinn et al., "Simultaneous determination of proteins using an element-tagged immunoassay coupled with ICP-MS detection," Journal of Analytical Atomic Spectrometry 17:892-96, 2002; Sharma et al., "Sesbania drummondii cell cultures: ICP-MS determination of the accumulation of Pb and Cu," Microchemical Journal 81:163-69, 2005; and Giesen et al., "Multiplexed See "Immunohistochemical detection of tumor markers in breast cancer tissue using laser ablation inductively coupled plasma mass spectrometry." Plasma sources other than ICP are also within the scope of intended use.

[0025] Due to constraints imposed by nanopositioning stages, access requirements for high NA lenses and / or the desire to keep the sample as close as possible to the plasma in the IMC device, there are several different plasma and sampling orientations that are considered here and listed in the table below: [Table 1]

[0026] Rotating the plasma to an optimal orientation eliminates some of the undesirable characteristics of previous configurations (which resulted in transient broadening due to the 90-degree rotation and long transport length, and secondary contamination due to gravity returning large particles to the sample surface). Notably, vertical plasma was utilized in ICP-OES machines because the orientation made it easier to image along the axis of the torch (because the cylindrical symmetry of the plasma was preserved). In comparison, symmetry is broken in horizontal orientations due to convective lift of the plasma as it exits the torch body.

[0027] In certain embodiments, the torch can be a closed torch. A closed torch is airtight and has no fluid communication with the air outside the torch (e.g., other than the gas source supplying the gas flow). A closed torch can be vacuum-sealed. In certain embodiments, the laser ablation chamber is airtight and has no fluid communication with the air outside the instrument. In certain embodiments, the entire laser ablation ICP-MS system is airtight and prevents air from passing through the torch. Gas flow and gas dynamics within the closed torch can dominate over convective effects. A closed torch can behave nearly identically in any orientation (e.g., with similar ionization effects for the same gas flow, sample material, and induction conditions) and can be oriented vertically (e.g., facing away from or toward the gravity vector).

[0028] Transporting the removed material by gas flow poses several challenges in our application: strong plume rotation causes transient broadening, as does excessive total length of gas channel between sample and plasma. As a result, optimal pixel acquisition speed requires the plasma to be oriented as close as possible to the sample substrate perpendicular to it.

[0029] The limitation on the orientation choice is related to the specifications of the nanopositioning stage. Stages possessing the speed and dynamic positioning accuracy required for our application typically lack the maximum force required to support the sample and associated hardware (springs, fasteners, etc.) against gravity. This means that configurations involving vertically oriented samples are more challenging from an XYZ stage engineering perspective. However, such devices are relatively low risk in terms of plasma engineering, as we have already experienced configuring mass analyzers with horizontal plasmas.

[0030] Another option is to hold the sample horizontally and direct the plasma either upwards or downwards. Each of these configurations has its own potential advantages and disadvantages.

[0031] One advantage over an upwardly directed plasma is that the heat from the plasma rises away from the sample and into the interface region of the mass analyzer. Because of the liquid cooling already at the interface, this represents a small (possibly negligible) difference in the mechanical thermal budget. A drawback of this approach is that any removed material that is not captured by the gas flow (i.e., large particles) falls back onto the sample, causing crosstalk and contamination.

[0032] In the case of downward-directed plasmas, the risk of sample contamination from larger dislodged particles is not high because gravity pulls the particles away from the sample. However, heat rising from the plasma can pose a challenge as it rises toward the sample. This can be overcome by including a thermal break between the sample and the plasma containment, but this is done without potentially extending the gas path between the sample and the plasma, resulting in transient broadening. Convective forces can also create additional transient broadening in such plasmas.

[0033] The ablation light can, in principle, be incident on either side of the sample, however there are some trade-offs that must be considered.

[0034] If the ablation light is incident from the same side as the sample delivery / plasma, a compromise must be made between optical access to the ablation spot and sample delivery access, which may result in one or more of the following trade-offs: - Focusing optics significantly increase the minimum sample transfer distance. - Focusing optics are more difficult to fabricate and assemble. - The maximum achievable throughput is reduced. Any optical inspection of the sample area is a compromise between field of view, resolution, illumination uniformity, etc. - The fixtures will be less modular. On the other hand, having the ablation light incident from the opposite side of the sample delivery / plasma may result in one or more of the following trade-offs: - The ablation light must pass through the sample substrate or another substrate such as silica that is at least partially transparent to UV light, which means that a quartz microscope slide must be used for UV laser ablation. - Optical inspection of the sample area may be affected by light from the plasma if the sample transport passes through in a straight line. - The specimen holder and stage assembly must provide an aperture for the ablation / inspection optics, making the assembly more expensive / complex.

[0035] In certain embodiments, the laser ablation can be non-UV laser ablation (e.g., in the visible or infrared spectrum, such as a green laser ablation source). The non-UV laser can have characteristics, such as frequency and / or power, similar to the UV laser ablation sources described herein. This can allow laser radiation to pass through glass slides (commonly used for microscopy). However, biological samples, such as tissue sections, cell smears, or cell cultures, can be ablated better in the ultraviolet spectrum than in the visible (e.g., green) or infrared spectrum.

[0036] In certain embodiments, the sample can be treated with a compound (e.g., after staining with mass-tagged SBP) that aids in laser ablation (e.g., to reduce the laser ablation threshold). The compound can be a dye that absorbs at wavelengths of laser ablation, such as non-UV wavelengths (e.g., green or infrared). The compound can be dispersed nonspecifically throughout the sample.

[0037] In certain embodiments, counter-side ablation can be used to remove layers below the sample to remove portions of the sample from the slide, such as described by U.S. Patent Publication No. 20160194590, which is incorporated herein by reference. In certain embodiments, a compound for transferring kinetic energy to the sample (e.g., transitioning to a gaseous state under ablation) can be embedded within the sample itself.

[0038] As described herein, the laser can be a femtosecond (fs) laser. For example, a near-infrared fs laser can be operated at its second harmonic to provide laser illumination in the green range, or at its third harmonic to provide laser illumination in the ultraviolet range. Low wavelengths, such as green or ultraviolet, can enable high resolution (e.g., small spot size). When laser illumination is transmitted across a sample support to affect a sample, the sample support must be transparent to the laser illumination. Glass and silica are transparent to green wavelengths, while glass is opaque to UV, but silica is transparent. To enable high resolution while allowing the use of glass slides, an infrared fs laser can be operated at its second harmonic (e.g., approximately 50% conversion efficiency) to provide green laser illumination. It is worth noting that commercially available objective lenses are often best corrected in the green range. The resolution achieved with green or ultraviolet fs lasers can be sub-1 μm, 800 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, or 100 nm spot size.

[0039] For example, the ablation frequency of the laser system may be in the range of 200 Hz to 100 MHz, 200 Hz to 10 MHz, 200 Hz to 1 MHz, 200 Hz to 100 kHz, 500 Hz to 50 kHz, or 1 kHz to 10 kHz. The ablation frequency of the laser should be matched to the scanning speed of the laser scanning system, as described above.

[0040] Figure 2 shows several configurations where the plasma (e.g., ICP) is horizontal. The laser illumination is indicated by the shaded triangle. The sample is shown mounted on a wide sample stage.

[0041] Figure 2A shows a horizontal stage injecting into a horizontal plasma, as has been done previously. Figure 2A shows a horizontal sample sampled from above, where the sample is held on a horizontally oriented slide (also referred to as a cover glass or substrate) and removed (either from above or through the substrate from below) with the plume directed upward. The plume is trapped in the gas flow, rotated orthogonally, and enters the horizontal plasma, which can result in gravitational redeposition.

[0042] Figure 2B shows a bottom-sampling configuration in which a plume is directed downward from a horizontal substrate by ablation from below or ablation through the substrate from above. The plume is captured by the gas flow, rotated at right angles, and enters the horizontal plasma. Redeposition (e.g., due to gravity) cannot occur in this configuration.

[0043] Figure 2C shows a vertical sample. The ablation light can be directed either through the substrate (from behind) or from the sample side. The plume is directed horizontally, captured, and transported directly into the plasma by the gas flow without the need for rotation.

[0044] FIG. 3 shows several configurations similar to FIG. 2, but where the plasma (eg, ICP) is vertical.

[0045] Figure 3A shows a horizontal sample sampled from above, with the plasma positioned above the sample and the gas flow directed upward. The ablation light is directed either above or below the sample, and a plume is emitted upward. The plume is captured and carried to the plasma by the gas flow.

[0046] 3B shows a horizontal sample sampled from below, where the gas flow toward the plasma is directed downward, and the plasma is therefore positioned below the sample. The ablation light is applied from either above or below, and the ablated plume exits the sample ejected downward, is captured by the gas flow, and is carried directly into the plasma.

[0047] 3C shows a vertical sampler that samples from the side (e.g., through the sample). The plume leaves the substrate surface and can be captured by the gas flow and directed either upwards or downwards before being turned 90 degrees and entering the plasma.

[0048] The apparatus for the intended application may include one or more of the components described below.

[0049] Aspects include methods and systems for laser ablation mass cytometry analysis, in which pulses of a laser beam are directed at the sample to generate a plume of sample for each pulse, distinctly capture each plume for each pulse, transport each distinctly captured plume to an ionization system, ionize each distinctly captured and transported plume with the ionization system, and generate ions for mass analysis, and an apparatus for performing the method. In various embodiments, the apparatus includes a laser ablation system for generating an ablated plume from the sample and a transfer conduit adapted to couple the laser ablation system to the ionization system of the apparatus. In some embodiments, the transfer conduit can have an inlet positioned within the laser ablation system such that the inlet can be configured to capture the ablated plume as it is generated. A gas inlet can be coupled to the inlet of the transfer conduit for passing a gas therebetween to transport the captured ablated plume into the ionization system. If the ionization system is an ICP, the transfer conduit may be referred to as an injector if the output of the conduit is directly into the plasma of the ICP. The components of the laser ablation system, ionization system, and mass spectrometer are described individually in more detail below. As noted above, the focus of this invention is on improvements to the transfer conduit that connects the laser ablation system to the ionization system.

[0050] transfer conduit The transfer conduit forms a link between the laser ablation system and the ionization system, transporting the plume of sample material generated by the laser ablation system from the laser ablation system to the ionization system. Part (or all) of the transfer conduit can be formed, for example, by drilling a suitable material to create a lumen (e.g., a lumen with a circular, rectangular, or other cross-section) for transporting the plume. The transfer conduit can have an inner diameter ranging from 0.2 mm to 3 mm. In some embodiments, the inner diameter of the transfer conduit varies along its length. For example, the transfer conduit can be tapered at its end. The transfer conduit can have a length ranging from 1 centimeter to 100 centimeters. In some embodiments, the length is no more than 10 centimeters (e.g., 1 to 10 centimeters), no more than 5 centimeters (e.g., 1 to 5 centimeters), or no more than 3 cm (e.g., 0.1 to 3 centimeters). In some embodiments, the lumen of the transfer conduit is straight along the entire distance from the ablation system to the ionization system, or along nearly the entire distance. In some embodiments, the lumen of the transfer conduit is not linear over its entire distance but has a different orientation. For example, the transfer conduit may be rotated 90 degrees in increments. This configuration allows the plume generated by ablation of a sample in the laser ablation system to initially travel in a vertical plane while the axis at the inlet of the transfer conduit points linearly upward, and then to travel horizontally as the plume approaches the ionization system (e.g., an ICP torch, which is typically oriented horizontally to utilize convective cooling). In some embodiments, the transfer conduit is linear for a distance of at least 0.1 centimeter, at least 0.5 centimeter, or at least 1 centimeter from the opening at the inlet where the plume enters or is formed. In some embodiments, the transfer conduit is adapted to minimize the time required to transport material from the laser ablation system to the ionization system.

[0051] The injector of the device may include a transfer conduit as described herein.

[0052] Sample cone inlet The transfer conduit comprises an inlet within the laser ablation system that receives sample material removed from the sample in the laser ablation system and transports it to the ionization system. In some cases, the inlet of the laser ablation system is the source of all gas flow along the transfer conduit to the ionization system. In some cases, the inlet of the laser ablation system that receives material from the laser ablation system is an opening in the wall of a conduit through which a second "transport" gas flows from a separate transport stream inlet (e.g., as disclosed in WO2014146724 and WO2014147260). In this case, the transport gas forms a significant proportion, and in many cases, the majority, of the gas flow to the ionization system. The component that comprises the transport stream inlet, the inlet of the laser ablation system, and is the beginning of the transfer conduit that transports removed sample material toward the ionization system may also be referred to as a flow cell (as described in WO2014146724 and WO2014147260).

[0053] The carrier flow accomplishes at least three tasks: directing the plume entering the transfer conduit toward the ionization system, preventing plume material from contacting the sidewalls of the transfer conduit, forming a "protective zone" above the sample surface to ensure atmospheric control of the ablation plume, and increasing the flow velocity within the transfer conduit. In some embodiments, the viscosity of the trapping gas is lower than that of the primary carrier gas. This helps to confine the plume of sample material within the trapping gas to the center of the transfer conduit, minimizing diffusion of the sample material plume downstream of the laser ablation system (because the transport velocity is more constant and approximately flat at the center of the flow). The gas can be, for example, without limitation, argon, xenon, helium, nitrogen, or mixtures thereof. In some embodiments, the carrier gas is argon. Argon is particularly suitable for stopping the diffusion of the plume before it reaches the walls of the transfer conduit (and also helps improve instrument sensitivity when the ionization system is an argon-based ICP). The trapping gas is preferably helium. However, the trapping gas may be replaced by or include other gases, such as hydrogen, nitrogen, or water vapor. The dynamic viscosity (dynamic viscosity / density) of argon is approximately 1.3E at 25°C. -5 m 2 / sec, and in helium, it is about 1.2E -4 m 2 / sec. Thus, the difference in kinematic viscosity values ​​between argon and helium can be at least a factor of 5, or at least a factor of 10. In some embodiments, the trapping gas is helium and the carrier gas is helium.

[0054] The use of a sample cone can minimize the distance between the target and the conduit containing the gas transport stream. This improves sample material capture with less turbulence because the distance the capture gas must flow at the point of the cone is reduced, thereby reducing the dispersion of the plume of ablated sample material. The inlet of the transfer conduit is therefore the opening at the tip of the sample cone. The cone projects into the ablation chamber.

[0055] A further type of asymmetry is a cone formed from two halves of an ellipse that share a common height (z) and one base diameter (x diameter) but differ in the other base (y diameter) (or one ellipse and one circular half).

