Plasma and Sampling Geometry for Imaging Mass Cell Analysis

Abstract

Systems and methods for imaging mass spectrometry, including imaging mass cell analysis, are described herein. Aspects of the present application include apparatus and methods for imaging mass spectrometry (IMS) that improve sample acquisition rate, signal sensitivity, and / or signal stability. Systems and methods described herein can minimize the delivery time of a plume of sample material excised from a sample to be delivered to a component of an imaging mass spectrometer or mass cell analyzer that ionizes and analyzes the sample material, and / or minimize diffusion.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 114,313, filed November 16, 2020, and U.S. Provisional Application No. 62 / 951,556, filed December 20, 2019, the contents of all of which are incorporated herein by reference for all purposes.

[0003] Technology field

[0004] The present invention relates to an apparatus and method for laser ablation-based imaging mass spectrometry, comprising imaging mass cell analysis. Background Technology

[0005] Imaging mass spectrometry applications, such as imaging mass spectrometry, benefit from the rapid acquisition of distinct ablation plumes. For example, laser ablation ICP-MS systems can ablate spots approximately 1 micron in diameter and individually detect elemental or isotopic compositions consisting of millions of spots within a single sample. Rapid delivery of the ablation plume while minimizing transient spreading enables the rapid analysis of many ablation spots and improves sensitivity. Turbulence resulting from changes in plume transport orientation relative to the plume expansion direction during plume formation can increase transient spreading. System components, such as optical components, can block the rapid injection of the plume into the plasma source. Additionally, modifying the gas flow of the traditional composition can improve signal sensitivity and / or stability.

[0006] In the present invention, the inventors have devised a number of inventions related to conventional laser ablation-based imaging mass cell analyzers and imaging mass spectrometers. In particular, these inventions relate to modifications that minimize the delivery time and / or diffusion of a plume of ablated sample material from a sample to be delivered to a component of an imaging mass spectrometer or mass cell analyzer that ionizes and analyzes the sample material.

[0007] The device of the present invention, such as an imaging mass spectrometer or an imaging mass cell analyzer, generally comprises three components. The first is a laser ablation system for generating a plume of vapor and particulate matter from a sample for analysis. Before atoms within the plume of the ablated sample material (including any detectable labeled atoms as described below) can be detected by the mass spectrometer component (MS component; third component), the sample must be atomized and ionized (although some ionization of the sample material may occur during ablation, the device requires a separate ionization component because space charge effects neutralize the charge long before it is detected). Accordingly, the device comprises a second component, namely an ionization system, which ionizes atoms to form elemental ions, thereby enabling the MS component to detect the elemental ions based on the mass-to-charge ratio. Between the laser ablation system and the ionization system, there is a transfer conduit configured to connect the laser ablation system to the ionization system; The transfer conduit has an inlet located within the laser ablation system, which is configured to capture the ablated plume when it is generated and to transfer the captured ablated plume to the ionization system (in some cases, for example, when the ionization system is a plasma such as inductively coupled plasma (ICP), the transfer conduit is the same conduit that introduces the sample directly into the ICP torch through the central injection tube, in which case the transfer conduit may be referred to as an injector). Thus, during operation, the sample is introduced into the device and ablated to generate vapor / particulate matter, which is ionized by the ionization system, and the ions from the sample are transferred into the MS component. Although the MS component can detect many ions, most of these will be ions of atoms that naturally constitute the sample. For some applications, such as geological or archaeological applications (e.g., mineral analysis), this may be sufficient.In imaging mass spectrometry applications such as imaging mass cell analysis, the MS component may be time-of-flight (TOF) or magnetic sector MS.

[0008] Accordingly, the present invention provides a device comprising the following:

[0009] (i) A laser ablation system configured to generate a plume of sample material from a sample;

[0010] (ii) a plasma source configured to receive the material removed from the sample by the laser ablation system and to ionize the material to form elemental ions;

[0011] (iii) a mass spectrometer that receives elemental ions from the above ionization system and analyzes the elemental ions,

[0012] Here, the laser ablation system and the ionization system are combined together by a transfer conduit configured to transport a gas flow containing a plume of the ablated sample material from the laser ablation system to the ionization system, and

[0013] The plasma is not oriented on the same axis as the sample stage.

[0014] The present invention also provides a device comprising the following:

[0015] A sample stage configured to move a sample in at least two directions, a laser ablation source configured to ablate a sample placed on the sample stage, a plasma source, and an injector configured to deliver an ablation plume generated 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 may be oriented more than 60 degrees, more than 70 degrees, or more than 80 degrees, e.g. more than 90 degrees, in a direction away from any axis of the sample stage (the axis of the flat sample mounted on the sample stage).

[0016] In a given embodiment, the injector is rigid and / or linear. The inner diameter of the injector may be less than 2 mm, less than 1 mm, or less than 0.5 mm. The length of the injector is less than 20 cm, less than 10 cm, less than 5 cm, or less than 3 cm. The device may be configured to guide a laser along a path that does not pass through the injector.

[0017] Here, the device can be operated to deliver at least 500, 1000, 5000, 10000, or 50000 discrete ablation plumes per second to an ICP source. In a given embodiment, a short plasma (e.g., less than 5 mm, less than 3 mm, less than 2 mm in length) can help prevent excessive diffusion and keep ions distinct in separate ablation plumes until they are detected by a mass spectrometer.

[0018] The device may additionally include a mass spectrometer (MS) coupled to the plasma source, such as a time-of-flight or magnetic sector mass spectrometer. The MS is configured to receive a vertical ion beam.

[0019] In a given embodiment, the sample stage may be vertical and may be operable to be executed in a vertical position (e.g., controlled by a motor capable of resisting gravity while still providing a step for scaling the ablation spot diameter).

[0020] In a given embodiment, the plasma source may be oriented vertically. The plasma source may be vacuum sealed (e.g., excluding the injector inlet to the plasma source).

[0021] The plasma source can be an inductively coupled plasma torch (ICP source). For example, the device can be an LA-ICP-MS system.

[0022] The method may include the step of analyzing a sample by LA-ICP-MS using the apparatus described herein. The sample is a biological sample and may contain a labeled atom (such as a labeled atom of SBP attached to an analyte of the biological sample). The method may further include the step of labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS.

