Plasma and Sampling Geometries for Imaging Mass Spectrometry

Abstract

Described herein are systems and methods for imaging mass spectrometry, including imaging mass cytometry. Aspects of interest include devices and methods for imaging mass spectrometry (IMS) that improve sample capture speed, signal sensitivity, and / or signal stability. The systems and methods described herein may minimize the transport time and / or minimize the diffusion of a plume of removed sample material from a delivered sample to components of an imaging mass spectrometer or mass cytometer that ionize and analyze the sample material.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 114,313, filed on November 16, 2020, and U.S. Provisional Patent Application No. 62 / 951,556, filed on December 20, 2019, and incorporates by reference herein in its entirety the disclosures of both applications for all purposes.

[0002] Background of the Invention Imaging mass spectrometry applications such as imaging mass cytometry gain advantages by rapidly acquiring different ablation plumes. For example, a laser ablation ICP - MS system can remove spots on the order of 1 micron in diameter and separately detect the elemental or isotopic composition of millions of spots within a single sample. If the ablation plume is rapidly delivered with minimal transient diffusion, many ablation spots can be analyzed quickly and sensitivity can be improved. Turbulent flow can increase transient diffusion due to changes in the orientation of plume transport during formation compared to the direction of plume expansion. Components of the system, such as optical components, can impede the rapid injection of the plume into the plasma source. Additionally, by changing the conventional composition of the gas flow, signal sensitivity and / or stability can be improved.

[0003] The present invention relates to an apparatus and method for laser ablation - based imaging mass spectrometry, including imaging mass cytometry.

Summary of the Invention

[0004] In the present invention, the inventors have devised many developed products of existing laser ablation - based imaging mass spectrometers and imaging mass analyzers. In particular, these developed products relate to improvements that minimize the transport time and / or the diffusion of the removed sample material plume from the transported sample to the components of an imaging mass analyzer or mass cytometer that ionize and analyze the sample material.

[0005] The apparatus of the present invention, such as an imaging mass spectrometer or an imaging mass cytometer, typically comprises 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 the atoms in the plume of removed sample material (including any detectable labeled atoms described below) can be detected by the components of the mass spectrometer (the MS component; the third component), the sample must be atomized and ionized (although some ionization of the sample material may occur during ablation, charge neutralization occurs well before the charge becomes detectable due to the space charge effect, and thus the apparatus requires a separate ionization component). Accordingly, the apparatus comprises a second component, an ionization system, which ionizes the elements to form elemental ions and enables the detection of the elemental ions by the MS component based on the mass / charge ratio. Between the laser ablation system and the ionization system, a transfer conduit adapted to couple the laser ablation system to the ionization system is provided. The transfer conduit has an inlet positioned within the laser ablation system, and the inlet is configured to capture the removed plume when the removed plume is generated and to convey the captured and removed 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 pipe. In this case, the transfer conduit may be referred to as an injector). Thus, the sample is conveyed to the apparatus during operation, removed to generate vapor / particulate material, ionized by the ionization system, and the ions of the sample are passed through the MS component. Although the MS component can detect many ions, most of these will be ions of the atoms that make up the sample in nature. In some applications for the typical analysis of minerals in geological or archaeological applications, etc., this may be sufficient. In applications of imaging mass spectrometry such as imaging mass cytometry, the MS component may be a time-of-flight (TOF) or magnetic sector MS.

[0006] Accordingly, the present invention provides (i) a laser ablation system adapted to generate a plume of sample material from a sample, (ii) a plasma source adapted to receive the material removed from the sample by the laser ablation system and ionize the material to form elemental ions, (iii) a mass spectrometer adapted to receive the elemental ions from the ionization system and analyze the elemental ions, and provides an apparatus comprising 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 removed sample material from the laser ablation system to the ionization system. The plasma is not oriented on the same axis as the sample stage.

[0007] The present invention also provides an apparatus comprising a sample stage configured to move the sample in at least two directions, a laser ablation source configured to remove the sample mounted on the sample stage, a plasma source, and an injector configured to convey 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 is oriented orthogonally to each other. 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 linear. 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 apparatus can be configured to direct the laser along 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 different ablation plumes per second to an ICP source. In certain embodiments, if the plasma is short (e.g., less than 5 mm in length, less than 3 mm in length, less than 2 mm in length), it helps prevent transient diffusion, and ions from separate ablation plumes may remain distinct as detected by mass spectrometry.

[0010] The device may further comprise a mass spectrometer (MS) coupled to a 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., while still providing steps on the scale of the diameter of the ablation spot, and adjusted by a motor capable of counteracting gravity).

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

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

[0014] The method may include analyzing a sample by LA-ICP-MS using the device described herein. The sample may be a biological sample and may include labeled atoms (such as labeled atoms of SBP adhered to the analyte of the biological sample). The method may further include labeling the sample with labeled atoms before analyzing the sample by LA-ICP-MS.

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

Brief Description of the Drawings

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

[0017]

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DETAILED DESCRIPTION OF THE INVENTION

[0018] It is to be understood that the indefinite articles "a" or "an" used in conjunction with the present description in relation to various elements include "one or more" or "at least one" unless the context clearly indicates otherwise.

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

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

[0021] The term "substantially" does not exclude "completely", e.g., a composition "substantially free of" Y may possibly be completely free of Y. Optionally, the term "substantially" may be omitted from the definition of the present invention.

[0022] The term "invention" refers to a particular aspect, example, or embodiment of the present invention, rather than referring to all embodiments of the present invention.

[0023] While the foregoing invention has been described in some detail for purposes of clarity and understanding, those skilled in the art will appreciate that various changes in form and detail may be made without departing from the scope of the invention as defined in the appended claims, once they become familiar with this disclosure. Accordingly, the invention is not limited to the exact components or details of the above-described methods or structures. Except to the extent necessary or inherent in the process itself, the steps or stages of the methods or processes described in this disclosure, including the drawings, are not intended or implied to be in a particular order. In many cases, the order of process steps may be changed without altering the purpose, effect, or impact of the described method. All publications and patent documents cited herein are incorporated by reference as if each such publication or patent document were specifically and individually indicated to be incorporated by reference. The 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 content or the date of issue 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). LA-ICP-MS has been described for the measurement of endogenous elements in biological materials and, more recently, for imaging by detection of element-tagged antibodies. For example, U.S. Patent Publication No. 2012 / 0061561 to Antonov, A. and Bandura, D., published in 2012, incorporated herein by reference; "Combination of immunohistochemistry and laser ablation ICP mass spectrometry for imaging of cancer biomarkers" by Seuma et al., Proteomics 8:3775-3784, published in 2008, incorporated herein by reference; "Imaging and spatial distribution of β-amyloid peptide and metal ions in Alzheimer’s plaques by laser ablation-inductively coupled plasma-mass spectrometry" by Hutchinson et al., Analytical Biochemistry 346.2:225-233, published in 2005, incorporated herein by reference; and "Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in elemental imaging of biological tissues and in proteomics" by Becker et al., Journal of Analytical Atomic Spectrometry 22.7:736-744, published in 2007, incorporated herein by reference.See, for example, 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 immunohistochemical detection of tumor markers in breast cancer tissue using laser ablation inductively coupled plasma mass spectrometry," Anal. Chem. 83:8177-8183 (2011). Plasma sources other than ICP are also within the scope of the intended use.

[0025] Due to constraints on the nanopositioning stage, access requirements for high-NA lenses, and / or the requirement to hold the sample within the IMC device as close as possible to the plasma, there are several different sampling orientations considered herein and described in the following table: [Table 1]

[0026] By rotating the plasma to an optimal orientation, some of the undesirable characteristics of previous configurations (resulting in transient broadening due to 90-degree rotation and long transfer lengths and secondary contamination due to gravity returning large particles to the sample surface) can be eliminated. As a point of note, vertical plasma is utilized in ICP-OES machines because it is easier to image along the axis of the torch due to the plasma's cylindrical symmetry being maintained by the orientation. In comparison, when the plasma exits the torch body, the symmetry is broken in the horizontal orientation due to the convective lift of the plasma.

[0027] In certain embodiments, the torch can be a sealed torch. A sealed torch is airtight and not in fluid communication with air outside the torch (e.g., other than the gas source supplying the gas flow). The sealed torch can be vacuum-sealed. In certain embodiments, the laser ablation chamber is airtight and not in fluid communication with air outside the instrument. In certain embodiments, the entire laser ablation ICP-MS system is sealed to prevent air from passing through the torch. The gas flow and aerodynamics within the sealed torch can be dominant over convective effects. The sealed torch can behave substantially the same in any orientation (e.g., having similar ionization effects for the same gas flow, sample material, and induction conditions) and can be oriented vertically (e.g., away from or towards the gravity vector).

[0028] Several challenges are presented in our applications for transporting the removed material by a gas flow. That is, when the plume is rotated strongly, a transient broadening occurs as would be the case for an excessive overall length of the gas channel between the sample and the plasma. As a result, for the optimal pixel acquisition speed, it is required to orient the plasma perpendicular to the sample substrate and bring them as close as possible.

[0029] The limitation in the choice of orientation is related to the specifications of the nanopositioning stage. Stages that possess the speed and dynamic positioning accuracy required for our applications typically have insufficient maximum force to support the sample and related hardware (springs, fasteners, etc.) against gravity. This means that the configurations involved in vertically oriented samples are more challenging from the perspective of XYZ stage engineering. However, such devices are relatively low-risk in terms of plasma engineering because it has already been experienced that a mass spectrometer can be constituted by a horizontal plasma.

[0030] As another option, the sample may be kept horizontal and the plasma directed either upward or downward. Each of these configurations has its own potential advantages and disadvantages.

[0031] For a plasma directed upward, one advantage is that heat from the plasma may rise away from the sample and into the interface region of the mass spectrometer. Since the interface is already liquid-cooled, this results in a small (presumably negligible) difference in the heat balance of the machine. The disadvantage of this approach is that any removed material that is not captured by the gas flow (i.e., large particles) may fall back onto the sample, causing crosstalk and contamination.

[0032] In the case of a plasma directed downward, the risk of sample contamination from larger removed particles is not high because gravity pulls the particles away from the sample. However, the heat rising from the plasma towards the sample can potentially pose a problem. This can be resolved by including thermal breakdown between the sample and plasma confinement, but this is done without potentially extending the gas path between the sample and the plasma, resulting in transient spreading. Convective forces can also cause additional transient spreading in such a plasma.

[0033] The ablation light can, in principle, be incident from either side of the sample. However, there are several trade-offs to consider.

[0034] If the ablation light is incident from the same side as the sample conveyance / plasma, it is necessary to compromise to achieve consistency between the optical access to the ablation spot and the sample conveyance access, and one or more of the following trade-offs may occur. - The focusing optical system significantly increases the minimum sample conveyance distance. - The focusing optical system is more difficult to fabricate and assemble. - The maximum achievable processing capacity is reduced. - Any optical inspection of the sample area is compromised to achieve consistency in terms of field of view, resolution, illumination uniformity, etc. - The instrument has a lower degree of modularity. On the other hand, if the ablation light is incident from the opposite side of the sample conveyance / plasma, one or more of the following trade-offs may occur. - The ablation light must pass through a sample substrate or another substrate such as silica that at least partially transmits ultraviolet light, which means that a quartz microscope slide must be used for UV laser ablation. - The optical inspection of the sample area may receive light from the plasma if the sample conveyance passes linearly. - The sample holder and stage assembly must provide an opening for the ablation / inspection optical system, making the assembly more expensive / more complex.

[0035] In certain embodiments, the laser ablation can be non-UV laser ablation (e.g., it can be in the visible or infrared spectrum such as a green laser ablation source, etc.). The non-UV laser can have characteristics such as frequency and / or output similar to those of the UV laser ablation source described herein. Thereby, the laser irradiation can pass through a slide glass (commonly used in microscopy). However, biological samples such as tissue sections, cell smear specimens, or cell cultures can be better removed in the ultraviolet spectrum rather than in the visible (e.g., green) or infrared spectrum.

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

[0037] In certain embodiments, ablation on the opposite side can be used to remove the layer under the sample to remove portions of the sample as described, for example, in U.S. Patent Publication No. 20160194590, which is incorporated herein by reference. In certain embodiments, a compound for transmitting kinetic energy to the sample (e.g., transition 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, an fs laser in the near-infrared range can be operated at the second harmonic to provide laser irradiation in the green range, or at the third harmonic to provide laser irradiation in the ultraviolet range. The lower wavelengths, such as green or ultraviolet, can enable high resolution (e.g., small spot size). When the laser irradiation is transmitted across the sample support for the laser irradiation to act on the sample, the sample support needs to be transmissive to the laser irradiation. Glass and silica transmit the green wavelength, while glass does not transmit UV but silica does. To enable high resolution while using a slide glass, an infrared fs laser can be operated at the second harmonic (e.g., about 50% conversion efficiency) to provide green laser irradiation. Notably, commercially available objective lenses often have the best correction in the green range. The resolution achieved with a green or ultraviolet fs laser can be a spot size of 1 μm or less, 800 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, or 100 nm.

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

[0040] Figure 2 shows a plurality of configurations where the plasma (e.g., ICP) is horizontal. Laser irradiation is indicated by the shaded triangles. The sample is shown mounted on a wide sample stage.

[0041] Figure 2A shows a horizontal stage for injection into a horizontal plasma as previously done. Figure 2A shows a horizontal sample sampled from above, where the sample is held on a horizontally oriented slide (also described as a cover glass or substrate), removed either from above or through the substrate from below, and the plume is directed upward. The plume is captured in the gas flow, rotated orthogonally, and enters the horizontal plasma. Thereby, redeposition due to gravity can occur.