[0056] All of the above adaptations may be present within a single asymmetric sample cone for use with the present invention: for example, the cone may be asymmetrically truncated, may be formed from two halves of different elliptical cones, the cone may be asymmetrically truncated, may include one of multiple orifices, etc.

[0057] The sample cone is thus adapted to capture all or a portion of the plume of material removed from the sample within the laser ablation system. The sample cone is operably positioned adjacent to the sample, for example, by manipulating the sample within the laser ablation system on a movable sample carrier tray, as described in more detail below. As described above, the plume of removed sample material passes through an opening in the narrow end of the sample cone and enters the transfer conduit. In some embodiments, the diameter of the opening is a) adjustable, b) sized to prevent perturbation of the removed plume as it passes into the transfer conduit, and / or c) approximately equal to the cross-sectional diameter of the removed plume. In some embodiments, the diameter of the opening is between about 100 μm and 1 mm. For example, the diameter of the opening is between about 200 μm and 900 μm, such as between 300 μm and 800 μm. In some embodiments, the diameter of the opening is between about 500 μm and 700 μm. In some embodiments, the diameter of the opening is about 500 μm. In some embodiments, the diameter of the opening is about 700 μm.

[0058] Tapered Conduit Gas travels faster in tubing with a smaller internal diameter at the same flow rate. Therefore, by using tubing with a smaller internal diameter, the plume of ablated sample material carried in the gas stream can be transported more quickly over a specified distance at a given flow rate (e.g., from a laser ablation system to an ionization system in a transport conduit). One of the main factors in how quickly an individual plume can be analyzed is the amount of diffusion the plume experiences during the time between its creation by ablation and the time its constituent ions are detected as components in the instrument's mass spectrometer (detector transit time). Therefore, using a narrow transport conduit reduces the time between ablation and detection, thereby reducing diffusion because it has less time to occur, ultimately reducing the transit time of each ablation plume at the detector. A shorter transit time means that more plumes can be generated and analyzed per unit time, resulting in higher quality and / or faster images.

[0059] The taper can include a gradual change in the internal diameter of the transfer conduit along that portion of its length (i.e., the internal diameter of the tubing with a given cross-section decreases along the portion from the end of the portion toward the inlet (at the laser ablation system end) to the outlet (at the ionization system end). As shown in Figures 3B and 7C, the tapering of the transfer conduit is applicable to all embodiments of the apparatus described herein, regardless of whether they include a direct injector inlet, a sample cone, or any other structure at the end of the transfer conduit's inlet to the ionization system. A large volume of the conduit before tapering facilitates confinement of the material produced by ablation. When ablated particles fly away from the ablation spot, they travel at high velocity. Friction in the gas slows these particles, but the plume can still spread over submillimeter to millimeter scales. A sufficient distance to the wall helps confine the plume near the center of the flow.

[0060] Because the wide inner diameter section is simply shorter (on the order of 1-2 mm), the plume does not significantly contribute to the overall transient time if it remains in the longer section of the narrower inner diameter transport conduit for a longer period of time. Thus, the larger inner diameter section is used to capture the ablation products, keeping them near the central streamline where the gas flow velocity is more uniform, while the smaller inner diameter conduit is used to rapidly transport these particles to the ionization system. The plume expands a certain amount, determined at least in part by the particle size and the gas selected. The initial section of the injector tubing can be sized so that the majority of the plume falls near the central streamline, preventing the plume from spreading due to the development of a parabolic flow profile. The injector can have a taper that narrows the diameter of the injector tubing after the initial section, for example, to improve transport velocity.

[0061] In some embodiments, the taper begins within 50 mm of the ionization system inlet and continues to the transfer conduit. In some embodiments, the taper begins within 40 mm of the ionization system inlet, such as within 30 mm, 20 mm, 15 mm, or 10 mm of the ionization system inlet. In some embodiments, the taper begins within 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm downstream of the ionization system inlet. In some embodiments, the taper begins 1-2 mm downstream of the ionization system inlet.

[0062] The taper between the large inner diameter portion and the small inner diameter region can be made sufficiently gradual to avoid turbulence. For example, the taper can be at least 5 degrees. In some embodiments, the taper angle can be at least 10 degrees, such as at least 15 degrees, at least 20 degrees, at least 25 degrees, or 30 degrees or more, or even 60 degrees. In some embodiments, the taper is less than 40 degrees, such as less than 30 degrees, less than 25 degrees, less than 20 degrees, less than 15 degrees, or less than 10 degrees. In some embodiments, the taper is less than 8 degrees, such as less than 5 degrees, less than 4 degrees, less than 3 degrees, less than 2 degrees, or less than 1 degree. In some embodiments, the taper angle is between 10 and 30 degrees. In some embodiments, the taper angle can increase or decrease along the length of the taper.

[0063] In some embodiments, the length of the taper is at least 5 mm, e.g., at least 10 mm, at least 20 mm, at least 30 mm, at least 40 mm, or at least 50 mm, or at least 100 mm. In some embodiments, the length of the taper is less than 10 mm, e.g., less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or 1 mm or less.

[0064] The inner diameter of the transfer conduit may be x millimeters (mm) at the input end of the conduit but may taper 5 times to x / 5 mm near the output end (e.g., 4 mm at the input end and 800 μm at the output end). In some embodiments, the taper reduces the inner diameter of the transfer conduit by less than 5 times, such as 4 times or less, 3 times or less, or 2 times or less. The inner diameter is the longest cross-sectional measurement across the conduit. For example, if the conduit is circular, the inner diameter is simply the diameter of the circle, but if the conduit is rectangular, it is oblique. In some embodiments, the inner diameter of the conduit after the taper is narrower than 2 mm, e.g., narrower than 1.5 mm, narrower than 1.25 mm, narrower than 1 mm, narrower than 900 μm, narrower than 800 μm, narrower than 700 μm, narrower than 600 μm, or narrower than 500 μm. In some embodiments, the inner diameter of the conduit after the taper is narrower than 400 μm, narrower than 300 μm, narrower than 200 μm, or narrower than 100 μm.

[0065] The diameter of the narrow bore section is limited by the diameter corresponding to the occurrence of turbulence. The Reynolds number can be calculated for circular pipes and known flows. Generally, Reynolds numbers above 4000 indicate turbulent flow and should therefore be avoided. Reynolds numbers above 2000 indicate transitional flow (between non-turbulent and turbulent flow) and therefore may preferably be avoided. For a given gas mass flow, the Reynolds number is inversely proportional to the diameter of the conduit. Thus, in some embodiments, the inner diameter of the narrow bore section of the transfer conduit is narrower than 2 mm, e.g., narrower than 1.5 mm, narrower than 1.25 mm, or narrower than 1 mm, but larger than the diameter at which a flow of 4 liters per minute of helium into the conduit would have a Reynolds number of greater than 4000.

[0066] Rough or smooth, angular edges at the transition between the constant diameter portion of the transfer conduit and the taper can create turbulence in the gas flow. Therefore, in some embodiments, the transition to and from the taper should have smooth edges adapted to reduce the generation of turbulence. For example, the edges can be rounded or chamfered.

[0067] Devices with tapered conduits may also include a sample cone (optionally asymmetric). Those skilled in the art will appreciate that tapered conduits may be used in any of the devices described herein that use alternative transfer conduit arrangements, for example, as shown in Figures 2-10 and described in detail in subsequent sections of this specification.

[0068] Sacrificial Flow The risk of turbulence occurring within the conduit increases with higher flow velocities. This is particularly true when the transfer conduit has a small internal diameter (e.g., 1 mm or less). However, the inventors have discovered that high velocity transfer (300 m / s or greater) can be achieved within a small internal diameter transfer conduit if a light gas such as helium or hydrogen is used in place of the conventionally used argon as the gas carrier stream. In certain embodiments, a gas mixture containing primarily helium or hydrogen is used.

[0069] A problem arises if high-velocity transport forces a plume of removed sample material through the ionization system without generating an acceptable level of ionization. The level of ionization can decrease as the plasma temperature at the end of the torch decreases with increasing cooling gas flow. If the sample material plume does not ionize to a suitable level, its constituents (including any identifying atomic / elemental tags) will be undetectable by the mass spectrometer, and information from the removed sample material will be lost. For example, a sample may pass through the plasma at the end of the torch in an ICP ionization system so quickly that the plasma ions have insufficient time to interact with the sample material to ionize it. The inventors have discovered that this problem, caused by high flow rates and high-velocity transport within a narrow-bore transport conduit, can be solved by introducing a flow sacrificing system at the outlet of the transport conduit. The flow sacrificing system is adapted to receive the gas flow from the transport conduit and pass only the portion that flows forward (the central portion of the flow, including any plume of removed sample material) to the injector leading to the ionization system. To facilitate diffusion of gas from the transfer conduit within the flow sacrifice system, the outlet of the transfer conduit may be flared.

[0070] The flow rate sacrifice system is positioned near the ionization system such that the length of the piping (e.g., injector) leading from the flow rate sacrifice system to the ionization system is short (e.g., 1 cm long; compared to the length of the transfer conduit, which is typically on the order of tens of centimeters, e.g., 50 cm), so that low gas velocities in the piping leading from the flow rate sacrifice system to the ionization system do not significantly affect the total transfer time, since the relatively slow portion of the overall transfer system is very short.

[0071] Therefore, the present invention provides (i) a laser ablation system adapted to generate a plume of sample material from the sample; (ii) an ionization system adapted to receive material dislodged from the sample by the laser ablation system and to ionize the material to form elemental ions; (iii) a mass spectrometer for receiving elemental ions from the ionization system and analyzing the elemental ions; The laser ablation system and the ionization system are coupled together by a transfer conduit and a flow sacrificial system; the transfer conduit is adapted to convey a gas stream containing the plume of ablated sample material from an inlet in the laser ablation system to an outlet in the flow rate sacrifice system; Flow sacrificing systems are (a) an outlet of the transfer conduit; (b) an inlet of the ionization system positioned to receive the sample material from the transfer conduit outlet and introduce the sample material into the ionization system; (c) a sacrificial flow outlet; the flow sacrificial system is adapted to direct a portion of the gas flow entering the flow sacrificial system from the sacrificial flow outlet to reduce the gas flow entering the ionization system through the inlet of the ionization system compared to the gas flow entering the flow sacrificial system through the transfer conduit; The outlet of the transfer conduit in the flow sacrifice system is optionally flared.

[0072] In some embodiments, the inlet of the ionization system is positioned coaxially with the outlet of the transfer conduit to minimize transfer of material from the transfer conduit through the flow sacrificial system to the inlet of the ionization system, and thus to the injector of the ionization system (because the plume of sample material transported along the conduit is entrained within the center of the transport flow). In some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the inlet of the ionization system is less than 2:1, such as 1.5:1 or 1:1. In some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the injector of the ionization system is less than 2:1, such as 1.5:1 or 1:1. In some embodiments, the inner diameter of the injector of the ionization system (or inlet to the ionization system) has a larger inner diameter than the transfer conduit. For example, in some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the inlet of the ionization system is less than 1:1, such as 1:1.5 or 1:2. In some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the injector of the ionization system is less than 1:1, such as 1:1.5 or 1:2.

[0073] In most arrangements, it is undesirable, or in some cases not possible, to significantly increase the diameter of the tubing (e.g., injector) passing from the flow sacrificial system to the ionization system as a way to reduce the gas velocity at the volumetric flow rate. For example, if the ionization system is an ICP, the conduit from the flow sacrificial system forms the injector tubing at the center of the ICP torch. Signal quality decreases when using injectors with wider inner diameters because the plume of ablated sample material cannot be injected very precisely into the center of the plasma (the part of the plasma that ionizes most efficiently). Injectors with an inner diameter of 1 mm or even narrower (e.g., inner diameters of 800 μm or less, such as 600 μm or less, 500 μm or less, or 400 μm or less) are strongly preferred. Other ionization techniques rely on material being ionized within a relatively small volume in three-dimensional space (because the energy density required for ionization can only be achieved in a small volume), so a wider inner diameter conduit means that much of the sample material passing through the conduit is outside the area where the energy density is sufficient to ionize the sample material. For this reason, narrow-diameter tubing from the flow sacrificial system into the ionization system is also used in instruments with non-ICP ionization systems. As mentioned above, if the sample material plume does not ionize to a suitable level, information from the removed sample material is lost because its components (including any labeled atomic / element tags) are undetectable by the mass spectrometer.

[0074] Rough or smooth, angular edges at the transition between the constant diameter portion of the transfer conduit and the flared portion at the outlet can cause turbulence in the gas flow. Therefore, in some embodiments, the transition to the flared extension should have smooth edges adapted to reduce turbulence. For example, the edges can be rounded.

[0075] The use of pumping can help ensure a desired split ratio between the sacrificial stream and the stream passing to the inlet of the ionization system. Thus, in some embodiments, the flow sacrificial system comprises a pump attached to the sacrificial stream outlet. A control restrictor can be added to the pump to control the sacrificial stream. Thus, in some embodiments, the pump of the flow sacrificial system further comprises a restrictor adapted to control the gas flow through the sacrificial stream outlet. In some embodiments, the flow sacrificial system comprises a mass flow controller adapted to control the restrictor.

[0076] When expensive gases are used, the gas pumped from the sacrificial flow outlet can be purified and recycled back into the same system using known methods of gas purification. Helium, as noted above, is particularly suitable as a carrier gas, but is expensive; therefore, reducing helium loss within the system (i.e., when passed through an ionization system and ionized) is advantageous. The flow sacrificial system splits the helium flow into a paraxial flow and a sacrificial flow. The sacrificial flow can be purified and recycled within the system, while the paraxial flow (the central portion of the flow carrying entrained particles from the removed plume) is passed to an ionization system (e.g., the plasma of an ICP torch). Helium from the paraxial flow is lost for recovery. Therefore, in some embodiments, a gas purification system is connected to the sacrificial flow outlet of the flow sacrificial system. In some embodiments, the gas purification system provides a portion of the gas flowed to the device, for example, through an inlet to the ablation chamber of a laser ablation system and / or through an inlet in a transfer conduit.

[0077] As before, a larger carrier flow rate is sent along the transport conduit, and only the central portion of this flow can be the portion of the injector flow that enters the ICP torch plasma. Typically, helium gas is used as the carrier flow because, as noted above, its properties favor high velocity transport of plume material over long conduits (i.e., it is less likely to induce turbulence for the same flow velocity, compared to argon). Even the opportunity to incorporate a gas purification system into the ionization system to recycle helium from the sacrificial flow and the paraxial flow of helium continuing through the flow sacrificial system is missed.