[0023] Aspects of the present application also include an apparatus and method for introducing hydrogen-containing molecules into the ICP torch of an LA-ICP-MS, for example, for signal enhancement. The hydrogen-containing molecules may be gases, such as hydrogen gas, ammonia, or methane as described below. Alternatively or additionally, hydrogen-containing molecules such as water or alcohol (e.g., ethanol) may be introduced into the gas stream as vapor as described below. Brief explanation of the drawing

[0024] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the applicant's application in any way. Figure 1 is a schematic diagram of a laser ablation system showing sampling a laser-ablated plume through an aperture configured to deliver the plume into an injector. Figure 2 is a diagram of a sampling configuration using horizontal plasma. Figure 3 is a diagram of a sampling configuration using vertical plasma. Figure 4 is a schematic diagram of an exemplary LA-ICP-MS system in the industry. Figure 5 is a graph of sensitivity (average number of Lu ions / shot) as a function of humidity (microbar) of the argon transfer gas. Figure 6 is a diagram of a humidification system suitable for LA-ICP-MS. Figure 7 is a graph of the sensitivity (number of Lu ions / shot) for a sample run of a long time (60,000 seconds) while the humidity of the argon transfer gas is maintained. Figure 8 is a graph of sensitivity (average number of Lu ions / shot) as a function of hydrogen gas flow (L / min). Figure 9 compares the improvement in the imaging mass cell analysis signal when humidified (left) or when a gas source containing a 3% hydrogen / helium premix is ​​used as the capture gas. Specific details for implementing the invention

[0025] Detailed description of various implementation examples

[0026] It should be understood that singular expressions (“a” or “an”) used with this journal, by reference to various elements, include “one or more” or “at least one” unless the context clearly indicates otherwise.

[0027] The term “comprising” includes “consisting” as well as “including.” For example, a composition “comprising” X may consist only of X or may include additional elements such as X+Y.

[0028] The term “about” related to the numerical value x is arbitrary and, for example, means x ± 10%.

[0029] The term “substantially” does not exclude “completely”. For example, a composition “substantially free” of Y may be a composition that is completely free of Y. If necessary, the term “substantially” may be omitted from the definition of the invention.

[0030] The term “invention” refers to a specific aspect, embodiment, or embodiment of the invention rather than all embodiments of the invention.

[0031] Although the foregoing invention has been described in some detail for the sake of clarity and understanding, those skilled in the art will understand that after becoming familiar with this disclosure, various modifications in form and detail may be made without departing from the actual scope of the invention as defined in the appended claims. Accordingly, the invention is not limited to the exact components or details of the methodology or configuration presented above. Except to the extent necessary for the method or inherent in the method itself, no specific order of steps or stages of the method described in this disclosure, including the drawings, is intended or implied. In many cases, the order of the method steps may be changed without altering the purpose, effect, or import of the described method. All publications and patent documents cited herein are incorporated herein by reference as if each of these publications or documents were specifically and individually incorporated herein by reference. The citation of publications and patent documents (patents, published patent applications, and unpublished patent applications) is not intended to be an acknowledgment that any of these documents are relevant prior art, nor does it constitute any acknowledgment of the contents or dates of said documents.

[0032] The present invention relates to imaging mass spectrometry comprising 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 the detection of element-tagged antibodies. For example, U.S. Patent Publication No. 2012 / 0061561 of 2012 by Antonov, A. and Bandura, D. incorporated herein by reference; and the work of Seuma et al. “Combination of immunohistochemistry and laser ablation ICP mass spectrometry for imaging of cancer biomarkers” 2008, Proteomics 8:3775-3784; Hutchinson et al. [“Imaging and spatial distribution of β-amyloid peptide and metal ions in Alzheimer's plaques by laser ablation-inductively coupled plasma-mass spectrometry” Analytical biochemistry 2005, 346.2:225-233]; Becker et al. [“Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in elemental imaging of biological tissues and in proteomics.” 2007, Journal of Analytical Atomic Spectrometry 22.7:736-744]; Binet et al. [“Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry” Analytical Biochemistry 2003, 318:30-38]; Quinn et al. [“Simultaneous determination of proteins using an element-tagged immunoassay coupled with ICP-MS detection Journal of Analytical Atomic Spectrometry” 2002, 17:892-96]; Sharma et al., “Sesbania drummondii cell cultures: ICP-MS determination of the accumulation of Pb and Cu Microchemical Journal” 2005, 81:163-69; and the literature of Giesen et al. (“Multiplexed immunohistochemical detection of tumor markers in breast cancer tissue using laser ablation inductively coupled plasma mass spectrometry” 2011, Anal. Chem. 83:8177-8183), each of which is incorporated herein by reference. Plasma sources other than ICP are also included within the scope of this application.

[0033] Due to limitations of the nano-positioning stage, access requirements for high NA optics, and / or the desire to keep the sample as close to the plasma as possible in the IMC device, several different plasma and sampling orientations exist that are considered herein and described in the table below:

[0034]

[0035] By rotating the plasma to an optimal orientation, some of the undesirable characteristics of the previous configuration (such as excessive expansion due to 90-degree rotation and long delivery lengths, and cross-contamination caused by gravity pulling large particles back onto the sample surface) can be overcome. For reference, vertical plasma has existed in ICP-OES machines because this orientation allows for simpler imaging along the torch axis (since the cylindrical symmetry of the plasma is preserved). In contrast, symmetry is broken into a horizontal orientation due to the convective rise of the plasma as it exits the torch body.

[0036] In a given embodiment, the torch may be a sealed torch. The sealed torch is hermetically sealed and does not fluidly communicate with air outside the torch (except, for example, a gas source supplying gas flow). The sealed torch may be vacuum sealed. In a given embodiment, the laser ablation chamber is hermetically sealed and does not fluidly communicate with air outside the instrument. In a given embodiment, the entire laser ablation ICP-MS system is sealed to prevent air from passing into the torch. Gas flow and gas kinetics within the sealed torch may be dominant over convective effects. The sealed torch may behave nearly identically in all orientations (e.g., with similar ionization efficiency for the same gas flow, sample material, and induction conditions) and may be oriented vertically (e.g., toward a direction away from the gravity vector or toward gravity).

[0037] In our application, there are several difficulties in transporting ablated material by gas flow. Specifically, vigorously rotating the plume leads to excessive expansion, as seen when the total length of the gas channel between the sample and the plasma is excessive. Consequently, for optimal pixel acquisition speed, it is desirable for the plasmas to be oriented perpendicularly to the sample substrate and as close together as possible.