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

[0043] Figure 2C shows a vertical sample. The light for ablation can be directed either through the substrate (from the back) or from the sample side. The plume is directed horizontally, captured, does not require rotation, and is directly transported to the plasma by the gas flow.

[0044] Figure 3 shows a plurality of configurations similar to Figure 2, where the plasma (e.g., ICP) is vertical.

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

[0046] Figure 3B shows a horizontal sample being sampled from below, where the gas flow towards the plasma is directed downward, and thus the plasma is positioned below the sample. The ablation light is irradiated from either above or below, and the removed plume exits the sample being ejected downward, is captured by the gas flow, and is directly transported to the plasma.

[0047] Figure 3C shows a vertical sample being sampled from the side (e.g., passing through the sample). The plume exits the substrate surface, is rotated 90 degrees, and before entering the plasma, is captured by the gas flow and can be directed either upward or downward.

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

[0049] Aspects include methods and systems for laser ablation mass spectrometry analysis, where pulses of a laser beam generate a plume of a sample for each of the pulses, each plume is distinctly captured for each of the pulses, each distinctly captured plume is conveyed to an ionization system, each distinctly captured and conveyed plume is ionized in the ionization system, and ions for mass spectrometry and an apparatus for performing the method are generated for a sample. In various embodiments, the apparatus has 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 may have an inlet positioned within the laser ablation system such that the inlet is configurable to capture the ablated plume when it is generated. The gas inlet may be connectable to the inlet of the transfer conduit through which gas passes therebetween to convey the captured and ablated plume into the ionization system. When the ionization system is an ICP, the transfer conduit may be referred to as an injector if the output of the conduit is directly within the plasma of the ICP. The components of the laser ablation system, ionization system, and mass spectrometer are described in more detail individually below. As noted above, the focus of the present invention is an improvement in 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 and conveys 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 by boring a suitable material to create a lumen (e.g., a lumen with a circular, rectangular, or other cross-section) for conveying the plume, for example. The transfer conduit may have an inner diameter in the range of 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 the ends. The transfer conduit may have a length in the range of 1 centimeter to 100 centimeters. In some embodiments, the length is only 10 centimeters (e.g., 1 to 10 centimeters), only 5 centimeters (e.g., 1 to 5 centimeters), or only 3 cm (e.g., 0.1 to 3 centimeters). In some embodiments, the lumen of the transfer conduit is linear along or substantially along the entire distance from the ablation system to the ionization system. In some embodiments, the lumen of the transfer conduit is not linear with respect to the entire distance and has different orientations. For example, the transfer conduit can rotate gradually by 90 degrees. With this configuration, the plume generated by ablation of the sample in the laser ablation system has its axis in the straight-up direction at the inlet of the transfer conduit, while moving initially within a vertical plane and being able to move horizontally as the plume approaches the ionization system (e.g., an ICP torch that is generally horizontally oriented for using convective cooling). In some embodiments, the transfer conduit is linear at a distance of at least 0.1 centimeter, at least 0.5 centimeter, or at least 1 centimeter from the opening of the inlet where the plume enters or is formed. In some embodiments, the transfer conduit is adapted to minimize the time required to convey the material from the laser ablation system to the ionization system.

[0051] The injector of the device can include the transfer conduit described herein.

[0052] Sample cone inlet The transfer conduit comprises an inlet in the laser ablation system that receives sample material removed from a sample within the laser ablation system and conveys it to an ionization system. In some instances, the inlet of the laser ablation system is all sources of gas flow along the transfer conduit to the ionization system. In some instances, the inlet of the laser ablation system that receives material from the laser ablation system is an opening within the wall of a conduit through which a second “transfer” gas flows from a separate carrier flow inlet (as disclosed, for example, in WO2014146724 and WO2014147260). In this instance, the carrier gas forms a substantial proportion and, in many instances, the majority of the gas flow to the ionization system. The component that comprises the inlet of the carrier flow, the inlet of the laser ablation system, and is the beginning of the transfer conduit that conveys the removed sample material towards the ionization system may also be referred to as a flow cell (as described in WO2014146724 and WO2014147260).

[0053] The transport stream realizes at least three tasks, namely, flowing a plume into the transfer conduit in the direction of the ionization system to prevent the plume material from contacting the side wall of the transfer conduit, forming a "protection region" above the sample surface so that the ablation plume is reliably performed under the control of the atmosphere, and further increasing the flow velocity in the transfer conduit. In some embodiments, the viscosity of the capture gas is lower than the viscosity of the primary transport gas. This helps to keep the plume of sample material in the capture gas at the center of the transfer conduit and minimize the diffusion of the plume of sample material downstream of the laser ablation system (since at the center of the flow, the transport velocity is more constant and nearly horizontal). The gas can be, for example, without limitation, argon, xenon, helium, nitrogen, or a mixture thereof. In some embodiments, the transport gas is argon. Argon is particularly suitable for stopping the diffusion of the plume before reaching the wall of the transfer conduit (furthermore, it also helps to improve the sensitivity of the instrument of the device when the ionization system is an argon gas-based ICP). The capture gas is preferably helium. However, the capture gas can be replaced or included with other gases such as hydrogen, nitrogen, or water vapor. The kinematic viscosity (kinematic viscosity coefficient / density) of argon is about 1.3E -5 m 2 / s at 25°C, and for helium, it is about 1.2E -4 m 2 / s. Therefore, the difference in the kinematic viscosity values of argon and helium can be at least 5 times or at least 10 times. In some embodiments, the capture gas is helium and the transport gas is helium.

[0054] Using a sample cone can minimize the distance between the target and the conduit containing the gas transport stream. Since the distance that the capture gas flows at the tip of the cone is reduced, this use improves the capture of the sample material with less turbulent flow, and thus reduces the diffusion of the plume of the removed sample material. Therefore, the inlet of the transfer conduit is the opening at the tip of the sample cone. The cone protrudes 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 can be present within a single asymmetric sample cone used in the present invention. For example, the cone can be asymmetric and have its tip truncated, can be formed from two halves of different elliptical cones, the cone can be asymmetric and have its tip truncated, and can include one of a plurality of orifices, etc.

[0057] Accordingly, the sample cone is 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 positioned operably proximate to the sample, for example, by manipulating the sample within the laser ablation system on a tray for transporting the movable sample, as described in more detail below. As described above, the plume of removed sample material enters the transfer conduit through the opening at the narrow end of the sample cone. In some embodiments, the diameter of the opening is a) adjustable, b) sized to prevent perturbation to 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 from about 100 μm to 1 mm. For example, the diameter of the opening is from about 200 μm to 900 μm such as from 300 μm to 800 μm. In some embodiments, the diameter of the opening is from about 500 μm to 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 In a pipe with a smaller inner diameter, if the gas has the same flow rate, it moves at a higher speed. Therefore, by using a pipe with a smaller inner diameter, the plume of the removed sample material carried in the gas flow 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 transfer conduit). One of the main factors determining how quickly an individual plume can be analyzed is the amount by which the plume spreads during the time from its generation by ablation until the time when its constituent ions are detected as components of the mass spectrometer of the apparatus (transit time of the detector). Therefore, by using a narrow transfer conduit, the time between ablation and detection is reduced, thereby reducing the diffusion because the time during which it can occur is shortened, and ultimately meaning that the transit time of each ablation plume at the detector is reduced. A shorter transit time means an increase in the number of plumes that can be generated and analyzed per unit time, and for this reason, the image is produced with higher quality and / or at higher speed.

[0059] The taper can include a gradual change in the inner diameter of the transfer conduit along that portion of the length of the transfer conduit (i.e., the inner diameter of the pipe with the cross-section obtained thereby decreases along the portion from the end of the portion towards the inlet (at the laser ablation system end) to the outlet (at the end of the ionization system)). As shown in FIGS. 3B and 7C, the improvement of the tapering of the transfer conduit is applicable to all embodiments of the apparatus described herein, regardless of whether it includes a direct injector inlet, a sample cone, or any other structure at the end of the inlet of the ionization system of the transfer conduit. If the conduit before tapering has a large volume, it becomes easier to limit the material generated by ablation. When the removed particles scatter from the removed spot, they move at high speed. Due to friction in the gas, these particles decelerate, but the plume can still diffuse in sub-millimeter to millimeter units. Keeping a sufficient distance from the wall helps to confine the plume near the center of the flow.

[0060] Since the wide inner diameter portion is simply short (on the order of 1 - 2 mm), if the plume remains in the longer portion of the transfer conduit with a smaller inner diameter for a longer time, this portion does not significantly contribute to the overall transit time. For this reason, the portion with a larger inner diameter is used to capture the ablation product and keep it near the central streamline where the gas flow velocity is more uniform, and the conduit with a smaller inner diameter is used to quickly transport these particles to the ionization system. The plume expands by a specific amount, which is at least partially determined by the particle size and the gas selected. The initial portion of the injector tubing can be sized such that most of the plume falls near the central streamline so that the plume is not spread by the influence of the development of the parabolic flow profile. The injector can comprise a taper, for example, to narrow the diameter of the injector tubing after the initial portion to improve the transport speed.

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

[0062] The taper between the large inner diameter portion and the small inner diameter region can be made sufficiently smooth to avoid the generation of turbulent flow. For example, the taper can be at an angle of 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, and further up to 60 degrees. In some embodiments, the taper is at an angle of 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 at an angle of 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, for example, at least 10 mm, at least 20 mm, at least 30 mm, at least 40 mm, at least 50 mm, or at least 100 mm. In some embodiments, the length of the taper is less than 10 mm, for example, 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 can be x millimeters (mm) at the input end of the conduit, but can be tapered to 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 measurement of the longest cross-section 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 diagonal. In some embodiments, the inner diameter of the conduit after the tapered portion is narrower than 2 mm, for example, 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 tapered portion is narrower than 400 μm, narrower than 300 μm, narrower than 200 μm, or narrower than 100 μm.

[0065] The diameter of the narrow inner diameter portion is limited by a diameter corresponding to the generation of turbulent flow. The Reynolds number is calculable for circular pipes and known flows. Generally, a Reynolds number above 4000 indicates turbulent flow and should thus be avoided. A Reynolds number above 2000 indicates transitional flow (between non-turbulent and turbulent flow) and can thus preferably be avoided. For a given mass flow of gas, the Reynolds number is inversely proportional to the diameter of the conduit. Accordingly, in some embodiments, the inner diameter of the narrow inner diameter portion of the transfer conduit is narrower than 2 mm, for example, narrower than 1.5 mm, narrower than 1.25 mm, narrower than 1 mm, but the flow of helium at 4 liters per minute within the conduit has a diameter larger than the diameter having a Reynolds number above 4000.

[0066] If there are uneven or smooth angled edges at the transition portion between the portion of constant diameter and the taper of the transfer conduit, turbulent flow can occur in the gas flow. Accordingly, in some embodiments, the transition portions to and from the taper should have smooth edges adapted to suppress the generation of turbulent flow. For example, the edges can be rounded or chamfered.

[0067] An apparatus comprising a tapered conduit may also comprise a sample cone (optionally, asymmetric). Those skilled in the art will understand that a tapered conduit can be used in any of the apparatuses described herein that use an alternative arrangement of transfer conduits, as shown, for example, in FIGS. 2 - 10 and described in detail in the following sections of this specification.

[0068] Sacrificial flow The risk of turbulent flow occurring within the conduit increases with increasing flow velocity. This is particularly true when the transfer conduit has a small inner diameter (e.g., 1 mm or less). However, the inventors have found that if a light gas such as helium or hydrogen is used in place of argon, which has conventionally been used as the carrier gas flow, it is possible to achieve high-speed transport (300 m / s or more) within a transfer conduit with a small inner diameter. In certain embodiments, a gas mixture mainly containing helium or hydrogen is used.

[0069] As long as the plume of the removed sample material is passed through the ionization system by high-speed conveyance without ionization occurring at an acceptable level, no problem occurs. The level of ionization can decrease because the plasma temperature drops at the end of the torch as the flow rate of the cooling gas increases. If the plume of the sample material is not ionized at a suitable level, the components (including any labeled atoms / element tags) cannot be detected by the mass spectrometer, and thus information from the removed sample material is lost. For example, the sample can pass through the plasma very quickly at the end of the torch in the ICP ionization system, so the plasma ions have insufficient time to act on the sample material for ionizing it. The inventors have found that this problem caused by high-speed conveyance at a high flow rate in a transfer conduit with a narrow inner diameter can be solved by introducing a flow sacrifice system at the outflow of the transfer conduit. The flow sacrifice system receives the gas flow from the transfer conduit and is adapted to pass only the forward-flowing portion (including the central portion of the flow containing any plume of the removed sample material) leading to an injector that communicates with the ionization system. To facilitate the diffusion of gas from the transfer conduit within the flow sacrifice system, the outflow of the transfer conduit can be flared.

[0070] The flow sacrifice system is positioned in the vicinity of the ionization system such that the length of the pipe (e.g., injector) communicating from the flow sacrifice system to the ionization system is short (e.g., a length of 1 cm; compared to the length of the transfer conduit on the order of several tens of cm such as 50 cm). For this reason, if the gas velocity in the pipe communicating from the flow sacrifice system to the ionization system is low, since the relatively slow portion of the entire conveyance system is very short, there is no significant impact on the total conveyance time.