[0078] Thus, in some embodiments, the flow sacrifice system is adapted to reduce the gas flow passing into the inlet of the ionization system (e.g., the injector of an ICP torch ionization system) to 1 Lpm or less, such as 0.5 Lpm or less, 0.4 Lpm or less, 0.3 Lpm or less, or 0.2 Lpm or less. In some embodiments, the ICP injector includes a second inlet through which gas can be flowed to supplement the flow rate in the injector. In some embodiments, the second inlet includes a concentric tube around an injector attached to the inlet of the ionization system that introduces makeup gas from the flow sacrifice system as a sheath flow around the sample-containing gas stream. This makeup flow inlet is different from the argon gas flow also provided in the middle and outer concentric tubes that support the plasma. This injector may also be referred to as a dual concentric injector.

[0079] An apparatus comprising a flow sacrificial system can also comprise a sample cone (optionally asymmetric) or a tapered conduit, as described above. In some embodiments, an apparatus comprises a flow sacrificial system, a sample cone (optionally asymmetric), and a tapered conduit, as described above. Those skilled in the art will appreciate that a flow sacrificial system can be used with any of the apparatus described herein that uses alternative transfer conduit arrangements.

[0080] Laser Ablation System A laser ablation system, also referred to as an "ablation cell" or "laser ablation source," contains a sample during ablation. Typically, the ablation cell includes a laser-transparent window to allow laser energy to be directed at the sample. Optionally, the ablation cell includes a stage for holding the sample to be analyzed. In some embodiments, the stage is movable in the xy or xyz dimensions. In the figures and examples described herein, the laser ablation system may be shown as an open device. However, such configurations are for illustrative purposes only, and it will be understood that some form of suitable enclosure is present to prevent contamination or infiltration from the surrounding environment. For example, a chamber configured with a gas inlet and / or optical port may be arranged around the laser ablation system to provide a suitable closed environment for capturing and transporting the ablated plume for mass analysis. The gas inlet and optical port are positioned to accommodate laser beam orientation, sample, plume expansion, and transport conduits suitable for the methods and apparatus disclosed herein. It will be understood that the ablation cell is generally gas-tight (except for any designated outlets or ports). Even if the ablation chamber contains air prior to operation, the ablation chamber may be sufficiently enclosed from the environment such that gas flow (e.g., trapping gas through the sample chamber and / or carrier gas to the ablation cell's injector tubing) may be sufficient to reduce air contamination during sample transfer. However, the initial presence of air may provide a level of humidity that affects sensitivity and / or signal drift. Laser ablation systems for target applications may have gas flows as shown in one or more of Figures 2, 3, or 4.

[0081] Lasers used for laser ablation according to the present invention generally fall into three categories: femtosecond pulsed lasers, deep UV pulsed lasers, and pulsed lasers ("wavelength-selective lasers") with wavelengths selected for high absorption in the ablated material. Deep UV and wavelength-selective lasers may operate with nanosecond or picosecond pulses. Each category of laser has its drawbacks and advantages and can be selected based on the particular application. In some embodiments, the laser is a femtosecond pulsed laser configured to operate at a pulse rate of 10-10,000 Hz. Femtosecond lasers are well known (see, e.g., Jhanis et al., "Rapid bulk analysis using femtosecond laser ablation inductively coupled plasma time-of-flight mass spectrometry," J. Anal. At. Spectrom. 27:1405-1412, 2012).

[0082] Femtosecond lasers enable laser ablation of almost any material, with the only prerequisite for laser ablation being sufficient power density. This can be achieved with relatively low pulse energies if the beam is tightly focused, for example, to a diameter of 1 micrometer, and of short duration (focused in time). Deep-UV lasers can also ablate a large class of materials, since most commonly used materials absorb deep-UV photons. Wavelength-selective laser ablation utilizes lasers with specific laser wavelengths that target absorption within the substrate material. The advantage of wavelength-specific lasers can be the cost and simple structure of the laser and optics, but the spectrum of substrate materials is more limited. Suitable lasers can have different operating principles, such as solid-state (e.g., Nd:YAG lasers), excimer lasers, fiber lasers, and OPO lasers.

[0083] A useful property of femtosecond laser irradiation is that it is absorbed only when a threshold power density is reached. This allows focused femtosecond laser irradiation to pass through thicker portions of material without being absorbed or causing any damage, and still remove the same material right at the surface where the focal point occurs. The focal point can then be gradually moved deeper into the material as layers of the sample are removed. Nanosecond laser pulses can be partially absorbed by the substrate, but still function for ablation because the energy density at the focal point is greatest (as long as it is sufficient for ablation).

[0084] The spatial resolution of the signal generated in this way depends on two main factors: (i) the laser spot size when the signal is integrated over the entire area to be removed, and (ii) the speed at which the plume can be analyzed relative to the speed at which the plume is generated, as described above, to avoid overlap from continuous plumes. The distance referred to as the spot size corresponds to the longest internal dimension of the beam, e.g., for a circular beam, a 2 μm diameter beam, and for a rectangular beam, the length of the diagonal between opposite corners. The laser pulse can be shaped using an aperture, homogenized (if necessary) using a beam homogenizer, and focused, for example, using an objective lens, to create the desired spot size. Typically, the spot size is 100 μm or less, such as 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less. Typical spot sizes include diameters (or equivalently sized ablation areas in other shapes) ranging from 0.10 to 3 μm (e.g., about 0.3 μm), 1 to 5 μm (e.g., about 3 μm), 1 to 10 μm (e.g., about 1, about 2, about 3, about 4, or about 5 μm), less than 10 μm, and less than 5 μm. In certain embodiments, the laser system is configured to operate with well-focused laser pulses to ablate a sample area on the order of about 1 μm, e.g., from 100 nm to 1 μm.

[0085] To analyze individual cells, the laser in a laser ablation system has a spot size that is on the order of these cells. While this size depends on the particular cells in the sample, the laser spot generally has a diameter of less than 4 μm, e.g., in the range of 0.1-4 μm, 0.25-3 μm, or 0.4-2 μm. Thus, the laser spot may have a diameter of about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.5 μm or less, such as about 400 nm or less, about 300 nm or less, about 200 nm or less, about 100 nm or less, or less than 100 nm. To analyze cells with subcellular resolution, the present invention uses laser spot sizes that are on the order of these cells, and more specifically, laser spot sizes that can ablate material with subcellular resolution. Single-cell analysis may be possible using spot sizes larger than the size of cells, for example, with cells spread across a slide with spacing between each other. However, larger spot sizes can be used and single-cell characterization can be achieved because the additional ablated area around the target cell does not contain additional cells. The specific spot size used can therefore be appropriately selected depending on the size of the cell being analyzed. In biological samples, if subcellular resolution imaging is desired, cells are rarely all the same size, and if a consistent spot size is maintained throughout the ablation procedure, the ablation spot size should be smaller than the smallest cell. Small spot sizes can be achieved by using broad laser beam demagnification and near-field optics. A 1 μm laser spot diameter corresponds to a 1 μm laser focus (i.e., the diameter of the laser beam at the beam's focus), but the laser focus can vary by ±20% or more due to variations in the spatial distribution of energy on the target (e.g., Gaussian beam shape) and variations in total laser energy relative to the ablation threshold energy. For example, using a 25 μm diameter laser beam and demagnifying it 25 times on a tissue sample yields a spot size with a 1 μm diameter.

[0086] Ablation at this small scale creates a very small amount of plume material, which in turn ensures that the plume size remains small. A smaller plume is more likely to remain in the middle of the trapped flow without contacting the walls of the ablation cell or the transport conduit. Ablation on the 1-micrometer scale also means that the distance between the ablated surface and the region where the plume expansion slows and ambient gas becomes dominant is very short. This distance can range from a few micrometers to hundreds of micrometers. In some versions of the invention, the trapped flow exists when the plume stops expanding. Thus, for purposes of illustration and not limitation, some of the accompanying drawings show the distance between the ablated surface and the region with trapped flow as approximately 100 micrometers.

[0087] While ablation on the scale of 1 micrometer (or less) is advantageous for certain applications (e.g., imaging), the methods and apparatus of the present invention are also useful when larger ablation spots are created, such as ablation spots in the range of about 5 to about 35 microns in diameter, for example, in the ranges of 5-15 microns, 10-20 microns, 15-25 microns, 20-30 microns, and 25-35 microns. In some applications where large ablation spots are created, only a portion of the plume material is captured.

[0088] In some embodiments, the laser is located outside the ablation chamber, and the laser beam (laser energy) enters the ablation chamber, for example, through an optical window. As used herein, a laser beam may be described as emanating from a surface (e.g., a laser lens or mirror), which may be oriented to direct the beam to a particular location or pattern of locations. To facilitate the description of the present invention, a directed beam may be considered to have a particular orientation, and the beam orientation may refer to an imaginary line aligned with the beam and extending beyond the actual beam (e.g., when the beam strikes a non-transparent surface). As is clear from the context, reference to the orientation or position of a laser beam may refer to the orientation or position that a beam of a parasitic laser system may generate using a laser.

[0089] For rapid analysis of tissue samples, high ablation frequencies, e.g., greater than 20 Hz (i.e., greater than 20 ablations per second, providing greater than 20 plumes per second), are necessary. In some embodiments, the laser ablation frequency is at least 40 Hz, such as at least 50 Hz or at least 100 Hz. In some embodiments, the laser ablation frequency is in the range of 40-2000 Hz, 40-1500 Hz, 40-500 Hz, 40-200 Hz, 40-150 Hz, or 75-150 Hz. Ablation frequencies greater than 40 Hz allow imaging of typical tissue samples in a reasonable amount of time. The frequency at which a laser pulse can be directed at a spot on the sample (assuming complete ablation of material at the spot) and still be individually resolved determines how quickly image pixels can be acquired. Thus, if the duration of the laser pulse required to ablate material at a given point means that fewer than five pulses per second can be directed at the sample, the time required to investigate a 1 mm x 1 mm area by ablation with a 1 μm spot size would exceed two days. At a 40 Hz rate, this could be approximately 6–7 hours, with further increases in pulse frequency further reducing the analysis time. At these frequencies, if it is desired to individually resolve each ablated plume, the instrumentation must be able to analyze the ablated material quickly enough to avoid significant signal overlap between successive ablations. Preferably, the overlap between signals resulting from successive plumes is <10% in intensity, more preferably <5%, and ideally <2%. The time required for plume analysis depends on the cleaning time of the ablation chamber (see the Ablation Chamber section below), the transit time of the plume of sample material to and through the ionization system (as discussed above for optimizing delivery to the ionization system), and the time required to analyze the ionized material. Each laser pulse can be correlated to a pixel in a subsequently enhanced image of the sample, as described in more detail below.

[0090] Ablation Chamber Ablation chambers (which may be referred to herein as sample chambers) with short cleaning times (e.g., 100 ms or less) are advantageous for use in the devices and methods of the present invention. Cells with long cleaning times either limit the speed at which images can be generated or result in overlap between signals resulting from successive sample spots (e.g., Kindness et al. (2003) Clin Chem 49:1916-23, which had signal durations of greater than 10 seconds). Thus, the cleaning time of the plume of sample material from the laser ablation cell is a major limiting factor for achieving high resolution without increasing total scan time. Ablation chambers with cleaning times ≦100 ms are known in the art. For example, Gurevich & Hergenroder (2007) J Anal. At. Spectrom. 22:1043-1050 disclose an ablation chamber with a cleaning time of ≦100 ms. An ablation chamber was disclosed in Wang et al. (2013) Anal. Chem. 85:10107-16 (see also reference WO2014 / 146724) that has a cleaning time of 30 ms or less, thereby allowing for high ablation frequencies (e.g., greater than 20 Hz) and therefore rapid analysis. Another such ablation chamber is disclosed in reference WO2014 / 127034. The ablation chamber of this document includes a sample capture cell configured to be operably arranged near a target (the sample capture cell described herein is an example of an improved inlet section of a transfer conduit that can be combined with the tapered and flow rate-sacrificial improvements of the transfer conduit described above), and the sample capture cell includes a capture cavity having an opening formed in the surface of the capture cell, configured to receive target material emitted or generated from the laser ablation site through the opening, and a guide wall exposed within the capture cavity and configured to direct the flow of carrier gas (also referred to herein as capture gas) within the capture cavity from the inlet section to the outlet section so that at least a portion of the target material received within the capture cavity can be transported to the outlet section as a sample. The volume of the capture cavity in the ablation chamber of Reference WO 2014 / 127034 can be less than 1 cm3 and 0.005 cm3 or less.The ablation chamber may have a cleaning time of 25 ms or less, such as 20 ms or 10 ms or less. The sample cone inlet of the transfer conduit, for example, an asymmetric sample cone, also helps reduce the cleaning time of the ablation chamber and is an alternative to the trapped cells described herein.

[0091] An existing LA-ICP-MS system including a laser ablation chamber is shown in FIG. 4. The ablation chamber may include a movable sample stage for positioning the sample. A gas source may provide a trapping gas that flows through the ablation chamber and transports the ablation plume to an injector tubing (also referred to herein as an injector or transfer conduit) leading to the ICP torch. The injector tubing may have flexible tubing between the ablation chamber and the ICP torch, or may be rigid and / or straight. In certain embodiments, the injector may be perpendicular to the sample and collinear with the gas flow shown in FIG. 2 or 3. The LA-ICP-MS system may include one or more additional gas sources for supplying carrier gas and / or internal and external gases (also referred to as auxiliary gas and plasma gas). In certain embodiments, the injector tubing may receive makeup gas downstream of the laser ablation plume introduced into the injector tubing. One or more gas streams may include hydrogen. For example, the gas stream may contain water or alcohol vapor, as further described herein. Alternatively or additionally, the gas stream may be from a premixed compressed gas source that contains hydrogen (such as hydrogen gas, ammonia, or methane), as further described herein.

[0092] In certain embodiments, the laser ablation chamber may include a trapping gas (which enters the chamber and carries the ablation plume to the injector piping) and may optionally further include a carrier gas (i.e., which enters the injector upstream of the ablation plume). The injector may further include a make-up gas (i.e., which supplements gas within the injector downstream of where the ablation plume enters the injector). The ICP torch may include an internal gas (i.e., an auxiliary gas flowing through the internal piping of the ICP torch) and an external gas (i.e., a plasma gas flowing through the external piping of the ICP torch). Additional gas flows may be present. In certain embodiments, hydrogen (e.g., hydrogen gas or water vapor) may be introduced into one or more of the above gas flows, as further described herein.