[0038] One limitation regarding orientation selection is related to the specifications of the nanopositioning stage. Stages with the speed and dynamic positioning precision required for the application generally do not possess sufficient maximum force to support the sample and associated hardware (springs, clamps, etc.) against gravity. From an engineering perspective, this means that configurations involving vertically oriented samples are more difficult compared to XYZ stages. However, these devices pose a relatively low risk in terms of plasma engineering, as we already have experience configuring mass spectrometers with horizontal plasma.

[0039] Another option is to place the sample horizontally and direct the plasma upward or downward. Each of these configurations has its own potential advantages and disadvantages.

[0040] In the case of upward-induction plasma, one advantage is that heat into the plasma rises away from the sample and into the interface region of the mass spectrometer. Since we have already cooled the interface with a liquid, this represents a small (perhaps negligible) change to the instrument's thermal budget. The disadvantage of this approach is that any ablated material (i.e., larger particles) not picked up by the gas flow falls back onto the sample, causing crosstalk and contamination.

[0041] In the case of downward-induced plasma, there is little risk of sample contamination from ablated large particles because gravity pulls particles away from the sample. However, since heat rising from the plasma will rise toward the sample, there is a possibility of problems. This can be overcome by incorporating thermal rupture between the sample and the plasma containment, but this increases the cost of potentially extending the gas path between the sample and the plasma and causes overexpansion. Convective forces may also cause additional overexpansion in such plasmas.

[0042] In principle, ablation light can be incident from both sides of the sample. However, there are some trade-offs to consider.

[0043] When the ablation light is incident from the same side as the sample transport / plasma, there must be a compromise between the optical access to the ablation spot and the access to the ablation sample transport, which may lead to one or more of the following trade-offs.

[0044] - The focusing optics will significantly increase the minimum sample delivery distance.

[0045] - Focusing optics will be more difficult to manufacture and assemble.

[0046] - The maximum achievable throughput will be lowered.

[0047] - Any engineering inspection of the sample area will be compromised in terms of field of view, resolution, lighting uniformity, etc.

[0048] - The device will be less modular.

[0049] Meanwhile, ablation light incident from the opposite side of the sample transport / plasma may lead to one or more of the following compromises.

[0050] - The ablation light must pass through the sample substrate, which means that a quartz microscope slide must be used for UV laser ablation, or another substrate such as silica that is at least partially transparent to UV light must be used.

[0051] - If sample delivery is in a straight line, optical inspection of the sample area may be damaged by the light from the plasma.

[0052] - The assembly of the sample holder and stage must provide a transparent aperture for the ablation / inspection optics, which makes the assembly more expensive and complex.

[0053] In certain embodiments, laser ablation may be non-UV laser ablation (e.g., may be in the visible spectrum or IR spectrum, such as a green laser ablation source). A non-UV laser may have characteristics similar to the UV laser ablation source described herein, such as frequency and / or power. This allows the laser radiation to pass through a glass slide (commonly used in microscopes). However, biological samples, such as tissue sections, smeared cells, or cell cultures, may be ablated better in the UV spectrum than in the visible light (e.g., green light) or IR spectrum.

[0054] In a given embodiment, the sample may be treated with a compound that assists laser ablation (e.g., a compound that lowers the laser ablation threshold) (e.g., after staining with mass-tagged SBP). The compound may be a dye that absorbs at the wavelength of laser ablation, for example, at non-UV wavelengths (e.g., green or IR). The compound may be nonspecifically distributed throughout the sample.

[0055] In a specific embodiment, as described in U.S. Patent Publication No. 20160194590 incorporated herein by reference, a portion of the sample may be lifted from the slide by using a counter-slide ablation to ablate the layer beneath the sample. In a specific embodiment, a compound for transferring kinetic energy to the sample (e.g., a compound that transitions to a gaseous state upon ablation) may be embedded in the sample itself.

[0056] As described herein, the laser may be a femtosecond (fs) laser. For example, a near-IR fs laser may operate at the second harmonic to provide laser radiation in the green range, or operate at the third harmonic to provide laser radiation in the UV range. Lower wavelengths, such as green or UV, may allow for higher resolution (e.g., smaller spot sizes). When laser radiation travels across a sample support and strikes the sample, the sample support must be transparent to the laser radiation. Glass and silica are transparent to green wavelengths; silica is transparent to UV, but glass is not. To increase resolution while allowing the use of glass slides, an IR fs laser may operate at the second harmonic (e.g., about 50% conversion efficiency) to provide green laser radiation. For reference, commercially available objective lenses are often best calibrated in the green range. The resolution achieved by a green or UV fs laser can be a spot size of 1 µm, 800 nm, 500 nm, 400 nm, 300 nm, 200 nm, 150 nm, or less than 100 nm.

[0057] For example, the ablation frequency by the 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. As mentioned above, the ablation frequency of the laser must match the scanning speed of the laser scanning system.

[0058] FIG. 2 illustrates a plurality of configurations in which the plasma (e.g., ICP) is horizontal. Laser radiation is illustrated by shaded triangles. The sample is shown mounted on a wider sample stage.

[0059] FIG. 2a illustrates a horizontal stage injected into a horizontal plasma as previously performed. FIG. 2a illustrates a horizontal sample when sampled from above—a sample fixed on a horizontally oriented slide (also described as a coverslip or substrate) is ablated (from above or through the substrate), and the plume is guided upward. The plume is captured by the gas flow and rotates orthogonally to enter the horizontal plasma. This can result in redeposition due to the force of gravity.

[0060] FIG. 2b illustrates the configuration when sampling from below—the plume is induced downward from the horizontal substrate by ablation from below or by ablation through the substrate from above. The plume is captured by the gas flow and rotates orthogonally to enter the horizontal plasma. In this configuration, redeposition (e.g. due to gravity) may not occur.

[0061] FIG. 2c illustrates a vertical sample. Light for ablation can be guided through the substrate (from the back) or from the sample side. The plume is guided horizontally, captured by the gas flow without the need for rotation, and delivered directly into the plasma.

[0062] Figure 3 is similar to Figure 2, but illustrates a plurality of configurations in which the plasma (e.g., ICP) is vertical.