[0071] Therefore, the present invention provides (i) a laser ablation system adapted to generate a plume of sample material from a sample, and (ii) an ionization system that receives the material excluded from the sample by the laser ablation system and is adapted to ionize the material to form elemental ions, (iii) providing an apparatus comprising a mass spectrometer that receives elemental ions from the ionization system and analyzes the elemental ions; The laser ablation system and the ionization system are coupled together by a transfer conduit and a flow sacrifice system. The transfer conduit is adapted to convey a gas stream containing a plume of removed sample material from an inlet within the laser ablation system to an outlet within the flow sacrifice system. The flow sacrifice system (a) an outlet of the transfer conduit, and (b) an inlet of the ionization system positioned to receive sample material from the transfer conduit outlet and introduce the sample material into the ionization system, and (c) a sacrificial flow outlet, and comprises a chamber. The flow sacrifice system is adapted to reduce the gas stream entering the ionization system through the inlet of the ionization system compared to the gas stream entering the flow sacrifice system through the transfer conduit by directing a portion of the gas stream entering the flow sacrifice system from the sacrificial flow outlet. The outlet of the transfer conduit within the flow sacrifice system is optionally flared.

[0072] In some embodiments, the inlet of the ionization system is positioned coaxially at the outlet of the transfer conduit to minimize the transfer of material from the transfer conduit through the flow sacrifice system to the inlet of the ionization system and thus to the injector of the ionization system (since the plume of sample material conveyed along the conduit is entrained within the center of the conveying 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, for example. 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, for example. In some embodiments, the inner diameter of the injector of the ionization system (or the 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, for example. 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, for example.

[0073] In most installations, it is not preferred or, in some cases, not possible to significantly increase the diameter of the tubing (e.g., injector) passing from the flow sacrifice system to the ionization system as a means of reducing the gas velocity in terms of volumetric flow rate. For example, when the ionization system is an ICP, the conduit from the flow sacrifice system forms an injector tube at the center of the ICP torch. When using an injector with a wider inner diameter, the signal quality deteriorates because the plume of the removed sample material cannot be injected very precisely into the center of the plasma (the most efficiently ionizing part of the plasma). Injectors with an inner diameter of 1 mm or even narrower (e.g., an inner diameter of 800 μm or less such as 600 μm or less, 500 μm or less, or 400 μm or less) are strongly preferred. Other ionization techniques rely on materials 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 if the inner diameter of the conduit is wider, it means that much of the sample material passing through the conduit is outside the region where the energy density is sufficient to ionize the sample material. For this reason, tubing with a narrow diameter from the flow sacrifice system into the ionization system is also used in devices equipped with non-ICP ionization systems. As described above, if the plume of the sample material is not ionized at a suitable level, the components (including any labeled atoms / element tags) cannot be detected by the mass spectrometer, and thus information from the removed sample material is lost.

[0074] If there are rough or smooth angled edges at the transition between the portion of the transfer conduit with a constant diameter and the flare at the outflow, turbulence can occur in the gas flow. Thus, in some embodiments, the transition to the flare expansion should have a smooth edge adapted to suppress the generation of turbulence. For example, the edge can be rounded.

[0075] Using pump conveyance can help ensure that the split ratio between the sacrificial flow and the flow passing into the ionization system inlet is as desired. Thus, in some embodiments, the flow rate sacrificial system includes a pump attached to the sacrificial flow outlet. A control restrictor can be added to the pump to control the sacrificial flow. Thus, in some embodiments, the pump of the flow rate sacrificial system further includes a restrictor adapted to control the gas flow through the sacrificial flow outlet. In some embodiments, the flow rate sacrificial system includes a mass flow control device adapted to control the restrictor.

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

[0077] Similarly, when the transport flow rate is larger, only the central portion of the flow that is sent along the transfer conduit can become the portion of the injector flow that enters the plasma of the ICP torch. Typically, helium gas is used as the transport flow, as its properties are suitable for high-speed transport of plume material over long conduits as described above (i.e., it is less likely to induce turbulent flow compared to argon for the same flow velocity). Even the opportunity to incorporate a gas purification system that recycles helium from the sacrificial flow and the near-axis flow of helium that continues through the flow rate sacrifice system into the ionization system is lost.

[0078] Thus, in some embodiments, the flow rate sacrifice system is adapted to reduce the gas flow passing through the inlet of the ionization system (e.g., the injector of the 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 comprises a second inlet through which gas can be flowed to supplement the flow rate within the injector. In some embodiments, the second inlet comprises concentric tubing around an injector attached to the inlet of the ionization system that introduces makeup gas as a sheath flow around the sample-containing gas flow from the flow rate sacrifice system. The inlet of this makeup flow is different from the argon gas flow similarly provided in the intermediate and outer concentric tubing that supports the plasma. This injector may also be referred to as a double concentric injector.

[0079] An apparatus comprising a flow rate sacrifice system may also comprise a sample cone (optionally, an asymmetric shape) or a tapered conduit as described above. In some embodiments, the apparatus comprises a flow rate sacrifice system, a sample cone (optionally, an asymmetric shape), and a tapered conduit as described above. One of ordinary skill in the art will understand that the flow rate sacrifice system can be used in any of the apparatuses described herein that use an alternative arrangement of transfer conduits.

[0080] Laser ablation system A laser ablation system, also referred to as an "ablation cell" or "laser ablation source", houses a sample during ablation. Typically, the ablation cell includes a laser transparent window for directing laser energy onto 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 x-y or x-y-z dimensions. In the drawings and examples described herein, the laser ablation system may be shown as an open apparatus. However, such a configuration is for illustrative purposes only, and it will be understood that there is some form of suitable enclosure to prevent contamination or ingress from the ambient environment. For example, a chamber configured with a gas inlet and / or optical ports may be arranged around the laser ablation system to provide a closed environment suitable for capturing and transporting the plume removed for mass spectrometry. The gas inlet and optical ports are positioned such that the orientation of the laser beam, the sample, the plume expansion, and the transfer conduit are suitable for the methods and apparatus disclosed herein. It will be understood that the ablation cell is generally gas-tight (except for designed outlets and ports). Even if the ablation chamber contains air prior to operation, the ablation chamber can be sufficiently enclosed from the environment such that a gas flow (e.g., a capture gas passing through the sample chamber and / or a carrier gas to the injector tubing of the ablation cell) can be sufficient to reduce contamination by air during sample movement. However, the initial presence of air can provide a level of humidity that affects sensitivity and / or signal drift. The laser ablation system for the intended application may have a gas flow as shown in one or more of FIGS. 2, 3, or 4.

[0081] The lasers used for laser ablation according to the present invention are generally classified into three types: femtosecond pulsed lasers, deep ultraviolet pulsed lasers, and pulsed lasers, which have wavelengths selected for high absorption in the removed material ( "wavelength-selective lasers"). Deep ultraviolet and wavelength-specific lasers can operate with nanosecond or picosecond pulses. Lasers of each classification have their disadvantages and advantages and can be selected based on specific applications. In some embodiments, the laser is a femtosecond pulsed laser configured to operate at a pulse rate of 10 to 10,000 Hz. Femtosecond lasers are well known (see, for example, "Rapid bulk analysis using femtosecond laser ablation inductively coupled plasma time-of-flight mass spectrometry" by Jhanis et al., J. Anal. At. Spectrom. 27: 1405-1412, published in 2012).

[0082] Femtosecond lasers enable laser ablation of almost all materials with only the preconditions for laser ablation that provide sufficient output density. This can be achieved, for example, with a relatively low pulse energy when the beam is strongly focused to a diameter of 1 micrometer and has a short duration (focused within a time). Deep ultraviolet lasers can also remove a large class of materials, which is generally because most of the materials used absorb deep ultraviolet photons. Wavelength-selective laser ablation can utilize a laser with a specific laser wavelength that targets absorption within the substrate material. The advantage of wavelength-specific lasers can be the cost and simple structure of the laser and optical system, although the spectrum of the substrate material is more restricted. 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 the laser irradiation is absorbed only when the threshold output density is reached. For this reason, focused femtosecond laser irradiation can pass through thicker portions of the material without being absorbed or causing any damage, and can further remove the same material on exactly the surface where the focus occurs. Thereafter, the focus can gradually move inside the material when the sample layer is removed. Nanosecond laser pulses can be partially absorbed by the substrate, but because the energy density at the focus is the maximum (as long as it is sufficient for ablation), they can still function for ablation.

[0084] The spatial resolution of the signal thus generated depends on two main factors: (i) the spot size of the laser when the signal is integrated over the entire area where it is removed, and (ii) as described above, the rate at which the plume can be analyzed relative to the rate at which the plume is generated to avoid overlap from consecutive plumes. The distance referred to as the spot size corresponds to the longest internal dimension of the beam. For example, for a circular beam, it is a beam with a diameter of 2 μm, and for a rectangular beam, it corresponds to the length of the diagonal between opposite corners. The laser pulse is homogenized (if necessary) using a beam homogenizer in a shape using an aperture to create the desired spot size, and can be focused, for example, using an objective lens. 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 ablation areas of equivalent size in other shapes) in the range of 0.10 - 3 μm (e.g., about 0.3 μm), 1 - 5 μm (e.g., about 3 μm), 1 - 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 laser pulses that are sufficiently focused to remove the sample area, for example, on the order of about 1 μm in the range of 100 nm - 1 μm.

[0085] To analyze individual cells, the laser in a laser ablation system has a spot size on the order of these cells. This size depends on the particular cells in the sample, but generally, the laser spot has a diameter of less than 4 μm, for example, in the range of 0.1 - 4 μm, 0.25 - 3 μm, or 0.4 - 2 μm. Thus, the laser spot can have a diameter of about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.5 μm or less than 0.5 μm, such as approximately 400 nm or less, approximately 300 nm or less, approximately 200 nm or less, approximately 100 nm or less than 100 nm. To analyze cells with intracellular resolution, the present invention uses a laser spot size on the order of these cells, and more specifically, a laser spot size capable of removing material with intracellular resolution. Single cell analysis can be performed, for example, by spreading cells on a slide with a spacing between each, using a spot size larger than the cell size. Here, a larger spot size can be used to achieve single cell characterization because the additional removed area around the target cell does not contain additional cells. The specific spot size to be used can thus be appropriately selected depending on the size of the cells to be analyzed. In a biological sample, if intracellular resolution imaging is desired, it is rare for all cells to be the same size, and if a constant spot size is maintained over the ablation procedure, the size of the ablation spot should be smaller than the smallest cell. The spot size can be made small by using the reduction of a wide laser beam 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 focus of the beam), but the laser focus can vary by ±20% or more due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and the variation of the total laser energy with respect to the ablation threshold energy. For example, using a 25 μm diameter laser beam and reducing it 25 times on a tissue sample gives a spot size with a 1 μm diameter.

[0086] At this small scale ablation, a very small amount of plume material is produced, and then the size of the plume is reliably kept small. A smaller plume is more likely to remain in the middle of the capture flow without contacting the walls of the ablation cell or the transfer conduit. Also, the fact that the ablation is at the 1 micrometer scale means that the distance between the removed surface and the region where the plume expansion decelerates and the ambient gas becomes dominant is very short. This distance can range from a few micrometers to several hundred micrometers. In some versions of the present invention, when the plume stops expanding, the capture flow is present. Thus, some of the accompanying drawings show, by way of illustration and not limitation, the distance between the removed surface and the region with the capture flow indicated at about 100 micrometers.

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

[0088] In some embodiments, the laser is installed outside the ablation chamber, and the laser beam (laser energy) enters the ablation chamber, for example, through an optical window. As used herein, the laser beam may be described as emitted from a surface (e.g., a laser lens or mirror), and that surface may be oriented to direct the beam to a particular location or location pattern. To facilitate the description of the present invention, the directed beam may be considered to have a particular orientation, and the orientation of the beam may refer to a virtual line that is aligned with the beam and extends beyond the actual beam (e.g., when the beam strikes a non-transmissive surface). As will be apparent from the context, when referring to the orientation or position of the laser beam, it may refer to the orientation or position that the beam of a battery-free laser system can generate if a laser is used.

[0089] For rapid analysis of tissue samples, for example, a high frequency of ablation above 20 Hz (i.e., more than 20 ablations per second, giving more than 20 plumes per second) is required. In some embodiments, the frequency of laser ablation is at least 40 Hz, such as at least 50 Hz, or at least 100 Hz. In some embodiments, the frequency of laser ablation is within the range of 40 - 2000 Hz, within the range of 40 - 1500 Hz, within the range of 40 - 500 Hz, within the range of 40 - 200 Hz, within the range of 40 - 150 Hz, or within the range of 75 - 150 Hz. An ablation frequency above 40 Hz enables imaging of typical tissue samples to be achieved in an appropriate time. The laser pulse can be directed at a spot on the sample (assuming complete ablation of the material at the spot), and the frequency at which individual resolution can still be achieved determines how quickly the pixels of the image can be acquired. Thus, if the duration of the laser pulse required to remove material at a point means that it can only be directed at the sample at less than 5 pulses per second, the time required to investigate a 1 mm × 1 mm area by ablation with a 1 μm spot size would exceed 2 days. This could be approximately 6 - 7 hours if the rate is 40 Hz, and the analysis time is further reduced for further increases in the frequency of the pulses. At these frequencies, if it is desired to resolve each removed plume individually, the instrument measurement must be able to analyze the removed material quickly enough to avoid significant signal overlap between successive ablations. Preferably, the overlap between signals due to 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 section on ablation chamber below), the transit time of the plume of sample material to and through the ionization system (as described above for optimization of transport to the ionization system), and the time required for analysis of the ionized material. Each laser pulse can correlate with the pixels of the image of the sample, which is enhanced subsequently as described in more detail below.