[0093] Ionization System Sample materials can be ionized by various techniques, such as in a plasma. The use of an ICP is suitable for IMS and IMC analysis. An ICP is a plasma source in which energy is provided by a current generated by electromagnetic induction. Typically, the plasma source is argon gas-based. For example, the ionization system may include an ICP torch. IMC using an ICP in an ionization system has been reported, for example, in Giesen et al. (2014) Nature Methods. 11:417-422 and Wang et al. (2013) Anal. Chem. 85:10107-16.

[0094] To this end, the ionization system receives the sample material from the laser sampling system and converts it into elemental ions for detection by the mass spectrometer. If the sample material is not particulate (e.g., the plume of sample material is still in the form of an aerosol of molecular or even particulate material), the ionization system acts to break the material into elemental ions as part of the ionization process.

[0095] mass spectrometer As mentioned above, the third component of the device is a mass analyzer. Mass analyzers for use in the present invention can be selected based on the needs of the operator or the particular application. Typical types of mass analyzers include quadrupole, time-of-flight (TOF), magnetic sector, high-resolution, single-collector, or multi-collector based mass analyzers.

[0096] The time required to analyze the ionized material depends on the type of mass analyzer / mass spectrometer used to detect the ions. For example, instruments using Faraday cups may be too slow for analyzing fast signals, but not all analyses require fast signal analysis, so one skilled in the art can select a mass analyzer or mass spectrometer appropriately. Overall, the desired analysis speed (and therefore the frequencies at which the ablation plume can be matched) and degree of multiplexing (number of atoms monitored simultaneously / quasi-simultaneously) will dictate the type of mass analyzer to be used (or conversely, the mass analyzer selected will determine the achievable speed and multiplexing).

[0097] Typically, time-of-flight mass spectrometers are used for recording fast transient events due to the transient times expected from fast laser ablation setups.

[0098] TOF detectors can quasi-simultaneously register multiple masses within a single sample. While TOF mass analyzers are typically avoided for atomic analysis due to compromises in addressing space charge effects within the TOF accelerator and flight tube, the effectiveness of this technique can be improved by using it for limited detection ranges. For example, in mass cytometry and imaging mass cytometry, a range can be selected so that ions from labeled atoms used to label target molecules in biological samples are detected, thereby eliminating other atoms (e.g., atoms with atomic masses less than 80). This reduces the density of the ion beam, enriching it with masses (e.g., in the 80-210 Dalton range) for more efficient manipulation and focusing, thereby facilitating TOF detection and taking advantage of TOF's high spectral scanning speed. Thus, rapid analysis can be achieved by combining TOF detection with the selection of labeled atoms that are rare within the sample, ideally with masses above those found in the unlabeled sample, e.g., by using high-mass transition elements. Further details regarding mass cytometry can be found in Tanner et al., Cancer Immunol Immunother (2013) 62:955-965 and U.S. Patent No. 7,479,630, and in Giesen et al. (2014) Nature Methods. 11:417-422, which describes imaging mass cytometry.

[0099] Additional variations of the invention to which the improvements in the equipment used and the transfer conduits described above are applicable The device of the present invention can be used for analyzing or imaging biological samples, which may be on a transparent substrate. In imaging embodiments, the laser can generally be operated in successive pulses or in bursts of pulses directed to different locations on the sample, referred to as "target spots" or "ablation locations or regions." The pulses can be directed to the spots in a set pattern, such as a raster for two-dimensional imaging. Alternatively, multiple individual spots at different locations (e.g., corresponding to individual cells) can be ablated. In some embodiments, the laser emits bursts of pulses that generate plumes originating from the same pixel (i.e., the same location on the target). Ablation plumes generated by individual pulses within a burst are expected to dissolve into one plume and travel through the instrument such that the plume is distinct from plumes created from other pixels. To distinguish individual pixels, the duration between bursts (pixel interrogation, which can be only one or 100 pulses) is maintained beyond a certain limit determined by the time spread of the ion signal (at the detector) from each pixel.

[0100] In accordance with the present disclosure, each distinct sample plume can be specifically analyzed by a mass analyzer. In one embodiment, the apparatus is configured so that the diffusion of the plume within the ablation cell (ablation system) and transport conduit is less than the diffusion occurring within the ionization system and mass analyzer. In one embodiment, the plumes can be specifically analyzed by transporting each ablated plume to the ionization system and detecting the ions by a mass analyzer during the cumulative transit time of the plume to the ionization system. This can be achieved by passing each sample plume through a gas flow and capturing it with a transport arrangement such that the ratio between the plume extent during the transport period (i.e., transport of the ablation plume from the site of ablation to the plasma) and the extent during the ion transit period (i.e., transport of ions from the plasma to the mass analyzer) is less than or equal to 1.

[0101] Generally, the limit on the sample particle size that an ionization system (e.g., ICP) can effectively vaporize and ionize for analytical detection purposes is on the order of about 10 μm or less. Particles generated by laser ablation on the 1-micrometer scale are submicrometer and suitable for ICP ion sources. For individual particle analysis (such as performed using Fluidigm Canada's CyTOF® instrumentation), the typical rate at which these particles can be ionized and analytically detected can be a function of the cumulative time spread or diffusion of the sample's transit time within the plasma while the particle is vaporized and ionized, and the cumulative time spread or diffusion of the ion's transit time between the ICP and its detection by the mass analyzer. Typically, the cumulative time spread or diffusion can be on the order of about 200 μs in duration. As a result, for spatially separated particles of 10 μm or less, analyzing each individual particle can be achieved by delivering each particle to the ionization system (e.g., ICP) for a period on the order of 200 μs. In some embodiments, particles are delivered to an ionization system (e.g., an ICP) in less than 200 μs or less than 150 μs. Thus, in a sample introduction system in which imaging of a biological sample can be performed by laser ablation, the laser system can be configured to operate with well-focused laser pulses, e.g., femtosecond pulsed laser irradiation, to ablate sample regions on the order of about 1 μm. With this configuration, the ablated plume formed by each laser pulse can typically contain sample particles with dimensions of about 1 μm or less. Under certain conditions described herein, these particles can be captured and delivered to meet the required delivery period, and each separate plume can then be effectively vaporized and ionized by the ionization system.

[0102] Furthermore, while operating the laser in successive pulses, such as when rastering across a sample surface for two-dimensional imaging, the distinctiveness of each plume and the spatial separation between each subsequent plume can be maintained between the plume region where it forms and the point of evaporation and ionization within the ionization system ion source. For example, as the plume is transported through a conduit, particles within the plume can diffuse and expand radially outward before the plume enters the ionization system (e.g., the plasma of an ICP). The diffusion of particles generated within the plume can depend on their diffusion coefficient, carrier flow velocity profile, and particle density distribution as they form and grow during their transition to the ionization system. For example, a femtosecond laser ablation spot size of 1 μm can produce a plume with an initial cross-sectional diameter of approximately 100 μm or less before further diffusion during its transition. The degree of plume diffusion can also be a function of the size of the ablated particles; larger particles tend to diffuse less but have higher momentum, which can potentially be lost by contacting the interior walls of the transport conduit / injector tubing. For this reason, it is desirable to transport the plume to the ionization system with minimal plume diffusion and / or enough time for evaporation and ionization before the degree of diffusion has any significant effect.

[0103] Thus, in various embodiments, using a laser to remove a 1 μm sample spot and efficiently transport the plume so that diffusion remains within the inner diameter of the transfer conduit / injector tubing can be achieved with the exemplary arrangements described herein and in the accompanying drawings.

[0104] For a given laser ablation system and a given sample, the ablated plume expands after laser ablation until it reaches a characteristic volume referred to as the "sampling volume." It is desirable to configure the system to minimize the sampling volume and increase the velocity at which the gas flow transports the plume away from the sampling volume. Combining a small sampling volume with a high gas flow rate reduces the time spread of plume transport to the transfer conduit / injector. The sampling volume can be described by the plume envelope at the moment when the velocity of the plume expansion in any dimension is significantly reduced (approximately 10 times) below the speed of sound in the ambient gas medium. Typical sampling volumes can range, without limitation, from 10-6 mm3 to 10 mm3. Sampling volumes are often in the range of 0.001 mm3 to 1 mm3. A trapping flow, if present, flows through at least a portion of the sampling volume and transports at least a portion of the plume to the transfer conduit / injector, so that the trapping flow can be transported by the transport flow to the ionization system (e.g., ICP). It is desirable that the velocity of the trapped flow be substantial (e.g., >1 m / s, >10 m / s, >100 m / s, or >500 m / s) as it enters the sampling volume. In some embodiments, the velocity of the trapped flow can be assessed by measuring the velocity of the trapped flow into the transfer conduit / injector (e.g., through the transfer conduit / injector opening) as it enters the sampling volume. In some embodiments, this measured velocity is >1 m / s, >10 m / s, >100 m / s, or >500 m / s. In contrast to the present invention, if the plume is not rapidly swept away, it will continue to expand and diffuse, undesirably filling the entire ablation cell.

[0105] In one aspect, the present invention provides a laser ablation arrangement in which a laser beam is directed at a target. In one embodiment, the target comprises a substrate and a sample disposed on the substrate. In one embodiment, the substrate is transparent and the target is a transparent target.

[0106] In one aspect, the present invention provides a laser ablation configuration for "transmission target" ablation. In this configuration, pulses of a laser beam are directed through a transparent target, and a sample plume (the "ablated plume" or "plume") is formed downstream of the beam into a transfer conduit / injector. Transmission target illumination is advantageous for optimizing transient time spread by removing optical elements (windows, objective lenses, etc.) from the linear path of the plume. In one aspect, the present invention provides a laser ablation system comprising: (a) a laser capable of producing laser illumination; (b) a laser ablation cell (or laser ablation system) into which a transparent target can be introduced; and a transfer conduit / injector with an opening through which the ablated plume can enter, where the laser illumination originates from one side of the transparent target and the opening of the transfer conduit / injector is on the other side. Other features that may be included in the system are described throughout this disclosure, including the examples.

[0107] In certain embodiments, smaller ablation spot sizes (higher resolution) can be achieved with high numerical aperture lenses, such as oil immersion lenses, which can be configured for transmissive targeted ablation (e.g., of thin samples such as tissue sections less than 500 nm in diameter).

[0108] Thus, in operation of one apparatus according to the present invention, a sample is delivered to the apparatus and sampled using a laser system including a lens where laser radiation is directed through an oil immersion lens onto the sample to produce ionized material (the sampling may subsequently produce vaporous / specific material that is ionized by the ionization system), and the ions of the sample material are passed to a detector system.

[0109] The present invention overcomes the limitations of conventional IMC and IMS by utilizing an immersion medium. The immersion medium has a refractive index greater than 1.0 and is placed between the objective lens and the sample stage. In this way, the device of the present invention achieves a numerical aperture greater than 1.0, thereby enabling a laser spot size of less than 200 nm, less than 150 nm, or less than 100 nm. Thus, the present invention provides an apparatus for imaging mass cytometry with a spatial resolution of 200 nm or greater, 150 nm or greater, or 100 nm or greater.

[0110] Thus, the sample stage holds the sample during operation, typically the sample is on a sample carrier, the same stage holds the sample carrier, and laser radiation is then directed through the lens of the instrument, through the objective lens and the immersion medium, to the sample, whereby radiation removes material from the sample.

[0111] To achieve optimal focusing conditions for the laser, the immersion medium of the present invention has a refractive index greater than 1.00, such as 1.33 or greater, 1.50 or greater, 2.00 or greater, or 2.50 or greater.

[0112] Furthermore, to reconstruct images of a monolayer thickness (or less) of living cells, or to read thicker specimens layer by layer and generate 3D images as further described herein, the sample preferably has a thickness of 100 micrometers or less, such as 10 micrometers or less, 5 micrometers or less, 2 micrometers or less, or 100 nm or less, or 50 nm or less, or 30 nm or less. In some embodiments described in further detail herein, the combination of objective lens and immersion medium is referred to as an oil immersion lens.

[0113] When using a liquid immersion medium, the sample needs to be positioned on the opposite side of the sample carrier, up to the liquid medium (as shown in Figure 3), so that the carrier gas can collect the ablated material. Therefore, a transmissive sample carrier ablation technique must be applied here. This has the added advantage of shortening the achievable working distance for the ablation material collection hardware and eliminating the need for bending the transition conduit between the sample chamber and the detector. This also leads to a reduction in the transient time, thus increasing the achievable ablation rate per second at the spot.

[0114] Thus, the present invention provides an apparatus in which the solid immersion medium is a hemispherical solid oil immersion lens or a Weierstrass solid oil immersion lens. A biological sample can be attached to the solid immersion material on the opposite side of the sample stage. The stage on which the sample is attached can be made of a material with the same refractive index as the solid oil immersion lens, and the solid oil immersion lens can be made thinner by an amount equal to the thickness of the substrate to maintain the focus position.

[0115] In various embodiments, the target sample can be configured for laser ablation using a sample configured to be compatible with a transparent target. The sample can be mounted on a transparent substrate, incorporated into the transparent substrate, or formed as a transparent target. Suitable laser-transparent substrates can include glass, plastic, quartz, and other materials. Generally, the substrate is substantially planar or flat. In some embodiments, the substrate is curved. In certain embodiments, the substrate is 0.1 mm to 3 mm thick. In some embodiments, the substrate is coded (see, e.g., Antonov, A. and Bandura, D., 2012, U.S. Patent Publication No. 2012 / 0061561, incorporated herein by reference). In this configuration, pulses of a laser beam are directed through the transparent target, and a sample plume ("ablated plume" or "plume") is formed downstream of the beam to a transfer conduit / injector.

[0116] The transfer conduit (i.e., injector tubing) can have an inlet configured to capture the removed plume, such as an inlet formed as a sample cone with a small opening or aperture. In this configuration, the sample cone can be positioned near the region where the plume is formed. For example, the opening of the sample cone can be positioned 10 μm to 1000 μm away from the transparent target, such as about 100 μm away from the transparent target. As a result, the removed plume can be generated or formed at least partially within the expansion region of the cone. In some embodiments, the diameter and / or spacing (including angle) of the aperture can be adjusted to allow optimization under various conditions. For example, while the plume has a cross-sectional diameter on the scale of 100 μm, the diameter of the aperture can be sized on the order of 100 μm, with sufficient clearance to prevent perturbation of the plume as it passes through.

[0117] A transfer conduit can continue downstream of the sampling cone to receive the removed plumes in a configuration that promotes plume movement and maintains the spatial distinctiveness of each subsequent plume as a function of the laser pulse. Thus, a gas flow can be introduced through the opening of the sampling cone to specifically capture each plume (capture flow) and help direct the plumes, while an additional gas flow can be introduced into the transfer conduit / injector to transport each specifically captured plume toward the ionization system (carrying flow or sheath flow). Another function of the carrying flow or sheath flow is to prevent particles generated within the plumes from contacting the walls of the transfer conduit / injector. The gas can be, for example, without limitation, argon, xenon, helium, nitrogen, or a mixture thereof. In some embodiments, the gas is argon. The capture flow gas and the carrying flow gas can be the same or different.