[0063] Figure 3a illustrates a horizontal sample when sampling from above—the plasma is positioned above the sample, and the gas flow is directed upward. The ablation light is directed from above or below the sample, and the plume protrudes upward. The plume is captured by the gas flow and transferred to the plasma.

[0064] FIG. 3b illustrates a horizontal sample when sampled from below—where the gas flow toward the plasma is directed downward, so the plasma is located below the sample. The ablation light is applied from above or below, and the ablation plume leaving the downwardly protruding sample is captured by the gas flow and delivered directly into the plasma.

[0065] FIG. 3c illustrates a vertical sample when sampling from the side (e.g., through the sample). The plume leaves the surface of the substrate, is captured by the gas flow, rotates 90 degrees, and enters the plasma, which can be induced upward or downward.

[0066] The device of the present application may include one or more of the components described below.

[0067] An embodiment comprises a method and system for laser ablation mass cytometry analysis, and an apparatus for carrying out said method, said system and method comprising the following steps: inducing pulses of a laser beam to a sample to generate a plume of the sample for each pulse; distinctly capturing each plume for each pulse; delivering each of the distinctly captured plumes to an ionization system; and ionizing each of the distinctly captured and delivered plumes in the ionization system to generate ions for mass analysis. In various embodiments, the apparatus comprises: a laser ablation system for generating an ablated plume from a sample; and a delivery conduit configured to connect the laser ablation system to the ionization system of the apparatus. In some embodiments, the delivery conduit may have an inlet located within the laser ablation system, and the inlet may be configured to capture the ablated plume when the ablated plume is generated. To deliver the captured ablated plume into the ionization system, a gas inlet may be connected to the inlet of the delivery conduit to allow gas to pass through. If the ionization system is an ICP, and the outlet of the conduit is located directly within the plasma of the ICP, the transfer conduit may be referred to as an injector. The components of the laser ablation system, the ionization system, and the mass spectrometer are discussed individually in more detail below. As previously mentioned, the focus of the present invention is a variation on the transfer conduit connecting the laser ablation system to the ionization system.

[0068] transfer conduit

[0069] A transfer conduit forms a link between a laser ablation system and an ionization system and enables the transport of a 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 may be formed, for example, by drilling through a suitable material to create a lumen for the passage of the plume (e.g., a lumen having a circular, rectangular, or other cross-section). The transfer conduit sometimes has 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 may taper at the ends. The transfer conduit sometimes has a length in the range of 1 cm to 100 cm. In some embodiments, the length is 10 cm or less (e.g., 1 to 10 cm), 5 cm or less (e.g., 1 to 5 cm), or 3 cm or less (e.g., 0.1 to 3 cm). In some embodiments, the transfer conduit lumen is straight along the entire or nearly entire distance from the ablation system to the ionization system. In some embodiments, the transfer conduit lumen is not straight over the entire distance and changes orientation. For example, the transfer conduit may rotate gradually 90 degrees. This configuration causes the plume generated by the ablation of the sample in the laser ablation system to initially move in a vertical plane, while the axis of the transfer conduit inlet moves straight upward, and moves horizontally when approaching the ionization system (e.g., an ICP torch typically oriented horizontally to utilize convective cooling). In some embodiments, the transfer conduit is straight for a distance of at least 0.1 cm, at least 0.5 cm, or at least 1 cm from the inlet opening where the plume enters or is formed. In some embodiments, the transfer conduit is configured to minimize the time required to transfer material from the laser ablation system to the ionization system.

[0070] The injector of the device may include the delivery conduit described herein.

[0071] sample cone inlet

[0072] A transfer conduit contains an inlet within a laser ablation system, which receives sample material ablated from the sample into the laser ablation system and transfers it to an ionization system. In some cases, the laser ablation system inlet is the source of all gas flow along the transfer conduit to the ionization system. In some cases, the laser ablation system inlet receiving material from the laser ablation system is an aperture in the conduit wall, and a second “transfer” gas flows along the conduit from a separate transfer flow inlet (as disclosed, for example, in WO2014146724 and WO2014147260). In such cases, the transfer gas forms a significant proportion, and in many cases, most of the gas flow flows to the ionization system. A component comprising the transfer flow inlet and the laser ablation system inlet, where the transfer piping carrying the ablated sample material toward the ionization system begins, may also be called a flow cell (as in WO2014146724 and WO2014147260).

[0073] The transfer flow satisfies at least three of the following tasks: the transfer flow equalizes the plume entering the transfer conduit in the direction of the ionization system and prevents the plume material from coming into contact with the sidewalls of the transfer conduit; the transfer flow forms a “protective zone” over the sample surface to ensure that the ablation plume is performed under a controlled atmosphere; and the transfer flow increases the flow rate within the transfer conduit. In some embodiments, the viscosity of the capture gas is lower than the viscosity of the primary transfer gas. This helps to confine the plume of the sample material within the capture gas at the center of the transfer conduit and minimize the diffusion of the plume of the sample material downstream of the laser ablation system (since at the center of the flow, the transport rate is more constant and nearly a single rate). The gas(s) may be, for example, argon, xenon, helium, nitrogen, or a mixture thereof, but are not limited thereto. In some embodiments, the transfer gas is argon. Argon is particularly well suited for stopping the diffusion of a plume before it reaches the walls of the delivery conduit (in addition, argon also helps improve instrument sensitivity in devices where the ionization system is an argon gas-based ICP). The capture gas is preferably helium. However, the capture gas may be replaced with or contain other gases such as hydrogen, nitrogen, or water vapor. At 25°C, the kinetic viscosity (dynamic viscosity / density) for argon is approximately 1.3E -5 m 2 While it is / s, helium is about 1.2E -4 m 2 / s. As such, the difference in kinetic viscosity values ​​for argon and helium can be at least 5 times, or at least 10 times. In some embodiments, the capture gas is helium, and the transfer gas is helium.

[0074] Using a sample cone minimizes the distance between the target and the conduit containing the transfer gas flow. Because the distance the capture gas travels through the cone point is reduced, sample capture is improved, turbulence is reduced, and consequently, plume diffusion of the ablated sample material is decreased. Therefore, the inlet of the transfer conduit is the aperture at the tip of the sample cone. The cone protrudes into the ablation chamber.

[0075] Another type of asymmetric structure is a cone formed of two elliptical halves, which share a common height (z) and one base diameter (x diameter), but have different bases (y diameter) (or one elliptical half and one circular half).