[0090] Ablation chamber An ablation chamber having a short wash time (e.g., 100 ms or less) (which may be referred to herein as a sample chamber) is advantageous for use in the apparatus and method of the present invention. Cells having a long wash time either limit the rate at which an image can be generated or cause overlap between signals resulting from consecutive sample spots (e.g., Kindness et al. (2003) Clin Chem 49:1916 - 23 having a signal duration exceeding 10 seconds). Thus, the wash time of the plume of sample material from the laser ablation cell is a major limiting factor for achieving high resolution without increasing the total scan time. Ablation chambers with a wash time ≦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 wash time of 100 ms or less. The ablation chamber has a wash time of 30 ms or less, thereby enabling high ablation frequencies (e.g., >20 Hz) and thus rapid analysis, as disclosed in the reference of Wang et al. (2013) Anal.Chem.85:10107 - 16 (see also reference WO2014 / 146724). 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 closest to the target (the sample capture cell described herein is an example of an improvement in the inlet of a transfer conduit that can be combined with a taper and the improvement in the flow sacrifice of the transfer conduit described above), the sample capture cell being a capture cavity having an opening formed on the surface of the capture cell, configured to receive the target material released or generated from the laser ablation site through the opening, a capture cavity, and at least a portion of the target material received in the capture cavity is exposed in the capture cavity so as to be conveyable as a sample to the outlet, and a guide wall configured to direct the flow of the carrier gas (also referred to herein as the capture gas) in the capture cavity from the inlet to the outlet. The volume of the capture cavity in the ablation chamber of reference WO2014 / 127034 is less than 1 cm3 and may be 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 is, for example, an asymmetric sample cone, which also helps to reduce the cleaning time of the ablation chamber and is an alternative to the capture cells described herein.

[0091] An existing LA-ICP-MS system including a laser ablation chamber is shown in FIG. 4. The ablation chamber may comprise a movable sample stage for positioning the sample. The gas source may provide a capture gas that flows through the ablation chamber and conveys the ablation plume to an injector pipe (also referred to herein as an injector or transfer conduit) that connects to the ICP torch. The injector pipe may have a flexible pipe between the ablation chamber and the ICP torch, or may be rigid and / or linear. In certain embodiments, the injector may be orthogonal to the sample and collinear with the gas flow shown in FIG. 2 or FIG. 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 pipe may receive makeup gas downstream of the laser ablation plume introduced into the injector pipe. One or more gas flows may include hydrogen. For example, the gas flow may include water or alcohol vapor as further described herein. Alternatively or in addition, the gas flow may be from a premixed compressed gas source that includes hydrogen (such as hydrogen gas, ammonia or methane).

[0092] In certain embodiments, the laser ablation chamber may contain a capture gas (which enters the chamber and conveys the ablation plume to the injector tubing) and, optionally, may further contain a carrier gas (i.e., which enters the injector upstream of the ablation plume). The injector may further contain a makeup gas (i.e., which supplements gas within the injector downstream where the ablation plume enters the injector). The ICP torch may contain an internal gas (i.e., an auxiliary gas flowing through the internal tubing of the ICP torch) and an external gas (i.e., a plasma gas flowing through the external tubing 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 within a plasma. The use of ICP is suitable for IMS and IMC analysis. ICP is a plasma source where energy is supplied by a current generated by electromagnetic induction. Typically, the plasma source is an argon gas system. For example, the ionization system may comprise an ICP torch. The use of ICP in IMC within an ionization system has been reported, for example, by Giesen et al. (2014) Nature Methods. 11:417 - 422 and Wang et al. (2013) Anal. Chem. 85:10107 - 16.

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

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

[0096] The time required for the analysis of the ionized material depends on the type of mass analyzer / mass spectrometer used for the detection of ions. For example, an instrument using a Faraday cup may be very slow for the analysis of rapid signals, but not all analyses require rapid signal analysis, and thus one skilled in the art can appropriately select a mass spectrometer or mass analyzer. Overall, the desired analysis speed (and thus the frequency at which the ablation plume can be interrogated) and the degree of multiplexing (number of atoms monitored simultaneously / quasi-simultaneously) dictate the type of mass analyzer to be used (or conversely, the speed and multiplexing achievable are determined by the mass analyzer selected).

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

[0098] A TOF detector can simultaneously register multiple masses within a single sample. Since a TOF mass spectrometer usually has to compromise to accommodate the TOF accelerator and the effects of space charge in the flight tube, it is avoided for atomic analysis. However, the effectiveness of this technique can be improved by using it, but only for some range of detection purposes. For example, in mass spectrometry and imaging mass spectrometry, ions from labeled atoms used to label target molecules in a biological sample are detected, so that the range can be selected only to the extent that other atoms (e.g., atoms with an atomic mass less than 80) can be removed. This results in a decrease in the density of the ion beam concentrated at masses in the range of, for example, 80 to 210 Daltons, which makes TOF detection easier and utilizes the high spectral scanning speed of TOF. Therefore, rapid analysis can be achieved by combining TOF detection with the selection of rare labeled atoms in the sample and aiming for masses exceeding those found in unlabeled samples, for example, by using high mass transition elements. Further details regarding mass spectrometry 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, where descriptions regarding imaging mass spectrometry can be found.

[0099] Additional variations of the present invention to which the apparatus used and the improvements to the transfer conduit described above are applicable The device of the present invention can be on a transparent substrate and can be used for the analysis or imaging of a biological sample. In an imaging embodiment, generally, the laser can be operated in continuous pulses, or in a burst of pulses directed at different positions of the sample, referred to as a "target spot" or "ablation position or area". The pulses can be directed at the spots in a set pattern such as a raster for two-dimensional imaging. Alternatively, a plurality of individual spots at different positions (e.g., corresponding to individual cells) can be ablated. In some embodiments, the laser emits a burst of pulses that generate a plume from the same pixel (i.e., the same position on the target). The ablation plumes generated by the individual pulses within the burst dissolve within one plume and are expected to travel through the instrument differently from plumes created from other pixels. To distinguish individual pixels, the duration between bursts (a pixel interrogation that can be one or 100 pulses) is maintained beyond a specific limit determined by the time spread of the ion signal (at the detector) from the individual pixels.

[0100] In accordance with the description herein, each separate sample plume can be clearly analyzed by a mass spectrometer. In one aspect, the device is configured such that the diffusion of the plume in the ablation cell (ablation system) and the transfer conduit is less than the diffusion that occurs in the ionization system and the mass spectrometer. In one aspect, the plume can be clearly analyzed by transporting each ablated plume to the ionization system within a period during the cumulative transit time to the plume ionization system and detecting the ions by the mass spectrometer. This can be achieved by capturing each sample plume by passing it through a gas flow and a transport configuration such that the ratio between the spread of the plume during the transport period (i.e., the transport of the ablation plume from the ablation site to the plasma) and the spread during the ion transit period (i.e., the transport of the ions from the plasma to the mass spectrometer) is 1 or less.

[0101] Generally, for an ionization system (e.g., ICP) for the purpose of analytical detection, the limit of the sample particle size that can be effectively evaporated and ionized is on the order of about 10 μm or less. Particles generated by laser ablation on the scale of 1 micrometer are less than 1 micrometer and are suitable for an ICP ion source. For separate particle analysis (such as performed using measurements by the CyTOF® instrument manufactured by Fluidigm Canada), the typical rate at which these particles can be ionized and analytically detected can be a function of the cumulative spread or diffusion of the transient time of the sample in the plasma while the particles are being evaporated and ionized, and the spread or diffusion of the transient time of the ions between the ICP and their detection by the mass analyzer. Generally, the cumulative spread or diffusion can be on the order of a duration of about 200 μs. As a result, for spatially separated particles of 10 μm or less, analyzing each separate particle can be achieved by transporting each particle to an ionization system (e.g., ICP) in a period on the order of 200 μs. In some embodiments, the particles are transported to the ionization system (e.g., ICP) in less than 200 μs, or less than 150 μs. Thus, in a sample introduction system where imaging of a biological sample can be performed by laser ablation, the laser system can be configured to operate with a laser pulse that is sufficiently focused to remove a sample region, for example, on the order of about 1 μm, such as by femtosecond pulse laser irradiation. With this configuration, the removed plume formed by each laser pulse can typically include sample microparticles having dimensions of about 1 μm or less. Under the specific conditions described herein, these microparticles can be captured and transported to meet the required transport period, and subsequently, each separate plume can be effectively evaporated and ionized by the ionization system.

[0102] Furthermore, during operation of the laser in continuous pulses, such as when rasterizing across a sample surface for two-dimensional imaging, the discrimination of each plume and the spatial isolation between each successive plume can be maintained between the formed plume region and the point of evaporation / ionization within the ionization system ion source. For example, when a plume is transported through a conduit, the particles within the plume can diffuse / expand radially outwards before the plume enters the ionization system (e.g., the plasma of an ICP). The diffusion of the particles generated within the plume can depend on its diffusion coefficient, the velocity profile of the carrier flow, and the distribution of the particle density as it forms and grows during its transit to the ionization system. For example, if the spot size of a femtosecond laser ablation is 1 μm, a plume with an initial cross-sectional diameter of approximately 100 μm or less can be generated before further diffusion during its transit. The degree of plume diffusion can also be a function of the size of the particles removed, with larger particles having a lower diffusion spread but a tendency for higher momentum that can cause potential losses by contacting the inner wall of the transfer conduit / injector tubing. For this reason, it is desirable to transport the plume to the ionization system within a sufficient time for evaporation / ionization before minimizing plume diffusion and / or before the degree of diffusion has any valuable effect.

[0103] Accordingly, in various embodiments, removing a 1 μm sample spot and using a laser to efficiently transport the plume such that diffusion is maintained within the inner diameter of the transfer conduit / injector tubing can be achieved as described herein and by typical arrangements 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 called the "sampling volume". It is desirable to configure the system to minimize the sampling volume and increase the rate at which the gas flow conveys the plume away from the sampling volume. Combining a small sampling volume with a high gas flow rate reduces the time dispersion of plume transport to the transfer conduit / injector. The sampling volume can be described by the plume envelope at the instant when the rate of plume expansion in any dimension significantly decreases (by about 10 times) below the speed of sound in the ambient gas medium. A typical sampling volume can range from 10 - 6 mm3 to 10 mm3 without limitation. The sampling volume is often in the range of 0.001 mm3 to 1 mm3. The capture flow, if present, flows into at least a portion of the sampling volume and conveys at least a portion of the plume to the transfer conduit / injector, and as a result, the capture flow can be conveyed by the carrier flow to an ionization system (e.g., ICP). It is desirable for the velocity of the capture flow to be substantial (e.g., >1 m / s, >10 m / s, >100 m / s, or >500 m / s) when it enters the sampling volume. In some embodiments, the velocity of the capture flow can be evaluated by measuring the velocity of the capture flow into the transfer conduit / injector (e.g., passing through the transfer conduit / injector opening) when 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 flushed away, it continues to expand and diffuse and undesirably fills the entire ablation cell.

[0105] In one aspect, the present invention provides a laser ablation configuration 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, the pulses of the laser beam are directed through a transparent target, and a sample plume (the "ablated plume" or "plume") is formed downstream of the beam to the transfer conduit / injector. Transmission target irradiation is advantageous for optimizing the spread of the transit time by removing optical elements (windows, objective lenses, etc.) from the direct path of the plume. In one aspect, the present invention provides a laser ablation system comprising: (a) a laser capable of producing laser irradiation; (b) a laser ablation cell (or laser ablation system) into which a transparent target can be introduced; and a transfer conduit / injector having an opening into which the ablated plume can enter, wherein the laser irradiation is generated 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 the present disclosure, including examples.

[0107] In certain aspects, a smaller ablation spot size (high resolution) can be achieved with a high numerical aperture lens such as an oil immersion lens. Such an oil immersion lens can be configured for transmission target ablation of thin samples (e.g., tissue sections less than 500 nm in diameter).

[0108] Thus, during operation of one device according to the present invention, a sample is transported to the device and sampled using a laser system including a lens through which the laser irradiation passes onto the sample to generate ionized material (the sampling can subsequently generate a vaporous / specific material that is ionized by an ionization system), and the ions of the sample material are passed through 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 installed between the objective lens and the sample stage. In this way, the apparatus of the present invention achieves a numerical aperture greater than 1.0, and therefore, the spot size of the laser is less than 200 nm, less than 150 nm, or less than 100 nm. For this reason, the present invention provides an apparatus for imaging mass spectrometry having a spatial resolution of 200 nm or more, 150 nm or more, or 100 nm or more.

[0110] Accordingly, the sample stage holds the sample during operation, and typically, the sample is on a sample carrier, and the same stage holds the sample carrier. The laser irradiation then passes through the lens of the apparatus, through the objective lens and the immersion medium, and is directed towards the sample, and by the irradiation, material is removed from the sample.

[0111] To achieve the optimal focus alignment conditions for the laser, the immersion medium of the present invention has a refractive index greater than 1.0, such as 1.33 or more, 1.50 or more, 2.00 or more, or 2.50 or more.