[0118] Selecting or determining suitable gas flow rates for the present invention is within the capabilities of those skilled in the art, as defined by this disclosure. The total flow rate through the transfer conduit is typically dictated by the requirements of the ionization source (e.g., an ICP ionization source). The laser ablation setup is necessary to provide a flow that will meet these requirements. For example, the transfer conduit may optionally have an inner diameter of 1 mm or less, coupled with a cumulative gas flow rate of approximately 1 liter per minute (a capture flow of 0.1 liter per minute plus a transport flow of 0.9 liters per minute). It is expected that transfer conduits of larger or smaller diameters can be adapted to various geometries, with correspondingly selected gas flow rates, providing similar expected results. Conditions that dynamically maintain non-turbulent gas flow within the transfer conduit are desirable to preserve the distinctiveness of each separate ablated plume.

[0119] As described herein, given the specific configuration of elements (e.g., the specific configuration of gas inlet locations, apertures, transport conduit characteristics, and other factors), the capture and delivery flow rates are selected to transport each removed plume to the ionization system (e.g., ICP) for a period within the plume's cumulative transit time between the ionization system and its detection by the mass analyzer. This can be achieved by passing each sample plume through the gas flow and capturing it with the transport configuration so that the ratio between the plume's extent during the transport period and its extent during the ion transit period is equal to or less than one. That is, the time spread (or time diffusion) of the transition signal is important. ICP-MS devices (such as the CyTOF® ICP-TOF instrument manufactured by Fluidigm Canada) are characterized by an inherent broadening of the signal. In the case of laser ablation, the act of injecting a single plume may or may not be rapid compared to the time diffusion in the ICP-MS itself. The plume's diffusion prior to ionization depends on the design of the laser ablation system, particularly the ablation chamber and transport conduit. It is desirable that the laser ablation system and transport conduits do not diffuse the initial ablation plume beyond the inherent extent of the remaining instrument. This condition ensures that the peak in the detected signal generated by the ablation plume is as sharp (in time) as possible for the chosen instrument. If the plume diffusion is significant, e.g., diffusion in an ICP-MS system, the laser ablation event from a single pulse will appear more widespread at the detector. However, if the diffusion within the laser ablation region is smaller than the diffusion due to the instrument, then the diffusion due to the instrument will dominate over the total diffusion. Therefore, calibration beads can be used to measure the diffusion due to the instrument, and then the total diffusion from a single laser pulse can be measured and these two figures compared. If the diffusion from laser ablation is smaller than the diffusion due to the instrument, then the total diffusion will be less than twice the diffusion due to the instrument.

[0120] The time spread of a characteristic instrument can be measured experimentally, for example, using labeled cells or calibration beads. Whenever a single bead enters a mass cytometer (e.g., a CyTOF® ICP-TOF instrument), it is vaporized and ionized in a plasma and then subjected to mass analysis until its signal reaches a detector. Transient events are detected and used to record information about the particular bead, such as the width of the transient signal (representing the time spread from a single event) and the value of the spread occurring starting at the ICP source and ending at the detector.

[0121] In some embodiments, the device is configured to allow a time spread of 10 to 1000 microseconds for a defined path between the sample and the ion detector of the mass analyzer.

[0122] Typical capture flow rates are in the range of 0.1 to 1 Lpm. The optimum capture flow rate can be determined empirically but is usually at the lower end of the range (e.g., about 0.1 Lpm). Typical delivery flow rates are in the range of 0.1 to 1 Lpm. The optimum delivery flow rate can be determined empirically but is usually at the upper end of the range (e.g., about 0.9 Lpm). In some embodiments, the capture flow rate is lower than the delivery flow rate. The delivery flow rate can be zero in some cases, for example, if the capture flow rate is about 1 Lpm. The delivery flow rate is often in the range of 0.4 to 1 Lpm (e.g., 0.4, 0.6, 0.8, or 1 Lpm).

[0123] While the subject matter herein is described in conjunction with various embodiments, it is not intended to be limited to such embodiments. On the contrary, the subject matter herein encompasses various modifications, variations, and equivalents, as will be understood by those skilled in the art. For example, in the various embodiments shown in the drawings, the transfer conduit / injector piping has generally been described as having an inner diameter of 1 mm, in conjunction with a cumulative gas flow rate of approximately 1 liter per minute (0.1 plus 0.9 liters per minute). It is expected that transfer conduits / injectors of larger or smaller diameters, in conjunction with gas flow rates selected accordingly, may be applicable to various geometries presented with similar expected results. However, conditions that dynamically maintain non-turbulent or nearly non-turbulent gas flow within the injector piping may be desirable to preserve the distinctiveness of each separate removed plume.

[0124] Furthermore, in some cases where the laser pulse rate is increased, two or more ablated plumes can be distinctly captured and delivered to an ionization system (e.g., an ICP) within the cumulative transient time spread described above. For example, at a 10 kHz repetition rate, a pulsed laser can generate two ablated plumes in 200 μs, which can subsequently be delivered to an ICP for ionization. The ions generated from the two separate plumes can be analyzed as a single, separate packet of ions by a mass analyzer. As a result, while the laser remains at the same ablation spot or moves across a trace of tiny, consecutive spots at a rate less than the repetition rate, the ablated plume and subsequent ions can provide a cumulative mass analysis at the same ablation spot or an average mass distribution along the trace, respectively. It should be noted that laser repetition rates as high as several MHz can be used to generate a signal representing the averaging of many laser pulses. The laser can also be fired in bursts to provide gaps in the data stream between individual sampling locations (or pixels).

[0125] It will be understood that the methods and devices of the present invention can be used with any of a variety of sample types, for example, biological samples. In one approach, the sample is cellular material, such as a tissue section, a cell monolayer, or a cell preparation. The sample can be thinly sectioned biological tissue up to 100 micrometers thick, a tissue sample on the order of millimeters thick, or an unsectioned tissue sample. In one example, a thin tissue section (such as a paraffin-embedded section) can be used. For illustration, some tissue sections have a thickness of 10 nanometers to 10 micrometers. In some cases, the sample is a cell population, or one or more selected cells from a cell population. See, e.g., Antonov, A. and Bandura, D., 2012, U.S. Patent No. 2012 / 0061561, incorporated herein by reference.

[0126] Image IMS and IMC can be configured to provide signals for multiple labeled atomic / element tags within the plume. Detecting the label within the plume reveals the presence of that cognate target at the location of ablation (or, correspondingly, the location of the slug of material desorption). By generating a series of plumes at known spatial locations on the sample surface, the MS signal reveals the location of the label on the sample, which can then be used to construct an image of the sample. By labeling multiple targets with distinguishable labels, the location of the labeled atoms can be correlated with the location of the cognate target, thereby enabling the present invention to construct complex images and achieve levels of multiplexing far beyond those achievable using existing techniques. For example, the GRAPHIS suite from Kylebank Software can be used, although other suites such as TERAPLOT, ImageJ, and CellProfiler can also be used. Imaging using MS data from techniques such as MALDI-MSI is known in the art; for example, Robichaud et al. (2013) J Am Soc Mass Spectrom 24(5):718-21 discloses the "MSiReader" interface for viewing and analyzing MS imaging files on a Matlab platform, and there are also instruments for rapid data exploration and visualization of both 2D and 3D MSI datasets at full spatial and spectral resolution, such as the "Datacube Explorer" program.

[0127] sample The present invention provides a method for imaging a sample. All types of samples can be analyzed by the method, including alloys, geological samples, and archaeological samples. Biological samples can also be analyzed. Such samples contain a plurality of cells, which can be processed by IMS and / or IMC to provide an image of those cells within the sample. In general, the present invention can be used to analyze tissue samples currently studied by IHC techniques, but using labels that are suitable for detection by IMC.

[0128] Any suitable tissue sample can be analyzed. For example, the tissue can be epithelial tissue, muscle tissue, nerve tissue, etc., and combinations thereof. For diagnostic or prognostic purposes, the tissue can be derived from a tumor. In some embodiments, the sample can be derived from a known tissue, but it may be unknown whether the sample contains tumor cells. Imaging can reveal the presence of targets indicative of the presence of a tumor, thus facilitating diagnosis. The tissue sample can include, for example, human breast cancer tissue or human mammary epithelial cell (HMLE) breast cancer tissue. The tissue sample can include formalin-fixed, paraffin-embedded (FFPE) tissue, frozen tissue, or tissue embedded in a suitable resin. The tissue can be obtained from any living multicellular organism, but is typically human.

[0129] The tissue sample is typically a section having a thickness in the range of 2 to 10 μm, e.g., 4 to 6 μm. Thinner tissue sections, e.g., less than 2 μm thick, e.g., less than 1 μm, less than 500 nm, less than 250 nm, or even less than 100 nm, can also be analyzed. Thinner tissue samples may produce lower signals due to the reduced volume of sample removed by the late pulse, but thinner sections allow for more sections to be generated from the tissue sample, providing advantages in terms of 3D imaging by imaging multiple sections. However, thinner sections (e.g., below the resolution of laser ablation) can be easily ablated through the slide (e.g., the full depth of the tissue section at the laser ablation spot can be removed). Techniques for preparing such sections are known from the field of IHC, e.g., using a microtome with a dehydration step, including embedding, etc. For this purpose, the tissue can be chemically fixed, after which sections can be prepared in the desired plane. Cryosection or laser capture microdissection can also be used to prepare tissue samples. The samples can be permeabilized, for example, to label reagents with intracellular targets (see above).

[0130] The size of the tissue sample to be analyzed is similar to current IHC methods, although the maximum size is dictated by the size of the sample that can fit into the laser ablation device, particularly its ablation chamber. Sizes up to 5 mm x 5 mm are typical, although smaller samples (e.g., 1 mm x 1 mm) are also useful (these dimensions refer to the size, not the thickness, of the section).

[0131] Labeling of tissue samples In some embodiments, as described above, the devices and methods of the present invention detect atoms added to a sample (i.e., atoms not normally present). Such atoms are referred to as labeled atoms (hence, labeled atoms represent elemental tags). The sample is typically a biological sample containing cells, and the labeled atoms are used to label target molecules within / on the cell surface. In some embodiments, two or more labeled atoms can be detected simultaneously, allowing for multiplexed detection of, for example, at least 3, 4, 5, 10, 20, 30, 32, 40, 50, or even 100 different labeled atoms. By labeling different targets with different labeled atoms, the presence of multiple targets in a single cell can be determined.

[0132] Labeled atoms that can be used in the present invention include any species that are detectable by MS and substantially absent in unlabeled samples. Thus, for example, 12C atoms would not be suitable as labeled atoms because they are abundant in nature, while 11C is an artificial isotope that does not occur in nature and therefore could theoretically be used. However, in a preferred embodiment, the labeled atoms are transition metals such as rare earth metals (the 15 lanthanides plus scandium and yttrium). These 17 elements provide many different isotopes that are easily distinguishable by MS. These various elements are available in enriched isotope form; for example, samarium has six stable isotopes and neodymium has seven stable isotopes, all of which are available in enriched form. The 15 lanthanide elements provide at least 37 isotopes with unique, non-overlapping masses. Examples of elements suitable for use as labeling atoms include lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), and yttrium (Y). In addition to rare earth metals, other metal atoms are suitable for detection by MS, such as gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), and bismuth (Bi). Radioisotopes are not preferred due to their inconvenient handling and instability; for example, Pm is not a preferred labeling atom among the lanthanides.

[0133] To facilitate TOF analysis (see above), it is useful to use labeled atoms with atomic masses in the range of 80-250, for example, in the range of 80-210, or in the range of 100-200. This range includes all of the lanthanides but excludes Sc and Y. The 100-200 range allows for analysis of a theoretical 101 building blocks using different labeled atoms, while the present invention allows for the high spectral scan speed of TOF MS. As noted above, by selecting labeled atoms whose masses lie in a window beyond those observed in unlabeled samples (e.g., in the range of 100-200), TOF detection can be used to provide rapid analysis at biologically significant levels.

[0134] Labeling a sample generally requires that a label atom be attached to one member of a specific binding pair (sbp). This label sbp is contacted with the sample so that it can interact with the other member of the sbp (the target sbp member), if present, thereby localizing the label atom to the target molecule in the sample. The method of the present invention then detects the presence of the label atom on the particle when the label atom is analyzed by a mass cytometer. Rare earth metals and other label atoms can be conjugated to sbp members using known techniques. For example, Bruckner et al. (2013) Anal. Chem. 86:585-91 describes the attachment of lanthanide atoms to oligonucleotide probes for MS detection, Gao & Yu (2007) Biosensor Bioelectronics 22:933-40 describes the use of ruthenium to label oligonucleotides, and Fluidigm Canada sells the MaxPar™ Metal Labeling Kit, which can be used to conjugate over 30 different label atoms to proteins (including antibodies).

[0135] Various numbers of label atoms can be attached to a single sbp member, and the achievable sensitivity increases when more label atoms are attached to any sbp member. For example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or even more than 100 label atoms can be attached to an sbp member. For example, monodisperse polymers containing multiple monomer units can be used to form elemental tags, each containing a chelator such as DTPA. DTPA, for example, binds to lanthanide ions with a dissociation constant of approximately 10 M [Tanner et al., Cancer Immunol Immunother (2013) 62:955-965]. These polymers can terminate in thiol-reactive groups (e.g., maleimides) that can be used to attach to sbp members. For example, the thiol-reactive groups can be attached to the Fc region of an antibody. Other functional groups can also be used for conjugation of these polymers, for example, N-hydroxysuccinimide esters, or amine-reactive groups such as groups reactive to carboxyl groups or to glycosylation of antibodies. Any number of polymers can be attached to each sbp member. Specific examples of polymers that can be used include linear ("X8") polymers or third-generation dendritic ("DN3") polymers, both available as MaxPar™ reagents. Metal nanoparticles can also be used to increase the number of atoms in the label.

[0136] As described above, a label atom is attached to an sbp member, and the labeled sbp member is contacted with a sample in which the target sbp member (if present) can be found, thereby forming a labeled sbp. The labeled sbp member can comprise any chemical structure that is attached to the label atom and is then suitable for detection according to the present invention.