[0076] All structures adopted above may exist as a single asymmetric sample cone as used in the present invention. For example, the cone may be asymmetrically cut and formed into two different elliptical cone halves, the cone may be asymmetrically cut and may include one or more orifices, etc.

[0077] Accordingly, the sample cone is configured to capture all or part of the plume of material ablated from the sample in a laser ablation system. The sample cone is positioned operably close to the sample by manipulating the sample within the laser ablation system, for example, using a movable sample carrier tray, as described in more detail below. As previously mentioned, the plume of the ablated sample material enters the delivery conduit through an aperture at the narrow end of the sample cone. In some embodiments, the diameter of the aperture is a) adjustable; b) has a size to prevent perturbation of the ablated plume as it passes into the delivery conduit; or c) is approximately equal to the cross-sectional diameter of the ablated plume. In some embodiments, the diameter of the aperture is about 100 μm to 1 mm. For example, the diameter of the aperture is about 200 μm to 900 μm, e.g., 300 μm to 800 μm. In some embodiments, the diameter of the aperture is about 500 μm to 700 μm. In some embodiments, the diameter of the aperture is about 500 μm. In some embodiments, the diameter of the aperture is about 700 μm.

[0078] tapered conduit

[0079] In a tube with a smaller inner diameter, gas traveling at the same flow rate moves at a faster speed. Therefore, by using a tube with a smaller inner diameter, the plume of ablated sample material carried by the gas flow can be transported more rapidly across a defined distance (e.g., from the laser ablation system to the ionization system within a transfer conduit) at a given flow rate. One of the key factors determining how quickly an individual plume can be analyzed is how much of the plume has diffused from the moment the plume is generated by ablation until the component ions of the plume are detected as mass spectrometer components of the device (transient time at the detector). Thus, by using a narrow transfer conduit, the time between ablation and detection is reduced, thereby reducing significant diffusion. This results in less time for diffusion to occur and ultimately reduces the transient time for each ablation plume at the detector. A shorter transient time means that more plumes can be generated and analyzed per unit time, thereby enabling the production of higher-quality images and / or faster generation.

[0080] The taper may involve a gradual change in the inner diameter of the delivery conduit along the aforementioned portion of the length of the delivery conduit (i.e., the inner diameter of the conduit was a cross-section taken along the portion where the inner diameter decreases from one end of the portion facing the inlet (at the end of the laser ablation system) to the outlet (at the end of the ionization system). As illustrated in FIGS. 3b and 7c, the tapering variation for the delivery conduit is applicable to all embodiments of the device described herein, regardless of whether it includes an injector inlet, a sample cone, or any other structure directly at the end of the ionization system inlet of the delivery conduit. The larger volume of the conduit prior to the taper facilitates the confinement of the material produced by ablation. When ablated particles fly from the ablation spot, they travel at high speeds. Friction within the gas slows these particles, but the plume can still diffuse to a scale of less than a millimeter to a millimeter. Allowing sufficient distance to the wall helps the flow confine the plume near the center.

[0081] Given that the plume spends more time in the longer section of the delivery conduit with a narrower inner diameter, the wide inner diameter section is only short (about 1 to 2 mm) and therefore does not contribute significantly to the overall transient time. Thus, the larger inner diameter section is used to capture ablation products and confine them near the central streamlined section where the gas flow velocity is more uniform, while the smaller inner diameter conduit is used to rapidly deliver these particles to the ionization system. The plume expands to a predetermined amount, at least partially determined by the particle size and the selection of the gas. The beginning of the injection conduit can be sized so that most of the plume falls close to the central streamlined section, so that the plume does not expand under the influence of the development of a parabolic flow profile. The injector may include a tapered section of the injection conduit after the beginning, where the diameter narrows, for example, to improve the delivery velocity.

[0082] In some embodiments, the tapered portion begins within 50 mm from the ionization system inlet in the direction of the delivery conduit. In some embodiments, the tapered portion begins within 40 mm from the ionization system inlet, e.g., within 30 mm, within 20 mm, within 15 mm, or within 10 mm from the ionization system inlet. In some embodiments, the tapered portion begins within 5 mm, within 4 mm, within 3 mm, within 2 mm, or within 1 mm downstream of the ionization system inlet. In some embodiments, the tapered portion begins 1 to 2 mm downstream of the ionization system inlet.

[0083] The tapered portion between the large inner diameter portion and the small inner diameter portion can be made smooth enough to avoid the occurrence of turbulence. For example, the tapered portion may have an angle of at least 5 degrees. In some embodiments, the angle of the taper may be at least 10 degrees, e.g., at least 15 degrees, at least 20 degrees, at least 25 degrees, or 30 degrees or more, e.g., 60 degrees. In some embodiments, the taper is less than 40 degrees, e.g., less than 30 degrees, less than 25 degrees, less than 20 degrees, less than 15 degrees, or less than 10 degrees. In some embodiments, the taper is less than 8 degrees, e.g., 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 angle of the taper is 10 to 30 degrees. In some embodiments, the angle of the taper may increase or decrease along the length of the taper.

[0084] In some embodiments, the length of the taper is at least 5 mm, e.g., at least 10 mm, at least 20 mm, at least 30 mm, at least 40 mm, or at least 50 mm, or at least 100 mm. In some embodiments, the length of the taper is less than 10 mm, e.g., less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, or 1 mm or less.

[0085] The inner diameter of the delivery conduit may be x millimeters (mm) at the inlet end of the conduit, but may taper fivefold to x / 5 mm near the outlet end (e.g., 4 mm at the inlet end and 800 μm at the outlet end). In some embodiments, the tapered portion reduces the inner diameter of the delivery conduit by less than five times, e.g., 4 times or less, 3 times or less, or 2 times or less. The inner diameter is a measure of the longest cross-section over the entire conduit. For example, if the conduit is circular, the inner diameter is simply the diameter of the circle, but if the conduit is rectangular, the inner diameter is the diagonal. In some embodiments, the inner diameter of the conduit after the tapered portion is less than 2 mm, e.g., less than 1.5 mm, less than 1.25 mm, less than 1 mm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, or less than 500 μm. In some embodiments, the inner diameter of the conduit after the tapered portion is 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less.