[0112] Furthermore, to reconstruct an image of the thickness (or less than the thickness) of a monolayer of living cells, or as further described herein, to read a thicker specimen layer by layer and generate a 3D image, 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 more detail herein, the combination of the objective lens and the 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 to the liquid medium (as shown in Figure 3) such that the carrier gas can recover the removed material. Therefore, the permeable sample carrier ablation technique must be applied herein. This has the additional advantage that the achievable working distance for the ablation material recovery hardware is short and there is no need to bend the transfer conduit between the sample chamber and the detector. This also leads to a reduction in the transit time and thus an increase in the achievable ablation rate per second at the spot.

[0114] Accordingly, the present invention provides an apparatus in which the solid immersion medium is a hemispherical solid oil immersion lens or a Welschtruss solid oil immersion lens. The 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 having 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 in order to maintain the focal 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 placed 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 has a thickness of 0.1 mm to 3 mm. 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, the laser beam pulse is directed through the transparent target, and a sample plume ("removed plume" or "plume") is formed downstream of the beam to the transfer conduit / injector.

[0116] The transfer conduit (i.e., the injector pipe) may have an inlet configured to capture the removed plume such as an inlet formed as a sample cone having a small opening or opening. 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 at least partially generated and formed within the expansion region of the cone. In some embodiments, the diameter of the opening and / or the size of the gap (including the angle) can be adjusted to allow optimization under various conditions. For example, while the plume has a cross-sectional diameter on the order of 100 μm, the diameter of the opening can be sized on the order of 100 μm with sufficient clearance to prevent perturbation to the plume as the plume passes through.

[0117] The transfer conduit can be continued downstream of the sampling cone for receiving the removed plume in a configuration that promotes the movement of the plume and maintains the spatial discrimination of each subsequent plume as a function of the laser pulse. Thus, a gas flow can be introduced to pass through the opening of the sampling cone (capture flow) to clearly capture each plume and to help direct the plume, while an additional gas flow can be introduced into the transfer conduit / injector for transporting each clearly captured plume toward the ionization system (transport flow or sheath flow). Another function of the transport flow or sheath flow is to prevent the particles generated within the plume from contacting the wall 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 transport flow gas can be the same or different.

[0118] Selecting or determining a gas flow rate suitable for the present invention is within the ability of one of ordinary skill in the art as defined by this disclosure. The total flow rate through the transfer conduit is typically defined by the requirements of the ionization source (e.g., ICP ionization source). Laser ablation settings are 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, in combination with a cumulative gas flow rate of about 1 liter per minute (0.1 liter per minute capture flow plus 0.9 liter per minute carrier flow). It would be expected that transfer conduits of various diameters, in combination with the gas flow rates selected accordingly, would be applicable to various geometries presented with similar expected results. Conditions for dynamically maintaining a laminar gas within the transfer conduit are desired to maintain the discriminability of each individually removed plume.

[0119] As described herein, considering the particular configuration of elements (e.g., a particular configuration of the gas inlet location, an aperture, transfer conduit characteristics, and other elements), the capture flow rate and the transport flow rate are selected such that each removed plume is transported to an ionization system (e.g., ICP) during a period that is within the cumulative transit time of the plume between the ionization system and its detection by the mass spectrometer. This can be achieved by capturing each sample plume through a gas flow and by the transport configuration such that the ratio between the spread of the plume during the transport period and the spread during the ion transit period is 1 or less. That is, the time spread (or time dispersion) of the transition signal is important. ICP-MS devices (such as the CyTOF® ICP-TOF instrument manufactured by Fluidigm Canada) are characterized by the inherent spread of the signal. In the case of laser ablation, the operation of ejecting a single plume can be fast or not, compared to the time dispersion in the ICP-MS itself. The spread of the plume prior to ionization depends on the laser ablation system, particularly the design of the ablation chamber and the transfer conduit. It is desirable that the laser ablation system and the transfer conduit do not spread the initial ablation plume beyond the inherent spread of the rest of the instrument. This condition ensures that the peak of the detection signal generated by the ablation plume is as sharp (in the time axis) as it can be for the selected instrument. If the spread of the plume is quite long, for example, the spread in an ICP-MS system, the event of laser ablation from a single pulse will appear more widely at the detector. However, if the spread within the laser ablation portion is smaller than the spread by the instrument, the spread by the instrument will be dominant over the total spread. For this reason, calibration beads can be used to measure the spread by the instrument, and then the total spread can be measured from a single laser pulse, and these two values can be compared. If the spread from laser ablation is smaller than the spread from the instrument, the total spread is less than twice the spread of the instrument.

[0120] The time spread by the characteristic instrument can be measured experimentally, for example, using labeled cells or calibration beads. Whenever a single bead enters a mass spectrometer (e.g., a CyTOF® ICP-TOF instrument), the bead is evaporated and ionized in the plasma and then subjected to a mass analyzer until its signal reaches the detector. Transient events are detected and used to record information about a particular bead, such as the width of the transient signal (representing the time spread from a single event) and the value of the spread that occurs starting from 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 path defined 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 optimal capture flow rate can be determined experimentally but is usually at the lower end of the range (e.g., about 0.1 Lpm). Typical carrier flow rates are in the range of 0.1 to 1 Lpm. The optimal carrier flow rate can be determined experimentally 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 carrier flow rate. The carrier flow rate can be, for example, 0 in some cases if the capture flow rate is about 1 Lpm. The carrier 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] Although the matters described in this specification are described in conjunction with various embodiments, such descriptions are not intended to be limited to such embodiments. On the contrary, the matters described in this specification include various variations, modifications, and equivalents, as will be understood by those skilled in the art. For example, in various examples shown in the drawings, the transfer conduit / injector pipe is generally described as having an inner diameter of 1 mm in combination with a cumulative gas flow rate of about 1 liter per minute (0.1 plus 0.9 liters per minute). It would be expected that transfer conduits / injectors of various diameters, in combination with the correspondingly selected gas flow rates, would be applicable to various outer shapes presenting similar expected results. However, conditions for dynamically maintaining non-turbulent or nearly non-turbulent gas within the injector pipe may be desirable in order to retain the discriminability of each individually removed plume.

[0124] Furthermore, in some cases where the laser pulse rate increases, two or more removed plumes can be clearly captured within the cumulative transient time spread and conveyed to an ionization system (e.g., ICP) as described above. For example, at a repetition rate of 10 kHz, a pulsed laser can generate two removed plumes in 200 μs that can subsequently be conveyed to the ICP for ionization. Ions generated from two separate plumes can be analyzed by a mass spectrometer as a single separate packet of ions. As a result, while the laser remains at the same ablation spot or the velocity of the laser movement over a trace of a small number of continuous spots is less than the repetition rate, the removed plumes, and subsequent ions, can each provide cumulative mass spectrometry at the same ablation spot or an average mass distribution along the trace. It should be noted that a laser repetition rate on the order of several MHz can be used to produce a signal representing the averaging of many laser pulses. The laser can also be emitted in bursts to provide gaps in the data flow between individual sampling positions (or pixels).

[0125] It will be understood that the methods and apparatus of the present invention can be used with any of a variety of types of samples, for example, biological samples. In one approach, the sample is a cellular material such as a tissue section, cell monolayer, cell preparation, etc. The sample can be a thinly sectioned biological tissue up to 100 micrometers in thickness, a tissue sample on the order of millimeter thickness, or an undivided tissue sample. In one embodiment, thin tissue sections (such as paraffin-embedded sections) can be used. For purposes of 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, for example, Antonov, A. and Bandura, D., 2012, U.S. Patent Application Publication No. 2012 / 0061561, which is incorporated herein by reference.

[0126] When constructing an image IMS and IMC, signals can be provided to a plurality of labeled atoms / element tags within the plume. Detecting the label within the plume reveals the presence of that same type of target at the ablation location (or, correspondingly, the location of desorption of the slag of the material). By generating a series of plumes at known spatial positions on the sample surface, the MS signal reveals the position of the label on the sample, and thus the signal can be used to construct an image of the sample. By labeling multiple targets with distinguishable labels, it is possible to associate the position of the labeled atoms with the position of the same type of target, and thus the present invention can construct a complex image and reach a level of multiplexing far exceeding the levels achievable using existing techniques. For example, the GRAPHIS package software manufactured by Kylebank Software can be used, but other package software such as TERAPLOT, ImageJ, and CellProfiler can also be used. Imaging using MS data by 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 disclose an "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 data sets at the full spatial and spectral resolution of the "Datacube Explorer" program.

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

[0128] Any suitable tissue sample is analyzable. 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 of tumor origin. In some embodiments, the sample can be from a known tissue, but it may be unknown whether the sample contains tumor cells. Imaging can reveal the presence of a target indicating the presence of a tumor, thus facilitating diagnosis. The tissue sample can include, for example, human breast cancer tissue or breast cancer tissue of human mammary epithelial cells (HMLE). The tissue sample can include formalin-fixed, paraffin-embedded (FFPE) tissue, can be frozen tissue, or can be tissue embedded in a suitable resin. The tissue can be obtained from any living multicellular organism, but is usually human.

[0129] The tissue sample is typically a section having a thickness in the range of 2 to 10 μm, such as 4 to 6 μm, for example. Thin tissue sections with a thickness less than 2 μm, such as less than 1 μm, less than 500 nm, less than 250 nm, or even less than 100 nm, are also analyzable. If the tissue sample is thin, the signal generated will be low due to the reduction in the volume of the sample removed by the later pulse, but the thinner the section, the more sections can be generated from the tissue sample, and imaging multiple sections provides advantages in terms of 3D imaging. However, if the section is thin (e.g., below the resolution of laser ablation), ablation can be easily performed through the slide (e.g., the entire 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, including using a microtome that includes a dehydration step, such as embedding. Thus, the tissue can be chemically fixed and then the section can be prepared in the desired plane. Cryosectioning or laser capture microdissection can also be used to prepare the tissue sample. The sample can be permeabilized, for example, to label intracellular targets with a reagent (see above).

[0130] The size of the tissue sample to be analyzed is defined by the maximum size that is compatible with the laser ablation device, particularly the ablation chamber thereof, but is similar to current IHC methods. A size of up to 5 mm × 5 mm is typical, although smaller samples (e.g., 1 mm × 1 mm) can also be 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 apparatus and method of the present invention detect atoms added to the sample (i.e., atoms that are not normally present). Such atoms are referred to as labeled atoms (thus, labeled atoms represent elemental tags). The sample is typically a biological sample containing cells, and the labeled atoms are used to label target molecules on the intracellular / cell surface. In some embodiments, for example, it is possible to simultaneously detect two or more many labeled atoms that enable multiplexed detection of 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] The labeled atoms that can be used in the present invention include any type that is detectable by MS and is substantially absent in the unlabeled sample. For this reason, for example, while the 12C atom would not be suitable as a labeled atom as it is abundantly present in nature, 11C is a synthetic isotope that does not occur in nature and is thus theoretically usable. However, in a preferred embodiment, the labeled atom is a transition metal such as a rare earth metal (in addition to the 15 lanthanides, scandium and yttrium). These 17 elements provide many different isotopes that can be easily distinguished by MS. These various elements are available in the form of enriched isotopes. For example, samarium has 6 stable isotopes, neodymium has 7 stable isotopes, and all of these are available in enriched form. The 15 lanthanide elements provide at least 37 isotopes with non-overlapping unique masses. Examples of elements suitable for use as labeled 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), bismuth (Bi), etc. Radioactive isotopes are not preferred for use as they lack convenience in handling and are unstable. For example, Pm is not a preferred labeled atom among the lanthanides.

[0133] To facilitate TOF analysis (see above), for example, it is useful to use labeled atoms having an atomic mass within the range of 80 - 250, within the range of 80 - 210, or within the range of 100 - 200. This range includes all of the lanthanides, excluding Sc and Y. The range of 100 - 200 allows for analysis with 101 theoretical building blocks by using different labeled atoms, while the present invention can utilize the high spectral scanning speed of TOF MS. As described above, by selecting labeled atoms within the window beyond what is observed in the unlabeled sample (e.g., within the range of 100 - 200), TOF detection can be used to provide rapid analysis at biologically significant levels.

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

[0135] Various numbers of labeled atoms can adhere to a single sbp member, and as more labeled atoms are adhered to any sbp member, the achievable sensitivity increases. For example, 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 labeled atoms can adhere to an sbp member. For example, a monodisperse polymer containing multiple monomer units can be used to form elemental tags, each containing a chelating agent such as DTPA. DTPA binds, for example, to 3+ lanthanide ions having a dissociation constant of about 10~6M [Tanner et al. Cancer Immunol Immunother (2013) 62:955-965]. These polymers can terminate within a thiol-reactive group (e.g., maleimide) that can be used to adhere to an sbp member. For example, the thiol-reactive group can bind to the Fc region of an antibody. Other functional groups can also be used for conjugation of these polymers, such as amine-reactive groups, for example, N-hydroxysuccinimide esters, or groups reactive to carboxyl groups or to the glycosylation of antibodies. Any number of polymers can bind to each sbp member. Specific examples of polymers that can be used include linear ("X8") polymers or third-generation dendritic ("DN3") polymers, both of which are available as MaxPar™ reagents. Metal nanoparticles can also be used to increase the number of atoms within the label.

[0136] As described above, the labeled atoms are adhered to an sbp member, and this labeled sbp member is contacted with a sample that can find the target sbp member (if present), thereby forming a labeled sbp. The labeled sbp member adheres to the labeled atoms and can then contain any chemical structure suitable for detection according to the present invention.

[0137] Generally, the method of the present invention can be used to determine the presence of a target molecule in a sample (e.g., used in IHC or fluorescence in situ hybridization, FISH) or can be based on any sbp known for fluorescence-based flow cytometry, but the sbp member contacted with the sample carries a labeled atom detectable by MS. For this reason, the present invention can be easily implemented using available flow cytometry reagents only by improving the previously used labels, for example, to improve FISH probes for carrying labels detectable by MS.