[0137] Generally, the methods of the invention can be based on any sbp known for use in determining the presence of a target molecule in a sample (e.g., as used in IHC or fluorescence in situ hybridization, FISH) or for fluorescence-based flow cytometry, but the sbp member contacted with the sample carries a label atom that is detectable by MS. Thus, the invention can be readily implemented using available flow cytometry reagents, simply by improving previously used labels, for example, to improve FISH probes to carry labels that are detectable by MS.

[0138] An sbp can include any of the following: a nucleic acid duplex, an antibody / antigen complex, a receptor / ligand pair, or an aptamer / target pair. Thus, a labeled atom can be attached to a nucleic acid probe that is subsequently contacted with a sample so that the probe can hybridize to a complementary nucleic acid therein to form, for example, a DNA / DNA duplex, a DNA / RNA duplex, or an RNA / RNA duplex. Similarly, a labeled atom can be attached to an antibody that is subsequently contacted with a sample so that it can bind to its antigen. A labeled atom can be attached to a ligand that is subsequently contacted with a sample so that it can bind to its receptor. A labeled atom can be attached to an aptamer ligand that is subsequently contacted with a sample so that it can bind to its target. Thus, labeled sbp members can be used to detect a variety of target molecules in a sample, including DNA sequences, RNA sequences, proteins, sugars, lipids, or metabolites.

[0139] In one exemplary embodiment, the labeled sbp member is an antibody. Labeling of the antibody can be achieved by conjugating one or more labeling atoms that link the molecule to the antibody, for example, using the MaxPar™ conjugation kit described above. The antibody's target molecule, referred to as its antigen, can be a protein, carbohydrate, nucleic acid, or the like. Antibodies that recognize cellular proteins useful in mass cytometry are already widely available for use in IHC. By using labeling atoms instead of current labeling techniques (e.g., fluorescence), these known antibodies can be easily adapted for use in the methods of the present invention, but with the advantage of enhanced multiplexing capabilities. Antibodies used in the present invention can recognize targets on the cell surface or intracellular targets. Antibodies can recognize various targets, for example, specific recognition of individual proteins, or recognition of multiple related proteins sharing a common epitope, or recognition of specific post-translational modifications of proteins (e.g., distinguishing between tyrosine and phosphotyrosine, distinguishing between lysine and acetyllysine, detecting ubiquitination, etc.) in a protein of interest. The label atom conjugated to the antibody is detectable after binding to its target to reveal the presence of the target in a sample.

[0140] The labeled sbp member typically interacts directly with the target sbp member in the sample. However, in some embodiments, the labeled sbp member can interact indirectly with the target sbp member; for example, a primary antibody can bind to the target sbp member, and a labeled secondary antibody can then bind to the primary antibody in a sandwich assay. However, the present invention typically relies on a direct interaction because this is more easily accomplished and allows for high multiplexing. In both cases, however, the sample is contacted with an sbp member that is capable of binding to the target sbp member in the sample, and at a later stage, the label attached to the target sbp member is detected.

[0141] One feature of the present invention is the ability to detect multiple (e.g., 10 or more, or 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 in a sample. To enable the differential detection of three target sbp members, each of those sbp members should carry a different label atom so that their signals are distinguishable by MS. For example, if 10 different proteins are to be detected, 10 different antibodies (each specific for a different target protein) can be used, each carrying a unique label so that the signals from the different antibodies are distinguishable. In some embodiments, it is desirable to use multiple different antibodies against a single target, e.g., recognizing different epitopes on the same protein.

[0142] It is desirable that the antibodies should have similar affinities for their respective antigens, as the use of two or more labeled antibodies helps ensure that the relationship between the amount of labeled atom detected by MS and the abundance of the target antigen is more consistent across different sbps (especially at high scanning frequencies).

[0143] If the target sbp member component is located intracellularly, it is typically necessary to permeabilize the cell membrane before or during contacting the sample with the label. For example, if the target is a DNA sequence but the labeled sbp member cannot permeabilize the membrane of a living cell, the sample cells can be fixed and permeabilized. The labeled sbp member then enters the cell and forms an sbp with the target sbp member.

[0144] Typically, the methods of the invention detect at least one intracellular target and at least one cell surface target. However, in some embodiments, the invention can be used to detect multiple cell surface targets while ignoring intracellular targets. Overall, the choice of target will be determined by the information desired from the method.

[0145] Sample labeling does not entirely rely on SBPs. In some cases, classical dyes can be used to highlight desired features on tissue. In several cases, dyes used for microscopy contain elements that are rare in the natural state of cells. Thus, in the process of staining tissue, the tissue is enriched with specific elements that are readable by the devices and methods described herein.

[0146] Thus, in some embodiments, the analytical methods described above include labeling the sample with at least one label atom, which atom is then detectable using the methods described above.

[0147] signal enhancement Aspects of the target application include signal enhancement by adding hydrogen in LA-ICP-MS, as further described herein. The LA-ICP-MS system can have one or more gas flows as shown in FIG. 4 , such as a trapping gas flow to the internal torch line, a carrier gas flow, an internal (auxiliary) gas flow, and / or an external (plasma) gas flow to the external torch line. Notably, the gas sources shown in FIG. 4 can be configured differently, such that premixed or humidified gas is supplied to any one or more of the trapping gas flow, carrier gas flow, internal gas flow, and / or external gas flow. In certain embodiments, the LA-ICP-MS system can include three gas sources, such as a separate gas source for the carrier flow, compared to the internal and external gas flows. Alternatively or additionally, the laser ablation system can include an injector orthogonal to the sample, as shown in FIGS. 2-3 . In certain embodiments, only one gas flow (e.g., the trapping gas) can flow through the injector. In certain embodiments, the injector includes a make-up gas flow downstream of the laser ablation plume.

[0148] Aspects of the subject applications include devices and workflows for imaging mass cytometry (IMS) that improve sample capture speed, signal sensitivity, and / or signal stability. Imaging mass cytometry (IMC) is the detection of mass tags by imaging mass cytometry with cellular or subcellular spatial resolution. IMC systems and methods can include any of the aspects described in the subject applications. In certain aspects, mass cytometry can include laser ablation (LA) inductively coupled plasma (ICP) mass spectrometry (MS). The use of non-intrinsic elements, such as heavy metal mass tags, allows for better detection than endogenous elements. Endogenous elements, such as carbon, oxygen, and nitrogen, and light metals, such as calcium, can be depleted by the mass spectrometer, such as by a high-pass mass filter (e.g., RF quadrupole). In certain aspects, argon dimers (a by-product of argon-based ICP) can also be depleted, such as by a high-pass mass filter with a cutoff of at least 80 amu.

[0149] Heavy metal mass tags can contain heavy metals greater than 80 amu, as further described herein. In certain embodiments, individual mass tags can contain multiple labeled atoms of enriched heavy metal isotopes. Such labeled atoms can be on a polymer that is subsequently conjugated to a specific binding partner (SBP) that binds a specific target, such as an antibody that binds a specific protein target, or can be attached by chelating a pendant group to the polymer. For example, Maxpar tags, available from Fluidigm, each contain a polymer equipped with multiple labeled atoms of a single isotope, such as a lanthanide isotope, and can be conjugated to an antibody that binds a specific protein expressed by a cell. Detection of the isotope by ICP-MS indicates the presence of the corresponding target (e.g., protein).

[0150] The use of enriched isotopes as labeling atoms increases the number of detectable different targets beyond those that can be detected by elemental mass tags containing natural mixtures of isotopes.However, when using more than 20, 30, or 40 isotope mass tags, isotopes that are 16 amu apart from each other are often included, so that the oxide of one mass tag can interfere with the detection of another mass tag containing an isotope with a mass greater than 16 amu.Oxides, such as lanthanides, used as mass tags are by-products of ICP-based atomization and ionization.In certain embodiments, methods or systems for increasing sensitivity or signal stability can be implemented in a way that avoids the formation of excess oxides.

[0151] Thus, oxidation can present unique challenges for atomic IMC, unlike other forms of IMS, such as MALDI, because atomic mass detection of metal isotopes that are 16 amu from other mass tags is susceptible to oxide spillover. This consideration is further complicated by the desire for sensitive and stable detection within each laser-ablated spot (pixel). Each ablation creates a small (e.g., micron- or submicron-sized) ablation crater in which a small number of given mass tags may reside. Furthermore, IMC relies on the ability to accurately compare the expression of different targets (e.g., measured as signals in different mass channels, each corresponding to an isotopic mass tag of a different mass) across a sample, such as a tissue section.

[0152] In certain embodiments, the IMC system can be used for suspension mass cytometry, such as by coupling the system's ICP torch to a spray chamber for introducing whole cells instead of the laser ablation source. Thus, the ICP torch can be capable of micronizing and ionizing whole cells (e.g., cells up to at least 15 microns in diameter, or up to at least 20 microns in diameter). Therefore, the plasma generated by the ICP torch cannot be specifically designed for efficient ionization and / or micronization of the laser ablation plume (e.g., it can have a longer path length than would be necessary for the laser ablation plume). Alternatively, designing an LA-ICP-MS system to have a short transient signal can involve reducing the plasma path length, resulting in inefficient ionization and / or micronization (e.g., if not improved by introducing hydrogen, as described herein). The inventors have found that humidity levels affect the sensitivity of the IMC system, potentially resulting in signal drift, such as when humidity in the system tends to decrease over the sample transfer (e.g., when some initial level of humidity is observed by the air in the ablation chamber prior to activation). Indeed, as described further herein, both water vapor and hydrogen gas have been found to increase signal sensitivity and may further improve signal stability. Thus, the efficiency of ionization and / or atomization (e.g., as measured by increased sensitivity in one or more mass channels, e.g., for one or more labeled atoms) may be improved by adding one or more hydrogen-containing molecules to the gas stream, as described further herein. For example, suitable hydrogen-containing molecules may be water or alcohol (e.g., ethanol) as vapor. Alternatively or additionally, suitable hydrogen-containing molecules may be hydrogen gas, methane, or ammonium, provided, for example, premixed with helium or argon.

[0153] In certain embodiments, portions of the LA-ICP-MS system are open to the atmosphere, allowing air to be present within the laser ablation chamber, fluidic optics, and / or ICP torch. Such air may ultimately be removed or consumed by the operation of the ICP-MS system. However, such air can alter humidity and / or oxygen levels, which can affect the efficiency of the ICP plasma, such as ionization efficiency and / or oxide formation. In certain embodiments, humidity can be controlled throughout the sample transfer, as described herein (e.g., so that mass signals are increased and / or stabilized, while oxidation is minimized). Alternatively or additionally, hydrogen can be premixed with a gas (such as helium or argon) to increase sensitivity and / or signal stability.

[0154] The inventors have found that adding water or hydrogen during LA-ICP-MS analysis of heavy metal mass tags can improve sensitivity and control signal stability. In certain embodiments, one or more hydrogen-containing gases, such as hydrogen gas, water vapor, methane, and / or ammonia, can be introduced into the LA-ICP-MS. In certain embodiments, a gas (such as hydrogen gas, methane, or ammonia) can be provided premixed with an ICP gas, such as helium or argon, in a pressurized gas source. In certain embodiments, water vapor can be mixed with a gas, such as argon gas.

[0155] Generally, hydrogen gas may be provided to the ICP source at a flow rate sufficient to provide signal enhancement and / or stability.

[0156] The signal enhancement can be for one or more mass channels, such as a metal isotope channel of a mass tag used by the device to label the biological sample being analyzed. In certain embodiments, the metal isotope comprises a lanthanide isotope. The signal enhancement can be at least 20%, at least 30%, at least 50%, at least 80%, or at least 100% compared to the signal without hydrogen gas. In certain embodiments, the signal enhancement is the average signal enhancement across the range of mass channels in which a signal (e.g., from a metal isotope mass tag) is detected. In certain embodiments, the average signal enhancement for a lanthanide isotope measured by at least 10 counts is at least 20%, at least 30%, or at least 50%. Signal enhancement, also referred to as sensitivity improvement, can be measured as an increment in counts (e.g., average counts) per laser ablation plume, such as when analyzing labeled atoms in a sample or when analyzing an elemental standard containing a known amount of the detectable atom.

[0157] In certain embodiments, the amount of hydrogen gas flowing to the ICP source in an amount that is robust to changes in hydrogen gas flow, such as a 20% or 50% change in amount, will have less than a 10% change in signal, such as less than a 5% change in signal (e.g., for a standard, one labeled atom, several labeled atoms, or for all labeled atoms detected at greater than 10 counts).

[0158] Described herein are devices and methods for introducing hydrogen-containing molecules (e.g., in a premixed gas or vapor) to improve signal sensitivity and / or stability. In certain embodiments, the introduction of hydrogen improves signal stability such that external humidity over a range (e.g., 0-2000 microbar or 0-4000 microbar) has less than a 10% or less than a 5% change in signal sensitivity.

[0159] Hydrogen gas for signal enhancement In certain embodiments, an apparatus, method, and / or premixed compressed gas source may be provided for introducing hydrogen gas into an ICP torch, such as to improve signal sensitivity and / or signal stability.

[0160] In certain embodiments, the apparatus comprises one or more of a sample stage configured to move a sample in at least two directions, a laser ablation source configured to ablate a sample mounted on the sample stage, an inductively coupled plasma (ICP) torch, an injector configured to convey an ablation plume created from the sample by the laser ablation source to the ICP torch, and a source of compressed premixed gas comprising hydrogen gas mixed with at least one of helium and argon.

[0161] The hydrogen gas in the compressed premixed gas source may be from 0.1% to 5% by volume, such as from 1% to 4% by volume.

[0162] The compressed premixed gas source can be at least 50% helium by volume or at least 50% argon by volume. The compressed premixed gas source can supply gas to an ablation chamber including a sample stage. The premixed gas source can provide a trapping gas that carries the ablation plume to the injector. Alternatively or additionally, the premixed gas source can provide a carrier gas (i.e., that enters the injector upstream of the ablation plume), a make-up gas (i.e., that supplements the gas in the injector downstream of where the ablation plume enters the injector), and / or an internal gas (i.e., an auxiliary gas that flows into the internal piping of the ICP torch).

[0163] The additional gas source may provide a carrier gas to the injector, such as where the trapping gas lifts the ablation plume into the carrier gas in the injector. The carrier gas may include argon (e.g., at least 50% argon). The trapping gas may include a mixture of helium and hydrogen gas (e.g., at least 50% helium) or a mixture of argon (e.g., at least 50% argon) and hydrogen gas.

[0164] In certain embodiments, there may not be separate trapping and carrier gases, such as when only trapping gas enters the injector. In certain embodiments, the injector is configured to direct the laser ablation plume toward a vertical ICP torch, such as when the apparatus includes a vertically oriented ICP torch.