[0086] The diameter of the narrow inner section is limited by the diameter corresponding to the occurrence of turbulence. The Reynolds number can be calculated for a round tube and a known flow. Generally, a Reynolds number exceeding 4000 indicates turbulence and should therefore be avoided. A Reynolds number exceeding 2000 indicates transient flow (between non-turbulent and turbulent) and may therefore be desirable to avoid. Given the mass flow of the gas, the Reynolds number is inversely proportional to the diameter of the conduit. Thus, in some embodiments, the inner diameter of the narrow section of the delivery conduit is less than 2 mm, e.g., less than 1.5 mm, less than 1.25 mm, or less than 1 mm, but the flow of 4 liters of helium per minute within the conduit is greater than the diameter having a Reynolds number greater than 4000.

[0087] Rough or even angled edges at the transition between sections of a conduit with a constant diameter and a tapered section can cause turbulence in the gas flow. Therefore, in some embodiments, the transition into and from the tapered section must have smooth edges suitable for suppressing the occurrence of turbulence. For example, the edges may be rounded or chamfered.

[0088] A device including a tapered conduit may also include a sample cone (optionally asymmetric). As understood by those skilled in the art, the tapered conduit may be used in any of the devices described herein, using alternative delivery conduit arrangements, such as those illustrated in FIGS. 2 through 10 and discussed in detail in the following section.

[0089] sacrificial flow

[0090] As the flow rate increases, the risk of turbulence in the conduit increases. This is particularly true when the transfer conduit has a small inner diameter (e.g., 1 mm or less than 1 mm). However, the inventors have discovered that it is possible to achieve high-speed transfer (up to 300 m / s and above) in transfer conduits having a small inner diameter when a light gas, such as helium or hydrogen, is used instead of argon, which has traditionally been used as the gas transfer flow. In a given embodiment, a mixture of gases, mainly comprising helium or hydrogen, is used.

[0091] High-speed delivery poses a problem insofar as the plume of the ablated sample material passes through the ionization system without an acceptable level of ionization occurring. The ionization level may drop because the increased flow of cooling gas reduces the temperature of the plasma at the tip of the torch. If the plume of the sample material is not ionized to an appropriate level, information is lost from the ablated sample material, as the corresponding components (including any labeled atoms / element tags) cannot be detected by a mass spectrometer. For example, a sample may pass through the plasma very quickly at the tip of the torch in an ICP ionization system, and the plasma ions do not have sufficient time to act on the sample material to ionize it. The inventors have discovered that these problems caused by high flow rates and high-speed delivery in a narrow-diameter delivery conduit can be resolved by introducing a flow sacrifice system at the outlet of the delivery conduit. The flow sacrifice system is configured to receive the flow of gas from the delivery conduit and to allow only a portion of that flow (the central portion of the flow containing any plume of the ablated sample material) to pass into an injector leading to the ionization system. To facilitate the dispersion of gas from the transfer conduit within the flow sacrifice system, the transfer conduit outlet can be flared out in a trumpet shape.

[0092] Since the flow sacrifice system is located close to the ionization system, the length of the tube (e.g., injector) leading from the flow sacrifice system to the ionization system is short (e.g., about 1 cm in length compared to the length of a transfer conduit, which is typically several tens of cm, e.g., about 50 cm). Therefore, the lower gas velocity within the tube leading from the flow sacrifice system to the ionization system does not significantly affect the overall transport time, because the relatively slower part of the overall transport system is much shorter.

[0093] Accordingly, the present invention provides a device comprising the following:

[0094] (i) A laser ablation system configured to generate a plume of sample material from a sample;

[0095] (ii) an ionization system configured to receive the material removed from the sample by a laser ablation system and to ionize the material to form elemental ions;

[0096] (iii) a mass spectrometer that receives elemental ions from the above ionization system and analyzes the elemental ions,

[0097] Here, the laser ablation system and the ionization system are combined together by a delivery conduit and a flow sacrifice system, and

[0098] A delivery conduit is configured to transport a flow of gas containing a plume of ablated sample material from the inlet of the laser ablation system to the outlet of the flow sacrifice system, and

[0099] The flow sacrifice system includes a chamber comprising the following:

[0100] (a) Outlet of the delivery conduit;

[0101] (b) an ionization system inlet positioned to receive a sample material from a transfer conduit outlet and introduce the sample material into the ionization system; and

[0102] (c) Sacrifice flow outlet,

[0103] Here, the flow sacrifice system is configured to reduce the gas flow entering the ionization system through the ionization system inlet compared to the gas flow entering the flow sacrifice system through the transfer conduit by diverting a portion of the gas flow entering the flow sacrifice system from the sacrifice flow outlet, and

[0104] The outlet of the delivery conduit within the flow sacrifice system unfolds arbitrarily into a trumpet shape.

[0105] In some embodiments, the ionization system inlet is positioned coaxially with the outlet of the transfer conduit (because a plume of sample material delivered along the conduit is entrained within the center of the transfer flow) to maximize the transfer of material from the transfer conduit, through the flow sacrifice system to the ionization system inlet, and then to the injector of the ionization system. In some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the ionization system inlet is less than 2:1, e.g., 1.5:1 or 1:1. In some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the ionization system injector is less than 2:1, e.g., 1.5:1 or 1:1. In some embodiments, the inner diameter of the ionization system injector (or the inlet of the ionization system) is larger than the inner diameter of the transfer conduit. For example, in some embodiments, the ratio of the inner diameter of the transfer conduit to the inner diameter of the ionization system inlet is less than 1:1, e.g., 1:1.5 or 1:2. In some embodiments, the ratio of the inner diameter of the delivery conduit to the inner diameter of the injector of the ionization system is less than 1:1, for example, 1:1.5 or 1:2.

[0106] In most arrangements, significantly increasing the diameter of the tube (e.g., injector) passing from the flow sacrifice system to the ionization system as a method of reducing the gas velocity by volumetric flow rate is undesirable or, in some cases, possible. For example, if the ionization system is ICP, the conduit forms an injection tube from the flow sacrifice system to the center of the ICP torch. When an injector with a wider inner diameter is used, signal quality is reduced because the plume of the ablated sample material cannot be precisely injected into the center of the plasma (the most efficient ionization part of the plasma). Strongly preferred is an injector with an inner diameter of 1 mm or even narrower (e.g., an inner diameter of 800 μm or less, e.g., 600 μm or less, 500 μm or less, or 400 μm or less). Other ionization techniques depend on the material to be ionized within a relatively small volume in three-dimensional space (since the energy density required for ionization can only be achieved in a small volume); therefore, a conduit with a wider inner diameter implies that a large portion of the sample material passing through the conduit is outside the range where the energy density is sufficient to ionize the sample material. Accordingly, a narrow-diameter tube connecting from the flow sacrifice system into the ionization system is also used in devices equipped with a non-ICP ionization system. As previously mentioned, if the plume of the sample material is not ionized to an appropriate level, information is lost from the excised sample material, because the components of the sample material (including any labeled atoms / element tags) cannot be detected by the mass spectrometer.