[0138] The sbp can hereinafter include any of a nucleic acid double strand, an antibody / antigen complex, a receptor / ligand pair, or an aptamer / target pair. For this reason, the labeled atom can be adherable to a nucleic acid probe subsequently contacted with the sample so that the probe can hybridize to a complementary nucleic acid therein, for example, to form a DNA / DNA double strand, a DNA / RNA double strand, or an RNA / RNA double strand. Similarly, the labeled atom can be adherable to an antibody subsequently contacted with the sample so that it can bind to its antigen. The labeled atom can be adherable to a ligand subsequently contacted with the sample so that it can bind to its receptor. The labeled atom can be adherable to an aptamer ligand subsequently contacted with the sample so that it can bind to its target. For this reason, the labeled sbp member can be used to detect various target molecules in a sample, including a DNA sequence, an RNA sequence, a protein, a saccharide, a lipid, or a metabolite.

[0139] In one typical embodiment, the labeled sbp member is an antibody. Labeling of the antibody can be achieved, for example, by conjugating one or more labeled atoms that bind the molecule to the antibody using the MaxPar™ conjugation kit described above. The target molecule of the antibody is referred to as its antigen and can be a protein, carbohydrate, nucleic acid, etc. Antibodies that recognize cell proteins useful for mass cytometry are already widely available for use in IHC, and by using labeled 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 and have the advantage of enhancing multiplexing capabilities. The antibodies used in the present invention are capable of recognizing targets on the cell surface or intracellular targets. Antibodies can recognize a variety of targets, for example, specifically recognize individual proteins, or recognize multiple related proteins sharing a common epitope, or recognize specific post-translational modifications of proteins (e.g., distinguish tyrosine and phosphotyrosine of the target protein, distinguish lysine and acetyllysine, detect ubiquitination, etc.). The labeled atoms conjugated to the antibody are detectable so as to reveal the presence of the target in the sample after binding to the target.

[0140] The labeled sbp member typically interacts directly with the target sbp member in the sample. However, in some embodiments, it is possible for the labeled sbp member to 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 method. However, generally, the present invention relies on direct interaction, which can be more easily achieved and enables high multiplexing. However, in both cases, the sample is contacted with an sbp member capable of binding to the target sbp member in the sample, and at a later stage, the label adhered to the target sbp member is detected.

[0141] One feature of the present invention is the ability to detect a plurality of (e.g., 10 or more, and even up to 100 or more) different target sbp members in a sample, such as detecting a plurality of different proteins and / or a plurality of different nucleic acid sequences in the sample. To enable the detection of three different target sbp members, each of their respective sbp members should carry different labeled atoms such that their signals are distinguishable by MS. For example, if 10 different proteins are detected, 10 different antibodies (each specific for a different target protein) can be used, and each of them carries a unique label such that the signals from the different antibodies are distinguishable. In some embodiments, it is desirable to use, for example, a plurality of different antibodies against a single target that recognize different epitopes of the same protein.

[0142] If two or more labeled antibodies are used, it is desirable for the antibodies to have similar affinities for their respective antigens, as this will help to ensure that the relationship between the amount of labeled atoms 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 permeate the membrane of living cells, the cells of the sample can be fixed and permeabilized. The labeled sbp member can then enter the cell and form an sbp with the target sbp member.

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

[0145] Sample labeling does not entirely depend on sbp. In some cases, classical dyes can be used to highlight desired features on tissues. In multiple cases, the dyes used for microscopy contain rare elements in the natural state of cells. Therefore, in the process of coloring the tissue, the tissue is concentrated with specific elements that can be interpreted by the devices and methods described herein.

[0146] Thus, in some embodiments, the above-described analysis method includes the step of labeling the sample with at least one labeled atom. This atom can then be detected using the methods described above.

[0147] Signal enhancement Aspects of the target application include signal enhancement by adding hydrogen with LA-ICP-MS as further described herein. The LA-ICP-MS system can have one or more gas flows as shown in Figure 4, such as a capture gas flow, a carrier gas flow, an internal (auxiliary) gas flow, and / or an external (plasma) gas flow to the external torch pipe. Notably, the gas sources shown in Figure 4 can be configured differently such that a premixed or humidified gas is supplied to any one or more of the capture gas flow, the carrier gas flow, the internal gas flow, and / or the external gas flow. In certain aspects, 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 in addition, the laser ablation system can include an injector orthogonal to the sample, as shown, for example, in Figures 2-3. In certain aspects, only one gas flow (e.g., the capture gas) can flow through the injector. In certain aspects, the injector includes a makeup gas flow downstream of the laser ablation plume.

[0148] Aspects of the target application include devices and workflows for imaging mass spectrometry (IMS) that improve the capture rate, signal sensitivity, and / or signal stability of samples. Imaging mass cytometry (IMC) is the detection of mass tags by imaging mass spectrometry with cellular or intracellular spatial resolution. IMC systems and methods can include any of the aspects described for the target application. In certain aspects, mass cytometry can include laser ablation (LA)-induced coupled plasma (ICP) mass spectrometry (MS). Using non-endogenous elements such as heavy metal mass tags enables detection superior to that of endogenous elements. Endogenous elements such as carbon, oxygen, nitrogen, and light metals such as calcium can be depleted by a mass spectrometer, such as by a high-pass mass filter (e.g., RF quadrupole). In certain aspects, argon dimers (by-products 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 include heavy metals above 80 amu, as further described herein. In certain aspects, individual mass tags can include a plurality of labeled atoms of an enriched heavy metal isotope. Such labeled atoms can be in a polymer that is bound, for example, by chelating a pendant group to the polymer, which is then conjugated to a specific binding partner (SBP) that binds a specific target, such as an antibody that binds a specific protein target. For example, the Maxpar tags provided by Fluidigm each include a polymer with a plurality of 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. When an isotope is detected by ICP-MS, it indicates the presence of the corresponding target (e.g., protein).

[0150] Using enriched isotopes as labeled atoms increases the number of distinguishable targets detectable with elemental mass tags that include natural mixtures of isotopes. However, when using more than 20, more than 30, or more than 40 isotope mass tags, there are often isotopes that are 16 amu apart such that the oxide of one mass tag can interfere with the detection of another mass tag that includes 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 problems for atomic IMC, unlike other forms of IMS such as MALDI, because the atomic mass detection of metal isotopes that are 16 amu from other mass tags is vulnerable to oxide spillover. This consideration is further complicated by the desire for highly sensitive and stable detection within each laser-ablated spot (pixel). Each ablation creates a small (e.g., micron or submicron sized) ablation crater where there can be a small number of predetermined mass tags. Additionally, IMC relies on the ability to accurately compare the expression of different targets across a sample such as a tissue section (e.g., measured as signals in different mass channels each corresponding to an isotope mass tag of a different mass).

[0152] In certain embodiments, the IMC system can be used for suspension mass spectrometry, such that the ICP torch of the system can be coupled to a spray chamber for introducing all cells, instead of a laser ablation source. Thus, the ICP torch can enable atomization and ionization of all cells (e.g., cells with a diameter of at least 15 microns at most, or at least 20 microns at most). Thus, the plasma generated by the ICP torch may not be specifically designed for efficient ionization and / or atomization of the laser ablation plume (e.g., it may have a longer path length than would be required for a laser ablation plume). Instead, designing the LA-ICP-MS system to have a short transient signal may involve reducing the path length of the plasma and may result in inefficient ionization and / or atomization (e.g., if not improved by introducing hydrogen as described herein). The inventors have found that humidity levels can affect the sensitivity of the IMC system and that signal drift can occur, such as when the humidity within the system tends to decrease over sample movement (e.g., when there is an initial level of humidity visible in the ablation chamber air prior to operation). In fact, as further described herein, both water vapor and hydrogen gas have been found to increase signal sensitivity and can further improve signal stability. Thus, the efficiency of ionization and / or atomization (e.g., as measured by an increase in sensitivity of one or more mass channels, such as for one or more labeled atoms) can be improved by adding one or more hydrogen-containing molecules to the gas stream, as further described herein. For example, suitable hydrogen-containing molecules can be water or alcohol (such as ethanol) as vapor. Instead of or in addition to this, suitable hydrogen-containing molecules can be, for example, hydrogen gas, methane, or ammonium, provided premixed with helium or argon.

[0153] In certain embodiments, portions of the LA-ICP-MS system are open to the atmosphere such that air can be present within the laser ablation chamber, the fluid optics, and / or the ICP torch. Such air can ultimately be removed or consumed by the operation of the ICP-MS system. However, changes in humidity and / or oxygen levels due to such air can affect the efficiency of the ICP plasma, such as ionization efficiency and / or oxide formation. In certain embodiments, humidity can be controlled (e.g., such that the mass signal increases and / or stabilizes while oxidation is minimized) across sample transfer as described herein. Alternatively or in addition, 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 sensitivity can be improved and signal stability can be controllable by adding water or hydrogen during LA-ICP-MS analysis of heavy metal mass tags. In certain embodiments, one or more hydrogen-containing gases such as hydrogen gas, water vapor, methane, and / or ammonia can be introduced during 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 within a pressurized gas source. In certain embodiments, water vapor can be mixed with a gas such as argon gas.

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

[0156] Signal enhancement can be for one or more mass channels, such as the metal isotope channels of mass tags used to label a biological sample that the device analyzes. In certain embodiments, the metal isotope includes lanthanide isotopes. 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 over the range of mass channels where the signal (e.g., from the metal isotope mass tag) is detected. In certain embodiments, the average signal enhancement for lanthanide isotopes measured by at least 10 counts is at least 20%, at least 30%, or at least 50%. Signal enhancement, also referred to as improved sensitivity, can be measured as an increment in the count number per laser ablation plume (e.g., average count number), such as in the case of analyzing labeled atoms of a sample or analyzing an elemental standard containing a known amount of detectable atoms.

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

[0158] Described herein are devices and methods for introducing hydrogen-containing molecules (such as 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 an external humidity over a range (e.g., 0 to 2000 microbars or 0 to 4000 microbars) has a change in signal sensitivity of less than 10% or less than 5%.

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

[0160] In certain embodiments, the apparatus comprises a sample stage configured to move a sample in at least two directions, a laser ablation source configured to remove a sample attached to 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 one or more of a source of compressed premixed gas containing hydrogen gas mixed with at least one of helium and argon.

[0161] The hydrogen gas in the compressed premixed gas source can 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% by volume of helium or at least 50% by volume of argon. The compressed premixed gas source can supply gas to an ablation chamber including the sample stage. The premixed gas source can provide a capture gas that conveys the ablation plume to the injector. Alternatively or in addition, the premixed gas source can provide a carrier gas (i.e., entering the injector upstream of the ablation plume), a makeup gas (i.e., downstream of where the ablation plume enters the injector and supplementing the gas in the injector), and / or an internal gas (i.e., an auxiliary gas flowing through the internal tubing of the ICP torch).

[0163] An additional gas source can provide carrier gas to the injector, such as when the capture gas draws the ablation plume into the carrier gas within the injector. The carrier gas can include argon (e.g., at least 50% argon). The capture gas can 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 be no separate capture gas and carrier gas, such as when only the capture gas enters the injector. In certain embodiments, the injector is configured to direct the laser ablation plume towards a vertically oriented ICP torch, such as when the device comprises a vertically oriented ICP torch.

[0165] The device may further comprise an additional gas source that provides 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 a liquid dewar, may be at least 50 volume % argon, and the premixed gas source may comprise at least 50 volume % helium. In contrast, the premixed gas source may not be in a liquid dewar because different gases may evaporate (and thus deplete) at different rates during operation.

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

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

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

[0169] The device can further comprise a mass spectrometer configured to detect ionized atoms generated 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 is configured to atomize and ionize all cells in a cell suspension mode in which the ICP torch is separated from the laser ablation source. For example, the ICP torch can be separated from the laser ablation source and is sufficient to atomize and ionize all cells introduced into the spray chamber in the suspension upstream of the ICP. The characteristics of the ICP torch, such as size and shape, can be suitable for atomizing and ionizing all cells. By considering such a design, the ICP torch can have a reduced efficiency of atomizing and / or ionizing the material in the laser ablation plume, such as a plume generated by a laser having a spot size of less than 2 microns, such as less than 1 micron.

[0171] Any of the devices of the above embodiments may further comprise a humidification system configured to humidify a gas stream (e.g., as further described herein).

[0172] The method for the target application may include analyzing a sample by LA-ICP-MS using any of the devices of the above aspects. The sample may be a biological sample as further described herein, such as a tissue section. The sample may include a labeled atom as further described herein, such as a labeled atom associated with an SBP that binds a target in the sample. Thus, the analysis method may further include labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS. In certain aspects, there is no labeled atom having an oxide spillover of more than 3%.

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

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

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

[0176] Premixed gas source and its use Aspects of the target application include a compressed premixed gas source for an inductively coupled plasma device. The premixed gas source may include at least 50 volume % helium or argon (e.g., at least 70 volume %, at least 90 volume %), and may further include at least 0.1 volume % of a gas containing elemental hydrogen. The gas may be, for example, hydrogen gas, ammonia, and / or methane.

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

[0178] The embodiment includes an LA-ICP-MS system including the above-described compressed premixed gas source. In certain embodiments, the mass spectrometer can alternatively be an elemental analyzer such as an emission spectroscopic analyzer.