[0165] The apparatus may further comprise an additional gas source providing at least one of a carrier gas, a makeup gas, an internal (auxiliary) gas, and / or an external (plasma) gas. The additional gas source may be a compressed gas or liquid dewar and may be at least 50% argon by volume, while the premixed gas source comprises at least 50% helium by volume. In contrast, the premixed gas source may not be in a liquid dewar because different gases may evaporate (and thus be depleted) at different rates during operation.

[0166] If the apparatus includes a make-up flow downstream of where the laser ablation plume enters the injector up to the injector, the make-up flow may optionally be further downstream of the sacrificial flow.

[0167] In certain aspects, the compressed premixed gas source provides at least one of a trapping gas, a carrier gas, and an internal torch gas.

[0168] The apparatus may be configured to provide a hydrogen gas flow of 0.001 L / min to 0.1 L / min to the ICP torch, such as a hydrogen gas flow of 0.001 L / min to 0.02 L / min. Alternatively or additionally, the hydrogen gas may be 0.002% to 1% of the total gas flow to the ICP torch, such as 0.01% to 0.1% of the total gas flow to the ICP torch. The total gas flow to the ICP torch may be 5 to 30 L / min, such as 10 to 25 L / min. For example, U.S. Patent No. 8,633,416, incorporated by reference, reports a gas flow of approximately 5 L / min. In certain embodiments, the hydrogen gas flow to the ICP torch may be varied by more than 20%, such as more than 50%, without reducing sensitivity (e.g., to one, some, or all of the target atoms) by more than 5%. In certain embodiments, the amount of hydrogen gas flow to the ICP torch during sample analysis may be varied by more than 10%, more than 20%, or more than 50%.

[0169] The apparatus may further comprise a mass spectrometer configured to detect ionized atoms produced by the ICP torch, as further described herein. In certain embodiments, the mass spectrometer comprises a high-pass filter configured to remove ions having a mass of at least 80 amu or less.

[0170] The ICP torch can be configured to micronize and ionize all cells in a cell suspension mode where the ICP torch is separated from the laser ablation source. For example, the ICP torch can be separated from the laser ablation source and be sufficient to micronize and ionize all cells introduced into the spray chamber in suspension upstream of the ICP. The characteristics of the ICP torch, such as its size and shape, can be suitable for micronizing and ionizing all cells. This design consideration can make the ICP torch less efficient at micronizing and / or ionizing material in a laser ablation plume, such as a plume generated by a laser with a spot size of less than 2 microns, such as a spot size of less than 1 micron.

[0171] The apparatus of any of the above embodiments may further comprise a humidification system (eg, as described further herein) configured to humidify the gas stream.

[0172] The method of the subject application can include analyzing a sample by LA-ICP-MS using any of the devices of the above-mentioned embodiments.The sample can be a biological sample as further described herein, such as a tissue section.The sample can contain a labeled atom as further described herein, such as a labeled atom associated with SBP that binds to a target in the sample.Therefore, the analysis method can further include labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS.In certain embodiments, no labeled atom has more than 3% oxide spillover.

[0173] In certain embodiments, hydrogen gas provides at least a 20% increase in sensitivity for at least some of the labeled atoms, such as labeled atoms with an average ion count of at least 10 per ablation plume, or at least a 50% increase in sensitivity for at least some of the labeled atoms.

[0174] In certain embodiments, the average sensitivity to the labeled atom (or elemental standard) in any given 5 minute period may not change by more than 10% over at least 1 hour of analyzing the sample.

[0175] While hydrogen gas is described above, other hydrogen-containing gases may be used in place of or in addition to hydrogen gas in any of the above embodiments. For example, methane or ammonia may be used in place of or in addition to hydrogen gas.

[0176] Premixed gas sources and uses thereof A target application embodiment includes a compressed premixed gas source for an inductively coupled plasma device. The premixed gas source can include at least 50% by volume helium or argon (e.g., at least 70% by volume, at least 90% by volume), and can further include at least 0.1% by volume of a gas including elemental hydrogen. The gas can be, for example, hydrogen gas, ammonia, and / or methane.

[0177] In certain embodiments, the gas comprises hydrogen gas. The premixed gas source may contain 0.1 to 5% by volume, such as 1% to 4% by volume, of hydrogen gas. The premixed gas source may contain hydrogen gas below its combustion point (e.g., approximately less than 4%). The premixed gas source may be a compressed gas cylinder for use in LA-ICP-MS.

[0178] Embodiments include LA-ICP-MS systems that include the compressed premixed gas source described above. In certain embodiments, the mass spectrometer can alternatively be an elemental analyzer, such as an optical emission spectrometer.

[0179] Gas humidification for signal enhancement Aspects of the target application include apparatus and methods for gas humidification for LA-ICP-MS.

[0180] In certain embodiments, the apparatus comprises one or more of a sample stage configured to move a sample in at least two directions, a laser ablation source configured to ablate a sample mounted on the sample stage, an inductively coupled plasma (ICP) source, an injector configured to convey an ablation plume created from the sample by the laser ablation source to the ICP torch, and a humidification system configured to humidify the gas flow.

[0181] The gas stream may include one or more of a carrier gas stream, a trapping gas stream, a make-up gas stream, and an auxiliary gas stream. In certain aspects, the gas stream is a carrier gas stream.

[0182] In certain embodiments, the gas stream comprises at least 50% argon (such as at least 70% or at least 90% argon), such as when the gas stream is a carrier gas. Alternatively, the gas stream can comprise at least 50% helium, such as when the gas stream is a trapping gas.

[0183] In certain embodiments, the humidification system has an adjustable range encompassing at least 500 μm (microbar) up to 5000 μB, such as 1000 μB to 4000 μB or 1500 μB to 3000 μB. This can produce humidified gas with a stability of 20% or greater, such as 20% or greater. In certain embodiments, humidity is not controlled within a range of less than 3%, such as less than 5%, as such control may not be necessary for signal stability.

[0184] The humidification system may include water diffusion piping. In certain aspects, the humidification system controls the temperature of the diffusion piping. Alternatively or additionally, the humidification system includes a variable splitter that is adjustable to redirect a gas flow (e.g., a flow of argon gas) around the water diffusion piping. The variable splitter may be adjusted by a controller coupled to a humidity sensor. In certain aspects, the controller and humidity sensor are both configured to redirect the gas flow around the diffusion piping to maintain humidity levels. Instead of diffusion piping, the humidification system may include a water pump configured to inject water directly into the gas flow.

[0185] The method may include analyzing the sample by LA-ICP-MS using an apparatus comprising any suitable humidification system, such as the humidification system described in one of the above embodiments.

[0186] The sample may be a biological sample, such as a tissue section, as further described herein. The sample may contain a labeled atom, such as a labeled atom associated with SBP that binds a target in the sample, as further described herein. Thus, the analytical method may further include labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS. In certain embodiments, no labeled atom has an oxide spillover greater than 3%. For example, high humidity can result in oxide spillover greater than 3%. Thus, humidity and / or plasma temperature may be maintained at a level that allows for improved sensitivity, as described below, but may be low enough to avoid oxide spillover greater than 3% for any labeled atom (e.g., average oxide spillover during any 5-minute period of sample transfer). For example, temperature may be controlled by adjusting the plasma temperature via makeup gas flow. Even small humidity values ​​can result in very large amounts of oxide if the temperature is not properly controlled.

[0187] In certain embodiments, humidification provides at least a 20% increase in sensitivity for at least some labeled atoms, such as labeled atoms with an average ion count of at least 10 per ablation plume (e.g., after pretreatment), or at least a 50% increase in sensitivity for at least some labeled atoms. In certain embodiments, the average sensitivity for labeled atoms (or elemental standards) over any given 5-minute period may not change by more than 10% over at least 1 hour of analyzing the sample. In certain embodiments, humidity may be changed by more than 20%, such as more than 50%, without reducing sensitivity (e.g., to one, some, or all of the labeled atoms) by more than 5%.

[0188] While gas humidification (with water vapor) is described above, other hydrogen-containing molecules may be introduced into the gas stream as a vapor instead of or in addition to water. For example, an alcohol such as ethanol may be introduced into the gas stream by any of the modes described above for water vapor, such as diffusion piping.

[0189] A typical laser ablation inductively coupled plasma (LAP) system uses a combination of gas flows to transport the ablated material from the ablation point to the plasma. Sensitivity is affected by the amount of moisture in the gas flow. Adding water to the gas flow via an atomized water mist disrupts the flow and, in imaging applications, can negatively impact transient signal and pixel speed. Humidifying the gas flow before it enters the instrument eliminates this problem. Ensuring stable humidity typically involves significant temperature stabilization. The humidity of the exiting gas flow can be measured to provide feedback to the humidifier to regulate humidity. The humidifier itself is a gas flow passing through a diffusion tubing. Because the flow through the tubing exits saturated, the inventors use a variable gas splitter to vary the flow through the diffusion tubing, thereby varying the moisture content in the gas flow after recombination. Varying the gas flow is a feedback mechanism.

[0190] Apparatus and method for supplying hydrogen for signal enhancement Aspects of the target application include an apparatus and method for introducing hydrogen-containing molecules into an ICP torch in a LA-ICP-MS. The hydrogen-containing molecules can be gases such as hydrogen gas, ammonia, or methane. Alternatively or additionally, hydrogen-containing molecules such as water or alcohols (e.g., ethanol) can be introduced into the gas stream as a vapor.

[0191] Embodiments include an apparatus comprising one or more of a sample stage configured to move a sample in at least two directions, a laser ablation source configured to ablate a sample mounted on the sample stage, an inductively coupled plasma (ICP) torch, an injector configured to convey an ablation plume created from the sample by the laser ablation source to the ICP torch, and a compressed premixed gas source comprising a hydrogen-containing gas mixed with at least one of helium and argon. In certain embodiments, the hydrogen-containing gas is methane, ammonia, or hydrogen gas.

[0192] Embodiments include an apparatus comprising one or more of a sample stage configured to move a sample in at least two directions, a laser ablation source configured to ablate a sample mounted on the sample stage, an inductively coupled plasma (ICP) source, and an injector configured to convey an ablation plume created from the sample by the laser ablation source to an ICP torch. The apparatus is configured to provide a vapor comprising a flow of hydrogen gas. The vapor can include water vapor or an alcohol vapor (e.g., ethanol, etc.).

[0193] Embodiments include an apparatus comprising one or more of a sample stage configured to move a sample in at least two directions, a laser ablation source configured to ablate a sample mounted on the sample stage, an inductively coupled plasma (ICP) torch coupled to a mass spectrometer, and an injector configured to deliver an ablation plume created from the sample by the laser ablation source to the ICP torch. The apparatus can be configured to supply hydrogen-containing molecules to the plasma (e.g., as described herein) of the ICP torch while operating for laser ablation ICP mass spectrometry but not in a cell suspension mode. In the cell suspension mode, the ICP torch can be separate from the laser ablation source and can be sufficient to atomize and ionize all cells introduced into the spray chamber in suspension upstream of the ICP.

[0194] Experimental results The inventors have found that signal stability and enhancement for lanthanide and other labeled atoms in biological or inorganic samples (e.g., elemental standards) can be achieved with small amounts of hydrogen. The hydrogen can be hydrogen gas (below the flammable limit), and the stability and enhancement can be robust to differences in the amount of hydrogen gas (e.g., by changing gas flows during operation to maintain a constant plasma). Alternatively, hydrogen can be introduced as water vapor, and signal stability and enhancement can similarly be robust to a range of humidity levels.

[0195] The inventors monitored the sensitivity of the IMC (a Fluidigm Hyperion imaging system) to see a decline during operation and recovery during shutdown. The inventors determined that this was due to small amounts of moisture diffusing into the system, accumulating during shutdown, and gradually drying and evaporating during operation. This led to the need to humidify the gas flow within the system as well as to propose methods to reduce moisture diffusion into the system. During their investigation, the inventors found that the amount of hydrogen in the system was a critical factor. The inventors initially rejected the idea of ​​mixing pure hydrogen into the injector gas due to the high cost associated with the extra gas handling considerations and safety measures. The inventors initially developed an argon flow humidifier device, which could consistently deliver H2O at the required levels. However, image analysis by biology experts revealed concerns about images on the mass channel being contaminated by oxide formation. The oxygen in this case originated from water molecules in the argon flow. The inventors then decided to explore a way to obtain the benefits of pure hydrogen mixed into the injector stream without the addition of oxygen, but in a configuration that eliminated flammability and safety concerns. The inventors realized that a premixed gas diluted to a non-flammable level could be managed with existing gas handling components, while providing sufficient hydrogen concentration to increase sensitivity and stabilize the signal.

[0196] The inventors initially found that the signal was sensitive to humidity and could vary with external humidity (e.g., that of the air within the LA-ICP-MS instrument space). As shown in Figure 5, the humidity range of 0-4000 uB (microbar) of the carrier gas accounts for approximately a 30% change in the sensitivity of the lutetium-containing elemental standard, measured as the average lutetium counts per ablation shot, across the humidity range. However, the inventors also noticed that the signal was consistently high within the humidity range (approximately 2000-3000 uB). The system used was similar to that shown in Figure 4.

[0197] The inventors have also found that adding water can cause oxide levels to become very high, but that the carrier gas flow rate can be adjusted to keep the oxides at a specific level, as opposed to simply adjusting for maximum sensitivity. Below are sample preparation results showing that limiting the oxide ratio to <3% (or less, such as <2% or <1%) while using a humidifier still achieves significant improvements in sensitivity over time: [Table 2]

[0198] One additional note is that Ce140 to Gd156 typically have the lowest oxide spillover of all mass channels, so limiting oxide spillover to <3% in a channel during conditioning can be much smaller in other channels. In certain embodiments, oxide spillover varies by less than 1%, such as less than 0.5% or less than 0.25% during sample transfer (e.g., over a period of more than 5, 10, or 30 minutes of sample transfer).

[0199] The inventors also confirmed that the addition of hydrogen (H) gas (3% hydrogen gas in a premixed gas source with helium) correlated strongly with signal enhancement / ion image brightness. Signal enhancement for both techniques is shown in Figure 9. Specifically, carrier gas humidification (left panel) shows improved signal for common markers, and premixed hydrogen-helium trapping gas (right panel) also shows improved signal for common markers.

[0200] The inventors also found that very faint channels showed less improvement in sensitivity than brighter channels, likely due to background effects, and therefore the improvement in sensitivity can be seen primarily for channels detected with an average of 10 or more ion counts per ablation shot.