[0107] Rough or even angled edges at the transition between the portions of the conduit with a constant diameter and the flared portion at the outlet can cause turbulence in the gas flow. Therefore, in some embodiments, the transition into the flared portion must have smooth edges suitable for suppressing the occurrence of turbulence. For example, the edges may be rounded.

[0108] Pumping may be used to help ensure a desired split ratio between the sacrificial flow and the flow passing into the inlet of the ionization system. Accordingly, in some embodiments, the flow sacrificial system includes a pump attached to the sacrificial flow outlet. A regulating restrictor may be added to the pump to regulate the sacrificial flow. Accordingly, in some embodiments, the pump of the flow sacrificial system further includes a restrictor configured to regulate the flow of gas through the sacrificial flow outlet. In some embodiments, the flow sacrificial system includes a mass flow controller configured to control the restrictor.

[0109] When expensive gases are used, the gas pumped from the sacrificial flow outlet can be cleaned using a known gas purification method and recycled back into the same system. As previously mentioned, helium is particularly suitable as a transport gas, but since it is expensive; therefore, it is advantageous to reduce the loss of helium in the system (i.e., when helium is delivered into the ionization system and ionized). The flow sacrificial system divides the helium flow into a paraxial flow and a sacrificial flow. The sacrificial flow can be cleaned and recycled within the system, whereas the paraxial flow (the central portion of the flow carrying aerosolized particles from the ablation plume) will be delivered into the ionization system (e.g., into the plasma of an ICP torch). The helium in the paraxial flow is lost and recovered. Thus, in some embodiments, a gas purification system is connected to the sacrificial flow outlet of the flow sacrificial system. In some embodiments, the gas purification system provides a portion of the gas flowing into the device, for example, into the ablation chamber of a laser ablation system through an inlet and / or into a delivery conduit through an inlet.

[0110] As before, a larger transfer flow rate is delivered down the transfer conduit, and only the central portion of this flow can become part of the injector flow that enters the plasma of the ICP torch. Typically, helium gas is used as the transfer flow because, as previously mentioned, the properties of helium gas are highly suitable for the high-speed transport of plume material through long conduits (i.e., compared to argon, it is less likely to cause turbulence at the same flow rate). Even if a gas purification system that recycles helium from the sacrificial flow is included, the paraxial flow of helium continuing to the ionization system through the flow sacrificial system is lost.

[0111] Accordingly, in some embodiments, the flow sacrifice system is configured to reduce the gas flow passing into the ionization system inlet (e.g., the injector of an ICP torch ionization system) to less than 1 Lpm, e.g., 0.5 Lpm or less, 0.4 Lpm or less, 0.3 Lpm or less, or 0.2 Lpm or less. In some embodiments, the ICP injector includes a second inlet into which gas can flow to supplement the flow rate within the injector. In some embodiments, the second inlet includes a concentric tube around the injector attached to the ionization system inlet, which introduces supplementary gas as a sheath flow around the sample-containing gas flow from the flow sacrifice system. This supplementary flow inlet is different from the flow of argon gas provided to the intermediate and outer concentric tubes supporting the plasma. Such an injector may also be referred to as a double concentric injector.

[0112] An apparatus comprising a flow sacrifice system may include a sample cone (optionally an asymmetric sample cone) or a tapered conduit as described above. In some embodiments, as described above, the apparatus comprises a flow sacrifice system, a sample cone (optionally an asymmetric sample cone), and a tapered conduit. As understood by those skilled in the art, the flow sacrifice system may be used in any of the apparatuses described herein using alternative delivery conduit arrangements.

[0113] Laser ablation system

[0114] A laser ablation system, also referred to as an “ablation cell” or “laser ablation source,” accommodates a sample during ablation. Generally, the ablation cell includes a laser-cleared window that allows laser energy to strike the sample. Optionally, the ablation cell includes a stage for fixing the sample to be analyzed. In some embodiments, the stage is movable in the xy or xyz dimensions. In the drawings and embodiments herein, the laser ablation system is sometimes depicted as an open arrangement. However, it will be understood that such configurations are merely for illustrative purposes, and that some form of appropriate enclosure exists to prevent contamination or infiltration from the surrounding environment. For example, a chamber comprising a gas inlet and / or an optical port may be arranged around the laser ablation system to provide an isolated environment suitable for capturing and delivering the ablated plume for mass analysis. The gas inlet and optical port(s) are positioned so that the orientation of the laser beam, sample, plume expansion, and delivery conduit is suitable for the method and apparatus disclosed herein. It will be understood that the ablation cell is generally hermetic (except for the designed outlet and port). Even if the ablation chamber contains air before operation, the ablation chamber can be sufficiently isolated from the environment so that the gas flow (e.g., capture of gas through the sample chamber and / or delivery of gas into the injector tube of the ablation cell) is sufficient to reduce contamination from the air driving the sample run. However, the initial presence of air may provide a level of humidity that affects sensitivity and / or signal drift. The laser ablation system of the present application may have the gas flow illustrated in one or more of FIG. 2, FIG. 3, or FIG. 4.

[0115] The lasers used for laser ablation according to the present invention generally fall into the following three categories: femtosecond pulsed lasers, deep UV pulsed lasers, and pulsed lasers having a wavelength selected for high absorption in the ablated material (“wavelength-selective lasers”). Deep UV lasers and wavelength-specific lasers may operate with nanosecond or picosecond pulses. Each class of lasers has unique disadvantages and advantages and can be selected based on the specific application. In some embodiments, the laser is a femtosecond pulsed laser configured to operate at pulse rates of 10 to 10,000 Hz. Femtosecond lasers are known (see, for example, the literature by Jhanis et al. (“Rapid bulk analysis using femtosecond laser ablation inductively coupled plasma time-of-flight mass spectrometry”).