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

[0180] In certain embodiments, the apparatus comprises one or more of a sample stage configured to move the sample in at least two directions, a laser ablation source configured to remove the sample attached to the sample stage, an inductively coupled plasma (ICP) source, an injector configured to transport 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 flow may include one or more of a carrier gas flow, a capture gas flow, a makeup gas flow, and an auxiliary gas flow. In certain embodiments, the gas flow is a carrier gas flow.

[0182] In certain embodiments, the gas flow includes at least 50% argon (such as at least 70% or at least 90% argon) when the gas flow is a carrier gas, for example. Alternatively, the gas flow may include at least 50% helium when the gas flow is a capture gas, for example.

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

[0184] The humidification system may include a water diffusion pipe. In certain embodiments, the humidification system controls the temperature of the diffusion pipe. Alternatively or in addition, the humidification system comprises a variable splitter that can be adjusted to redirect the gas flow (e.g., the flow of argon gas) around the water diffusion pipe. The variable splitter can be adjusted by a control device coupled to a humidity sensor. In certain embodiments, both the control device and the humidity sensor are configured to redirect the gas flow around the diffusion pipe to maintain the humidity level. Instead of the diffusion pipe, the humidification system may comprise a water pump configured to directly inject water into the gas flow.

[0185] The method may include analyzing a 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 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 related to an SBP that binds a target in the sample. Thus, the analytical method can further include labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS. In certain embodiments, there is no labeled atom having an oxide spillover of more than 3%. For example, when the humidity is high, the oxide spillover can exceed 3%. Thus, the humidity and / or the plasma temperature can be maintained at a level that allows for improvement in sensitivity as described below, but can be low enough to avoid an oxide spillover of more than 3% for any labeled atom (e.g., the average oxide spillover during any 5-minute period of sample movement). For example, the temperature can be controlled by adjusting the plasma temperature with a makeup gas flow. Even a small value of humidity can result in a very large amount 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 having 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 a labeled atom (or elemental basis) over any given 5-minute period cannot change by more than 10% over at least 1 hour of analyzing the sample. In certain embodiments, the humidity can be changed by more than 20%, such as more than 50%, without reducing the sensitivity by more than 5% (e.g., for one, some, or all of the labeled atoms).

[0188] While gas humidification (with water vapor) is described above, another hydrogen-containing molecule can be introduced into the gas stream as a vapor, either in place of or in addition to water. For example, an alcohol such as ethanol can be introduced into the gas stream by any of the manners such as a diffusion tube as described above for water vapor.

[0189] Typical laser ablation inductively coupled plasma devices use a combination of gas flows to transport the removed 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 by means of a spray mist can disrupt the flow and, in imaging applications, can negatively affect transient signals and pixel speed. This problem does not occur if the gas flow is humidified before entering the instrument. Usually, ensuring a stable humidity would involve a significant stabilization of temperature. The humidity of the gas flow at the outlet can be measured to provide feedback to a humidifier to regulate the humidity. The humidifier itself is a gas flow passing through a diffusion pipe. Since the flow passing through the pipe exits in a saturated state, the inventors use a variable gas splitter to vary the flow passing through the diffusion pipe and thus vary the moisture 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 subject application include apparatuses and methods for introducing hydrogen-containing molecules into an ICP torch within LA-ICP-MS. The hydrogen-containing molecules can be gases such as hydrogen gas, ammonia, or methane. Alternatively or in addition, hydrogen-containing molecules such as water or alcohol (e.g., ethanol) can be introduced into the gas flow as vapor.

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

[0192] Aspects 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 remove a sample attached to the sample stage; an inductively coupled plasma (ICP) source; and an injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch. The apparatus is configured to supply a vapor including a hydrogen gas stream. The vapor may include water vapor or alcohol vapor (e.g., ethanol, etc.).

[0193] Aspects 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 remove a sample attached to the sample stage; an inductively coupled plasma (ICP) torch coupled to a mass spectrometer; and an injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch. The apparatus may be configured to supply a hydrogen-containing molecule to the plasma of the ICP torch (e.g., as described herein) while operating for laser ablation ICP mass spectrometry but not in a cell suspension mode. In the cell suspension mode, the ICP torch may be separated from the laser ablation source and may be sufficient to atomize and ionize all cells introduced into the spray chamber in the suspension upstream of the ICP.

[0194] Experimental results The inventors have found that signal stability and enhancement for lanthanides and other marker 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 this stability and enhancement can be robust to differences in the amount of hydrogen gas (e.g., due to changes in the gas flow during operation to maintain a constant plasma). Alternatively, the hydrogen can be introduced as water vapor, and the signal stability and enhancement can similarly be robust to a range of humidities.

[0195] The inventor monitored the sensitivity of the IMC (Imaging System made by Fluidigm Hyperion), which weakens during operation and recovers during stoppage. The inventor determined that this was caused by a small amount of moisture diffusing into the system, accumulating during the stoppage time, and gradually drying and evaporating during operation. As a result, not only suggestions for reducing the method of moisture diffusion into the system were recognized, but also the necessity of humidifying the gas flow in the system. The inventor found during the investigation that an important factor was the amount of hydrogen in the system. Initially, the inventor rejected the idea of mixing pure hydrogen into the injector gas as being costly due to considerations for handling excess gas and safety measures. The inventor initially developed an argon flow humidifier device. The device can stably supply H2O at a required level. However, concerns about images on the mass channel contaminated by oxide formation became apparent through image analysis by biological experts. In this case, the oxygen was derived from water molecules in the argon flow. The inventor then decided to explore a method of obtaining the advantages of pure hydrogen mixed into the injector flow in a configuration that eliminates concerns about flammability and safety without the addition of oxygen. The inventor understood that a premixed gas diluted to a non-flammable level can be managed by the existing gas handling components, enhance the sensitivity, and provide a sufficient hydrogen concentration to stabilize the signal.

[0196] The inventor initially found that the signal was highly sensitive to humidity and could vary with external humidity (e.g., the air inside the space of an LA-ICP-MS instrument). As shown in Figure 5, the humidity range of 0 - 4000 uB (microbar) of the carrier gas was the main cause of an approximately 30% change in the sensitivity of the elemental standard containing lutetium, measured as the average lutetium count per ablation shot over the humidity range. However, the inventor also noticed that the signal was stable and 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 while adding water can result in very high oxide levels, the flow rate of the carrier gas can be adjusted to maintain the oxide at a specific level, as opposed to simply adjusting for maximum sensitivity. The following are sample adjustment results showing that while restricting the oxide ratio to <3% (e.g., <2% or <1% etc., 3% or less), further significant improvements in future sensitivity can be achieved with a humidifier:

Table 2

[0198] As an additional note, from Ce140 to Gd156, typically, oxide spillover is the lowest in all mass channels, so restricting the oxide spillover to <3% in a channel during adjustment can result in it being much smaller in other channels. In certain embodiments, the oxide spillover varies by less than 1% (e.g., less than 0.5% or less than 0.25% etc.) during sample movement (e.g., during a period of more than 5 minutes, more than 10 minutes, or more than 30 minutes of sample movement).

[0199] The inventors have also confirmed that the addition of hydrogen (H2) gas (from a premixed gas source with helium, where the hydrogen gas is 3%) shows a strong correlation with signal increase / ion image brightness. Signal enhancement for both methods is shown in Figure 9. Specifically, carrier gas humidification (left figure) shows improvement in the signal for common markers, and premixed hydrogen - helium capture gas (right figure) also shows improvement in the signal for common markers.

[0200] The inventors have also found that very faint channels have a lower improvement in sensitivity than brighter channels, presumably due to background effects. Thus, improvement in sensitivity can be confirmed mainly for channels where the average number of ion counts per ablation shot is detected to be 10 or more.

[0201] The results of comparing the trade - offs for these two methods are shown below.

Table 3

[0202] The inventors attempted the multi-humidification system shown in FIG. 6 and found that direct injection of water by a water pump (upper part of FIG. 6) caused certain stability problems related to wetting and droplet formation at the ends of the capillaries. To control the redirection of the gas flow around the diffusion pipe, it has been found that flowing the gas through the diffusion pipe (lower part of FIG. 6) by temperature and / or humidity feedback provides better humidification stability. By operating overnight at a constant rate and at a temperature that stabilizes the diffusion pipe, the humidity varies by + / - 10%, but a fairly stable signal is obtained (as shown in FIG. 7). However, sacrificing temperature stability and instead using feedback on the gas flow can result in better humidity control. The humidity is controlled by changing the gas flow passing through the diffusion pipe in response to the temperature detected by a sensor downstream of the pipe. Controlling the flow of gas through the diffusion pipe results in more consistent humidity and a better response time to changes.

[0203] Typical laser ablation inductively coupled plasma devices use a combination of gas flows to transport the removed material from the ablation point to the plasma. The sensitivity is affected by hydrogen in the gas flow.

[0204] The inventors determined the appropriate amount of hydrogen required for an LA-ICP-MS system similar to that shown in FIG. 4 using variable amounts of hydrogen gas. The mixing gas ratio for our helium-hydrogen gas flow, which provides the appropriate amount of hydrogen over a typical range of helium flows, was used. This allows switching to a hydrogen mixed gas system without making any other changes to the hardware or software other than sacrificing the use of pure helium and replacing it with a mixed hydrogen-helium gas. The total number of cylinders / regulators / mass flow controllers is the same. Unlike the humidifier system, the mixed gas system does not add oxygen and the oxide ratio detected by the MS system was not affected.