[0201] The trade-offs for these two approaches are compared below. [Table 3]

[0202] The inventors experimented with the multiple humidification system shown in Figure 6 and found that direct injection of water by a water pump (top of Figure 6) resulted in some stability issues related to wetting and droplet formation at the ends of the capillaries. Flowing gas through the diffusion tubing (bottom of Figure 6) with temperature and / or humidity feedback to control the redirection of gas flow around the diffusion tubing was found to provide better humidification stability. Overnight operation at a constant speed and stabilizing temperature for the diffusion tubing resulted in a fairly stable signal, with humidity varying by + / - 10% (as shown in Figure 7). However, forgoing temperature stability and instead using feedback on gas flow may provide better humidity control. Humidity is controlled by varying the gas flow through the diffusion tubing in response to the temperature detected by a sensor downstream of the tubing. Controlling the gas flow through the diffusion tubing results in more consistent humidity and better response time to changes.

[0203] A typical laser ablation inductively coupled plasma device uses a combination of gas flows to transport the ablated material from the ablation point to the plasma. Sensitivity is affected by hydrogen in the gas flow.

[0204] The inventors used varying amounts of hydrogen gas to determine the appropriate amount of hydrogen required for an LA-ICP-MS system similar to that shown in Figure 4. We used a mixed gas ratio for our helium-hydrogen gas flow that provided the appropriate amount of hydrogen over a typical range of helium flows. This allows switching to a hydrogen mixed gas system without any other changes to hardware or software other than abandoning the use of pure helium and substituting it with mixed hydrogen-helium gas. The total number of cylinders / regulators / mass flow controllers remains the same. Unlike the humidifier system, the mixed gas system does not add oxygen, and the oxide ratios detected by the MS system were not affected.

[0205] In an alternative configuration, hydrogen is premixed with argon (e.g., a gas source specific for carrier and / or trap gas). In some LA-ICP-MS systems, argon can be used as a trap gas for the ablated plume. In addition to LA-ICP-MS, in mass cytometry instruments, neon can also be considered for some applications, although it may be cost-prohibitive. In yet another alternative configuration, hydrogen premixed with argon is added to the carrier gas. The carrier gas is part of the total injector flow and is mixed with the ablation trap gas that carries the plume. Adding an H2 / argon premix to the carrier gas flow allows users to vary the ratio of pure argon to the H2 / Ar premix, controlling the overall flow of H2 to the injector while independently controlling the optimal argon flow. In other words, the premix flow controls the mass flow of H2, and the premix flow and the remaining argon flow control the total argon flow of the carrier gas. The total argon flow at the injector output must be carefully adjusted to achieve optimal plasma temperature and maximum sensitivity. The H2 level then provides a second dimension for sensitivity improvement. The proposed H2 / argon premix has a low concentration of hydrogen, below the flammability limit. This system has the disadvantage of requiring an additional cylinder of premix gas and an additional independent mass flow controller for the premix flow. However, the advantage of this system is the ability to independently control and adjust the hydrogen portion of the total flow for given instrument and plasma conditions. For example, this arrangement could be considered Plan B for a Deuterium system, with an H2 / helium premix as Plan A. The H2 / helium mixture is a low-cost, convenient solution due to the typically less significant limitation of the lack of independent H2 flow control.

Claims

1. 1. An apparatus comprising: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; a plasma source; and an injector configured to deliver an ablation plume created from the sample by the laser ablation source to the plasma source; The apparatus, wherein at least one of the plasma source and the sample stage are oriented orthogonal to one another.

2. The device of claim 1 , wherein the injector is rigid.

3. 3. The device of claim 1, wherein the injector is linear.

4. The device of any one of claims 1 to 3, wherein the injector has an inner diameter of less than 1 mm.

5. The device of any one of claims 1 to 4, wherein the injector has a length of less than 10 cm.

6. 6. The device of claim 5, wherein the injector has a length of less than 5 cm.

7. The apparatus of any one of claims 1 to 6, wherein the apparatus is configured to direct a laser on a path that does not pass through the injector.

8. The apparatus of any one of claims 1 to 7, wherein the apparatus is operable to deliver at least 1000 different ablation plumes per second to the ICP source.

9. The apparatus of any one of claims 1 to 8, further comprising a mass spectrometer.

10. The apparatus of claim 9 , wherein the mass spectrometer is a time-of-flight mass spectrometer.

11. 11. Apparatus according to claim 9 or 10, wherein the mass spectrometer is configured to receive a vertical beam of ions.

12. An apparatus according to any preceding claim, wherein the sample stage is vertical and operable to move into a vertical position.

13. The apparatus of any one of claims 1 to 11, wherein the plasma source is vertically oriented.

14. 14. The apparatus of claim 13, wherein the plasma source is vacuum sealed, except for an inlet of an injector to the plasma source.

15. The apparatus of any one of claims 1 to 14, wherein the plasma source is an ICP source.

16. A method comprising analysing a sample by LA-ICP-MS using an apparatus according to any one of claims 1 to 15.

17. 17. The method of claim 16, wherein the sample is a biological sample.

18. The method of claim 17 , wherein the sample comprises labeled atoms.

19. 19. The method of claim 18, further comprising labeling the sample with a labeling atom before analyzing the sample by LA-ICP-MS.

20. The method of any one of claims 16 to 19, wherein at least 1000 different ablation plumes are analyzed per second.

21. 1. An apparatus comprising: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to convey an ablation plume created from the specimen by the laser ablation source to the ICP torch; a source of compressed premixed gas comprising hydrogen gas mixed with at least one of helium and argon.

22. 22. The apparatus of claim 21, wherein the hydrogen gas in the compressed premixed gas source is greater than 0.1% and less than the flammability limit of hydrogen.

23. 22. The compressed premixed gas source of claim 21, wherein the hydrogen gas is between 1% and 4% by volume.

24. 24. The apparatus of claim 21, 22 or 23, wherein the source of compressed premixed gas comprises at least 50% helium by volume.

25. 24. The apparatus of claim 21, 22 or 23, wherein the source of compressed premixed gas comprises at least 50% argon by volume.

26. An apparatus according to any one of claims 21 to 25, wherein the compressed premixed gas source supplies gas to an ablation chamber containing the sample stage.

27. The apparatus of any one of claims 21 to 26, wherein the premixed gas source provides a trapping gas that carries the ablation plume into the injector.

28. 28. The apparatus of claim 27, wherein an additional gas source provides a carrier gas to the injector, and the trapping gas lifts the ablation plume into the carrier gas within the injector.

29. 30. The apparatus of claim 28, wherein the carrier gas comprises argon and the trapping gas comprises a mixture of helium and hydrogen gas.

30. 28. The apparatus of claim 27, wherein there are no separate trapping and carrier gases.

31. The apparatus of any one of claims 21 to 30, wherein the injector directs a laser ablation plume towards a vertical ICP torch.

32. 30. The apparatus of any one of claims 21 to 29, further comprising an additional gas source that provides the carrier gas to the injector.

33. 33. The apparatus of claim 32, wherein the additional gas source comprises at least 50% argon by volume and the premixed gas source comprises at least 50% helium by volume.

34. Apparatus according to any one of claims 21 to 33, wherein the additional gas source is a liquid dewar.

35. 35. The apparatus of any one of claims 21 to 34, wherein the compressed premixed gas source is configured to introduce a make-up flow into the injector downstream of where the laser ablation plume enters the injector.

36. 36. The apparatus of claim 35, wherein the makeup stream is introduced downstream of the sacrificial stream.

37. 37. The apparatus of claim 36, wherein the apparatus does not include a sacrificial stream.

38. The apparatus of any one of claims 21 to 34, wherein the compressed premixed gas source provides at least one of a trapping gas, a carrier gas, and an internal torch gas.

39. 39. An apparatus according to any one of claims 21 to 38, configured to provide a hydrogen gas flow to the ICP torch at between 0.001 L / min and 0.1 L / min.

40. 40. The apparatus of claim 39, configured to provide a hydrogen gas flow to the ICP torch at between 0.001 L / min and 0.02 L / min.

41. 41. The apparatus of any one of claims 21 to 40, further configured to provide hydrogen gas at 0.002% to 1% of the total gas flow to the ICP torch.

42. 42. The apparatus of claim 41, wherein the apparatus is configured to provide hydrogen gas at 0.01% to 0.1% of the total gas flow to the ICP torch.

43. 43. The apparatus of claim 41 or 42, wherein the total gas flow to the ICP torch is between 5 and 30 L / min.

44. 44. The apparatus of any one of claims 21 to 43, further comprising a mass spectrometer configured to detect ionized atoms produced by the ICP torch.

45. 45. The apparatus of claim 44, wherein the mass spectrometer comprises a high pass filter configured to remove ions having a mass of at least 80 amu or less.

46. 46. ​​The apparatus of any one of claims 21 to 45, wherein the ICP torch is configured to micronize and ionize all cells in a cell suspension mode in which the ICP torch is decoupled from the laser ablation source.

47. 47. The apparatus of any one of claims 21 to 46, further comprising a humidification system configured to humidify the gas flow.

48. 48. The apparatus of claim 47, wherein the humidified gas stream is a carrier gas stream.

49. A method comprising analysing a sample by LA-ICP-MS using an apparatus according to any one of claims 21 to 48.

50. 50. The method of claim 49, wherein the sample is a biological sample.

51. 51. The method of claim 50, wherein the sample comprises labeled atoms.

52. 52. The method of claim 51, further comprising labeling the sample with a labeling atom before analyzing the sample by LA-ICP-MS.

53. 53. The method of claim 52, further comprising: no labeled atoms having an average oxide spillover greater than 3%.

54. 54. The method of any one of claims 49 to 53, wherein the hydrogen gas provides at least a 20% increase in sensitivity to at least some of the labeled atoms.

55. 55. The method of claim 54, wherein the hydrogen gas provides at least a 50% increase in sensitivity to at least some of the labeled atoms.

56. 56. The method of any one of claims 49 to 55, wherein the average sensitivity to labeled atoms in any given 5 minute period does not change by more than 10% over at least 1 hour of analyzing the sample.

57. 1. A compressed premixed gas source for an inductively coupled plasma device, comprising: at least 50% by volume of helium or argon; and 0.1% to 5% by volume of hydrogen gas.

58. 58. The compressed premixed gas source of claim 57, comprising at least 50% by volume helium.

59. 58. The compressed premixed gas source of claim 57, comprising at least 50% by volume argon.

60. 58. The compressed premixed gas source of claim 55, 56, or 57, wherein the hydrogen gas is between 1% and 4% by volume.

61. 1. A compressed premixed gas source for an inductively coupled plasma device, comprising: at least 50% by volume of helium or argon; at least 0.1% by volume of a gas, the gas comprising elemental hydrogen.

62. 62. The compressed premixed gas source of claim 61, wherein the gas is methane, ammonia, or hydrogen gas.

63. 1. An apparatus comprising: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) source; an injector configured to convey an ablation plume created from the specimen by the laser ablation source to an ICP torch; a humidification system configured to humidify the gas stream.

64. 64. The apparatus of claim 63, wherein the gas flow comprises a carrier gas flow.

65. 65. The apparatus of claim 63 or 64, wherein the gas flow comprises a trapped gas flow.

66. 66. An apparatus according to any one of claims 63 to 65, wherein the gas flow comprises a make-up gas flow.

67. 67. Apparatus according to any one of claims 63 to 66, wherein the gas flow comprises an auxiliary gas flow.

68. 68. Apparatus according to any one of claims 63 to 67, wherein the gas flow comprises at least 50% argon.

69. 69. Apparatus according to any one of claims 63 to 68, wherein the humidification system comprises water diffusion piping.

70. 70. The apparatus of claim 69, wherein the humidification system controls the temperature of the water diffusion piping.

71. 71. The apparatus of claim 69 or 70, further comprising a variable splitter adjustable to redirect gas flow around the water diffusion piping.

72. 73. The apparatus of any one of claims 69 to 72, further comprising a controller and a humidity sensor together configured to redirect gas flow around the water diffusion piping to maintain a humidity level.

73. 69. Apparatus according to any one of claims 63 to 68, wherein the humidification system comprises a water pump configured to inject water directly into the gas stream.

74. 74. A method comprising analysing a sample by LA-ICP-MS using an apparatus according to any one of claims 63 to 73.

75. 75. The method of claim 74, wherein the sample is a biological sample.

76. 76. The method of claim 75, wherein the sample comprises labeled atoms.

77. 77. The method of claim 76, further comprising labeling the sample with a labeling atom before analyzing the sample by LA-ICP-MS.

78. 78. The method of claim 76 or 77, wherein no labeled atoms have an average oxide spillover of more than 3%.

79. 80. The method of claim 76, 77, or 78, wherein the humidification provides at least a 20% increase in sensitivity to at least some of the labeled atoms.

80. 80. The method of claim 79, wherein the humidification provides at least a 50% increase in sensitivity to at least some of the labeled atoms.

81. 81. The method of any one of claims 73 to 80, wherein the average sensitivity to labeled atoms in any given 5 minute period does not change by more than 10% over at least 1 hour of analyzing the sample.

82. 1. An apparatus comprising: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to convey an ablation plume created from the specimen by the laser ablation source to the ICP torch; a source of compressed premixed gas comprising a hydrogen-containing gas mixed with at least one of helium and argon.

83. 83. The apparatus of claim 82, wherein the hydrogen-containing gas is methane, ammonia, or hydrogen gas.

84. 1. An apparatus comprising: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) source; an injector configured to transport an ablation plume created from the specimen by the laser ablation source to an ICP torch; The apparatus is configured to supply hydrogen-containing molecules to the plasma of the ICP torch during operation for laser ablation ICP mass spectrometry.

85. 85. The apparatus of claim 84, wherein the apparatus is configured to provide a vapor comprising a hydrogen gas flow.

86. 86. Apparatus according to claim 84 or 85, wherein the vapor comprises water vapor or alcohol vapor.

87. 87. The apparatus of claim 86, wherein the vapor comprises water vapor.

88. 87. The apparatus of claim 86, wherein the vapor comprises alcohol.

89. 89. The apparatus of claim 88, wherein the alcohol is ethanol.

90. 90. A method comprising analysing a sample by LA-ICP-MS using an apparatus according to any one of claims 84 to 89.

91. 1. An apparatus comprising: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch coupled to a mass spectrometer; an injector configured to deliver an ablation plume created from the specimen by the laser ablation source to the ICP torch; the ICP torch is configured to micronize and ionize all cells in a cell suspension mode in which the ICP torch is separated from the laser ablation source; The apparatus is configured to supply hydrogen-containing molecules to the plasma of the ICP torch while the apparatus is in operation for laser ablation ICP mass spectrometry but not in the cell suspension mode.

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