[0205] In an alternative configuration, hydrogen is premixed with argon (such as a gas source specific to the carrier gas and / or scavenging gas). In some LA-ICP-MS systems, argon can be used as a scavenging gas for the removed plume. In addition to LA-ICP-MS, in mass spectrometry instruments, neon can also be considered for some applications, although it can be prohibitively expensive. In yet another alternative configuration, the hydrogen premixed with argon is added to the carrier gas. The carrier gas is a portion of the total injector flow and is mixed with the ablation scavenging gas that transports the plume. By adding the H2 / argon premix to the carrier gas stream, the user can vary the ratio of pure argon to the H2 / Ar premix and independently control the optimal argon flow while controlling the total flow of H2 to the injector. In other words, the flow of the premix controls the mass flow of H2, and the flow of the premix and the remaining argon flow control the total argon flow of the carrier gas. The total argon flow at the injector output needs to be carefully adjusted to achieve the optimal plasma temperature and maximum sensitivity. The level of H2 then provides a second dimension for improving sensitivity. The proposed H2 / argon premix has a low concentration of hydrogen and is below the flammability limit. This apparatus has the disadvantage that an additional cylinder for the premixed gas and an independent additional mass flow controller are required for the flow of the premix. However, the advantage of this apparatus is the ability to independently control and adjust the hydrogen portion of the total flow for a given instrument and plasma conditions. For example, this arrangement can be considered Plan B for a Deuterium plan with a H2 / helium premix as Plan A. The H2 / helium mixture is a low-cost and convenient solution due to typically less critical limitations of the lack of independent H2 flow control. Examples of the present invention are shown below. (Example 1) An apparatus comprising a sample stage configured to move a sample in at least two directions, a laser ablation source configured to remove a sample mounted on the sample stage, a plasma source, and an injector configured to convey 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 is oriented orthogonally to each other. (Example 2) The apparatus according to Example 1, wherein the injector is rigid. (Example 3) The apparatus according to Example 1 or 2, wherein the injector is linear. (Example 4) The apparatus according to any one of Examples 1 to 3, wherein the inner diameter of the injector is less than 1 mm. (Example 5) The apparatus according to any one of Examples 1 to 4, wherein the length of the injector is less than 10 cm. (Example 6) The apparatus according to Example 5, wherein the length of the injector is less than 5 cm. (Example 7) The apparatus according to any one of Examples 1 to 6, wherein the apparatus is configured to direct a laser along a path that does not pass through the injector. (Example 8) The apparatus according to any one of Examples 1 to 7, wherein the apparatus is operable to deliver at least 1000 different ablation plumes per second to an ICP source. (Example 9) The apparatus according to any one of Examples 1 to 8, further comprising a mass spectrometer. (Example 10) The apparatus according to Example 9, wherein the mass spectrometer is a time-of-flight mass spectrometer. (Example 11) The apparatus according to Example 9 or 10, wherein the mass spectrometer is configured to receive a vertical beam of ions. (Example 12) The apparatus according to any one of Examples 1 to 11, wherein the sample stage is vertical and operable to move to a vertical position. (Example 13) The apparatus according to any one of Examples 1 to 11, wherein the plasma source is vertically oriented. (Example 14) The apparatus according to Example 13, wherein the plasma source is vacuum-sealed, except for the inlet portion of the injector leading to the plasma source. (Example 15) The apparatus according to any one of Examples 1 to 14, wherein the plasma source is an ICP source. (Example 16) A method comprising analyzing a sample by LA-ICP-MS using the apparatus according to any one of Examples 1 to 15. (Example 17) The method according to Example 16, wherein the sample is a biological sample. (Example 18) The method according to Example 17, wherein the sample contains labeled atoms. (Example 19) The method according to Example 18, further comprising labeling the sample with labeled atoms before analyzing the sample by LA-ICP-MS. (Example 20) The method according to any one of Examples 16 to 19, wherein at least 1000 different ablation plumes are analyzed per second. (Example 21) An apparatus comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to remove a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch; and a compressed premixed gas source containing hydrogen gas mixed with at least one of helium and argon. (Example 22) The apparatus according to Example 21, wherein the hydrogen gas in the compressed premixed gas source is more than 0.1% and less than the flammable limit of hydrogen. (Example 23) The compressed premixed gas source according to Example 21, wherein the hydrogen gas is 1% to 4% by volume. (Example 24) The apparatus according to Example 21, 22 or 23, wherein the compressed premixed gas source contains at least 50% by volume of helium. (Example 25) The apparatus according to Example 21, 22 or 23, wherein the compressed premixed gas source contains at least 50% by volume of argon. (Example 26) The apparatus according to any one of Examples 21 to 25, wherein the compressed premixed gas source supplies gas to an ablation chamber including the sample stage. (Example 27) The apparatus according to any one of Examples 21 to 26, wherein the premixed gas source provides a capture gas for transporting the ablation plume into the injector. (Example 28) The apparatus according to Example 27, wherein an additional gas source provides a carrier gas to the injector, and the capture gas draws the ablation plume into the carrier gas in the injector. (Example 29) The apparatus according to Example 28, wherein the carrier gas contains argon and the capture gas contains a mixture of helium and hydrogen gas. (Example 30) The apparatus according to Example 27, wherein there is no separate capture gas and carrier gas. (Example 31) The injector is the device according to any one of Examples 21 to 30, which directs a laser ablation plume towards a vertical ICP torch. (Example 32) The device according to any one of Examples 21 to 29, further comprising an additional gas source for providing the carrier gas to the injector. (Example 33) The device according to Example 32, wherein the additional gas source contains at least 50% by volume of argon, and the premixed gas source contains at least 50% by volume of helium. (Example 34) The device according to any one of Examples 21 to 33, wherein the additional gas source is a liquid dewar. (Example 35) The device according to any one of Examples 21 to 34, wherein the compressed premixed gas source is configured to introduce a makeup flow to the injector downstream of where the laser ablation plume enters the injector. (Example 36) The device according to Example 35, wherein the makeup flow is introduced downstream of the sacrificial flow. (Example 37) The device according to Example 36, which does not include a sacrificial flow. (Example 38) The device according to any one of Examples 21 to 34, wherein the compressed premixed gas source provides at least one of a capture gas, a carrier gas, and an internal torch gas. (Example 39) The device according to any one of Examples 21 to 38, configured to provide a hydrogen gas flow to the ICP torch at 0.001 L / min to 0.1 L / min. (Example 40) The device according to Example 39, configured to provide a hydrogen gas flow to the ICP torch at 0.001 L / min to 0.02 L / min. (Example 41) The device according to any one of Examples 21 to 40, further configured to provide hydrogen gas at 0.002% to 1% of the total gas flow to the ICP torch. (Example 42) The device according to Example 41, configured to provide hydrogen gas at 0.01% to 0.1% of the total gas flow to the ICP torch. (Example 43) The device according to Example 41 or 42, wherein the total gas flow to the ICP torch is 5 to 30 L / min. (Example 44) The device according to any one of Examples 21 to 43, further comprising a mass spectrometer configured to detect the ionized atoms generated by the ICP torch. (Example 45) The device according to Example 44, wherein the mass spectrometer comprises a high-pass filter configured to remove ions with a mass of at least 80 amu or less. (Example 46) The ICP torch is configured to atomize and ionize all cells in a cell suspension mode in which the ICP torch is separated from the laser ablation source, according to the device of any one of Examples 21 to 45. (Example 47) The device according to any one of Examples 21 to 46, further comprising a humidification system configured to humidify the gas stream. (Example 48) The humidified gas stream is a carrier gas stream, according to the device of Example 47. (Example 49) A method comprising analyzing a sample by LA-ICP-MS using the device according to any one of Examples 21 to 48. (Example 50) The sample is a biological sample, according to the method of Example 49. (Example 51) The sample contains labeled atoms, according to the method of Example 50. (Example 52) The method according to Example 51, further comprising labeling the sample with labeled atoms before analyzing the sample by LA-ICP-MS. (Example 53) Furthermore, there are no labeled atoms having an average oxide spillover of more than 3%, according to the method of Example 52. (Example 54) The hydrogen gas provides at least a 20% increase in sensitivity to at least some labeled atoms, according to the method of any one of Examples 49 to 53. (Example 55) The hydrogen gas provides at least a 50% increase in sensitivity to at least some labeled atoms, according to the method of Example 54. (Example 56) The average sensitivity to labeled atoms over any given 5-minute period does not change by more than 10% over at least 1 hour of analyzing the sample, according to the method of any one of Examples 49 to 55. (Example 57) 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, a compressed premixed gas source. (Example 58) The compressed premixed gas source according to Example 57, comprising at least 50% by volume of helium. (Example 59) The compressed premixed gas source according to Example 57, comprising at least 50% by volume of argon. (Example 60) The hydrogen gas is from 1% to 4% by volume, according to the compressed premixed gas source of Examples 55, 56, or 57. (Example 61) A compressed premixed gas source for an inductively coupled plasma device, comprising at least 50% by volume of helium or argon, and at least 0.1% by volume of a gas containing elemental hydrogen, a compressed premixed gas source. (Example 62) The gas is methane, ammonia, or hydrogen gas, according to the compressed premixed gas source of Example 61. (Example 63) An apparatus comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to remove a sample mounted on the sample stage; an inductively coupled plasma (ICP) source; an injector configured to transport an ablation plume created from the sample by the laser ablation source to an ICP torch; a humidification system configured to humidify a gas flow. (Example 64) The apparatus according to Example 63, wherein the gas flow includes a carrier gas flow. (Example 65) The apparatus according to Example 63 or 64, wherein the gas flow includes a capture gas flow. (Example 66) The apparatus according to any one of Examples 63 to 65, wherein the gas flow includes a makeup gas flow. (Example 67) The apparatus according to any one of Examples 63 to 66, wherein the gas flow includes an auxiliary gas flow. (Example 68) The apparatus according to any one of Examples 63 to 67, wherein the gas flow includes at least 50% argon. (Example 69) The apparatus according to any one of Examples 63 to 68, wherein the humidification system includes a water diffusion pipe. (Example 70) The apparatus according to Example 69, wherein the humidification system controls the temperature of the water diffusion pipe. (Example 71) The apparatus according to Example 69 or 70, further comprising a variable splitter adjustable to redirect a gas flow around the water diffusion pipe. (Example 72) The apparatus according to any one of Examples 69 to 72, further comprising a control device and a humidity sensor configured together to redirect a gas flow around the water diffusion pipe to maintain a humidity level. (Example 73) The apparatus according to any one of Examples 63 to 68, wherein the humidification system includes a water pump configured to directly inject water into the gas flow. (Example 74) A method comprising analyzing a sample by LA-ICP-MS using the apparatus according to any one of Examples 63 to 73. (Example 75) The method according to Example 74, wherein the sample is a biological sample. (Example 76) The method according to Example 75, wherein the sample contains a labeled atom. (Example 77) The method according to Example 76, further comprising labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS. (Example 78) There is no labeled atom having an average oxide spillover of more than 3%, according to the method of Example 76 or 77. (Example 79) The humidification is the method according to Example 76, 77, or 78 that provides at least a 20% increase in sensitivity to at least some of the labeled atoms. (Example 80) The humidification is the method according to Example 79 that provides at least a 50% increase in sensitivity to at least some of the labeled atoms. (Example 81) The average sensitivity to the labeled atoms over any given five-minute period does not change by more than 10% over at least one hour of analyzing the sample, the method according to any one of Examples 73 - 80. (Example 82) An apparatus comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to remove a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch; a compressed premixed gas source comprising a hydrogen-containing gas mixed with at least one of helium and argon. (Example 83) The hydrogen-containing gas is methane, ammonia, or hydrogen gas, the apparatus according to Example 82. (Example 84) An apparatus comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to remove a sample mounted on the sample stage; an inductively coupled plasma (ICP) source; an injector configured to transport an ablation plume created from the sample by the laser ablation source to an ICP torch; the apparatus is configured to supply a hydrogen-containing molecule to the plasma of the ICP torch during operation for laser ablation ICP mass spectrometry. (Example 85) The apparatus is configured to supply a vapor comprising a hydrogen gas stream, the apparatus according to Example 84. (Example 86) The vapor comprises water vapor or alcohol vapor, the apparatus according to Example 84 or 85. (Example 87) The vapor comprises water vapor, the apparatus according to Example 86. (Example 88) The vapor comprises alcohol, the apparatus according to Example 86. (Example 89) The alcohol is ethanol, the apparatus according to Example 88. (Example 90) A method comprising analyzing a sample by LA-ICP-MS using the apparatus according to any one of Examples 84 - 89. (Example 91) An apparatus comprising: A sample stage configured to move a sample in at least two directions, A laser ablation source configured to remove a sample mounted on the sample stage, An inductively coupled plasma (ICP) torch coupled to a mass spectrometer, An injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch, and The ICP torch is configured to atomize 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 a hydrogen-containing molecule to the plasma of the ICP torch while the apparatus is operating for laser ablation ICP mass spectrometry but not in the cell suspension mode.

Claims

A method comprising analyzing a sample using an apparatus by LA-ICP-MS, wherein the apparatus comprises a sample stage configured to move the sample in at least two directions, a laser ablation source configured to remove the sample mounted on the sample stage, an inductively coupled plasma (ICP) torch, an injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch, a compressed premixed gas source comprising a hydrogen gas mixed with at least one of helium and argon, and the average sensitivity for labeled atoms over any given five-minute period does not change by more than 10% over at least one hour of analyzing the sample, the method. **Claim 2** The method according to claim 1, wherein the hydrogen gas in the compressed premixed gas source is more than 0.1% and below the flammable limit of hydrogen. **Claim 3** The method according to claim 1, wherein the hydrogen gas is from 1% to 4% by volume. **Claim 4** The method according to claim 1, wherein the compressed premixed gas source comprises at least 50% by volume of helium. **Claim 5** The method according to claim 1, wherein the compressed premixed gas source comprises at least 50% by volume of argon. **Claim 6** The method according to claim 1, wherein the compressed premixed gas source supplies gas to an ablation chamber including the sample stage. **Claim 7** The method according to claim 1, wherein the premixed gas source provides a capture gas for transporting the ablation plume into the injector. **Claim 8** The method according to claim 7, wherein an additional gas source provides a carrier gas to the injector, and the capture gas draws the ablation plume into the carrier gas within the injector. **Claim 9** The method according to claim 8, wherein the carrier gas comprises argon and the capture gas comprises a mixture of helium and hydrogen gas. **Claim 10** The method according to claim 7, wherein there is no separate capture gas and carrier gas. **Claim 11** The method according to claim 1, wherein the injector directs the laser ablation plume towards the vertical ICP torch. **Claim 12** The method according to claim 1, further comprising an additional gas source for providing a carrier gas to the injector. **Claim 13** The method according to claim 12, wherein the additional gas source contains at least 50% by volume of argon, and the premixed gas source contains at least 50% by volume of helium.

14. The method according to claim 1, wherein the compressed premixed gas source is configured to introduce a makeup flow into the injector downstream of where the laser ablation plume enters the injector.

15. The method according to claim 14, wherein the makeup flow is introduced downstream of the sacrificial flow.

16. The method according to claim 1, wherein the device does not include a sacrificial flow.

17. The method according to claim 1, wherein the compressed premixed gas source provides at least one of a capture gas, a carrier gas, and an internal torch gas.

18. The method according to claim 1, wherein the hydrogen gas flow to the ICP torch is configured to be provided at 0.001 L / min to 0.1 L / min.

19. The method according to claim 18, wherein the hydrogen gas flow to the ICP torch is configured to be provided at 0.001 L / min to 0.02 L / min.

20. The method according to claim 1, further configured to provide hydrogen gas at 0.002% to 1% of the total gas flow to the ICP torch.

21. The method according to claim 20, wherein the device is configured to provide hydrogen gas at 0.01% to 0.1% of the total gas flow to the ICP torch.

22. The method according to claim 20, wherein the total gas flow to the ICP torch is 5 to 30 L / min.

23. The method according to claim 1, further comprising a mass spectrometer configured to detect ionized atoms generated by the ICP torch.

24. The method according to claim 23, wherein the mass spectrometer comprises a high-pass filter configured to remove ions having a mass of at least 80 amu or less.

25. The method according to claim 1, wherein the ICP torch is configured to atomize and ionize all cells in a cell suspension mode in which the ICP torch is separated from the laser ablation source.

26. The method according to claim 1, further comprising a humidification system configured to humidify the gas flow.

27. The method according to claim 26, wherein the humidified gas flow is a carrier gas flow.

28. The method according to claim 1, wherein the sample is a biological sample.

29. The sample is the method according to claim 28, which contains labeled atoms.

30. The method according to claim 29, further comprising labeling the sample with labeled atoms before analyzing the sample by the LA-ICP-MS.

31. The method according to claim 30, wherein there are no labeled atoms having an average oxide spillover of more than 3%.

32. The method according to claim 1, wherein the hydrogen gas provides at least a 20% increase in sensitivity to at least some labeled atoms.

33. The method according to claim 32, wherein the hydrogen gas provides at least a 50% increase in sensitivity to at least some labeled atoms.

34. An apparatus comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to remove a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch coupled to a mass spectrometer; an injector configured to transport an ablation plume created from the sample by the laser ablation source to the ICP torch, wherein the ICP torch is configured to atomize 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 a hydrogen-containing molecule to the plasma of the ICP torch while the apparatus is operating for laser ablation ICP mass spectrometry but not in the cell suspension mode.

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