[0116] Femtosecond lasers enable the laser ablation of virtually any material, provided that power density is sufficient as the sole prerequisite for laser ablation. This can be achieved even with relatively low pulse energies when the beam is closely focused, for example, to a diameter of 1 μm and has a short duration (time-oriented). Additionally, deep UV lasers can ablate many classes of materials, as most commonly used materials absorb deep UV photons. Wavelength-selective laser ablation utilizes a laser with a specific wavelength that targets the absorbance of the substrate material. The advantages of wavelength-specific lasers may be the cost and simplicity of the laser and optical systems, although the spectrum of the substrate material is more limited. Suitable lasers may have different operating principles, such as solid-state lasers (e.g., Nd:YAG lasers), excimer lasers, fiber lasers, and OPO lasers.

[0117] A useful characteristic of femtosecond laser radiation is that it is absorbed only when it reaches a critical power density. Therefore, converging femtosecond laser radiation can pass through thicker parts of a material without being absorbed or causing any damage, while removing the same material at the surface where the focus occurs. The focus can then be gradually shifted within the material as the sample layer is ablated. Nanosecond laser pulses can be partially absorbed by the substrate, but they can still be used for ablation because the energy density at the focus is highest (as long as it is sufficient for ablation).

[0118] The spatial resolution of the signal generated in this manner depends on two main factors: (i) the laser spot size at which the signal is integrated across the total area being ablated; and (ii) the rate at which the plume can be analyzed relative to the rate at which the plume is generated, as discussed above, to avoid signal overlap in a continuous plume. The distance referred to as the spot size corresponds to the longest internal dimension of the beam, for example, to a beam with a diameter of 2 μm for a circular beam, and to the length of the diagonal between opposing corners for a square beam. The laser pulse can be shaped using an aperture, homogenized using a beam homogenizer (if necessary), and focused using, for example, an objective lens to produce the desired spot size. Typically, the spot size is 100 μm or less, e.g., 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less. Exemplary spot sizes include diameters ranging from 0.10 to 3 μm (e.g., about 0.3 μm), 1 to 5 μm (e.g., about 3 μm), 1 to 10 μm (e.g., about 1, about 2, about 3, about 4, or about 5 μm), less than 10 μm, and less than 5 μm (or ablation regions of equivalent size of other shapes). In a specific embodiment, the laser system is configured to operate with a sufficiently focused laser pulse to ablate a sample region of about 1 μm, e.g., from 100 nm to about 1 μm.

[0119] To analyze individual cells, the laser within the laser ablation system has a spot size smaller than the size of these cells. While this size varies depending on the specific cells in the sample, generally, the laser spot will have a diameter of less than 4 μm, for example, within the range of 0.1 to 4 μm, 0.25 to 3 μm, or 0.4 to 2 μm. Thus, the laser spot may have a diameter of about 3 μm or less, about 2 μm or less, about 1 μm or less, about 0.5 μm or less, for example, about 400 nm or less, about 300 nm or less, about 200 nm or less, about 100 nm or less, etc. To analyze cells at a resolution smaller than the cell size, the present invention uses a laser spot size not larger than these cells, and more specifically, uses a laser spot size capable of ablating material at a resolution smaller than the cell size. Occasionally, single-cell analysis may be performed using a spot size larger than the cell size, for example, when cells are spread out on a slide and there are spaces between cells. Here, a larger spot size is used, and single-cell characterization can be achieved because the additional ablated area around the cell of interest does not contain additional cells. Therefore, the specific spot size used can be appropriately selected depending on the size of the cell being analyzed. Since cells in biological samples are unlikely to all be the same size, if sub-cell size resolution imaging is desired, the ablation spot size must be smaller than the smallest cell, provided that a constant spot size is maintained throughout the ablation procedure. A small spot size can be achieved by reducing the size of a wider laser beam and using near-field optics.A laser spot with a diameter of 1 μm corresponds to a laser focus of 1 μm (i.e., the diameter of the laser beam at the focus of the beam), but the laser focus can vary by more than ±20% due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and the change in total laser energy relative to the ablation threshold energy. For example, if a laser beam with a diameter of 25 μm is used and reduced in size by 25 times and applied to a tissue sample, a spot size of 1 μm diameter will be provided.

[0120] Ablation using such a small scale generates a very small amount of plume material, which ultimately keeps the plume size small. The smaller the plume, the more likely it is to remain in the middle of the capture flow without coming into contact with the walls of the delivery ablation cell or the delivery conduit. Ablation using a 1 μm scale also implies that the distance between the region where plume expansion slows and the surrounding gas becomes dominant and the ablated surface is very short. This distance can range from several micrometers to several hundred micrometers. In some versions of the invention, the capture flow exists where the plume stops expanding. Thus, for the sake of non-limiting examples, some of the attached drawings show the distance between the region where the capture flow is depicted as about 100 μm and the ablated surface.

[0121] While ablation using a 1 μm (or lower) scale is advantageous for certain applications (e.g., imaging), the method and device of the present invention are also useful when larger ablation spots are generated, for example, when ablation spots with a diameter in the range of about 5 to about 35 μm, e.g., 5 to 15 μm, 10 to 20 μm, 15 to 25 μm, 20 to 30 μm, and 25 to 35 μm are generated. In some applications where large ablation spots are generated, only a portion of the plume material is captured.

[0122] In some embodiments, the laser is positioned 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 being emitted from a surface (e.g., a laser lens or a mirror), wherein the surface may be oriented to guide the beam toward a specific location or a pattern of locations. For ease of description of the invention, the guided beam may be considered to have a specific orientation; the orientation of the beam may refer to an imaginary line that is aligned with the beam and extends past the actual beam (e.g., when the beam strikes an opaque surface). As becomes apparent from the context, references to the orientation or location of the laser beam sometimes refer to the orientation or location that the beam of a laser system, when the laser is in use and power is not supplied, would produce.

[0123] For rapid analysis of tissue samples, a high frequency of ablation, for example exceeding 20 Hz (i.e., ablation exceeding 20 times per second, resulting in obtaining more than 20 plumes per second), is required. In some embodiments, the laser ablation frequency is at least 40 Hz, for example, at least 50 Hz, or at least 100 Hz. In some embodiments, the laser ablation frequency is within the range of 40 to 2000 Hz, 40 to 1500 Hz, 40 to 500 Hz, 40 to 200 Hz, 40 to 150 Hz, or 75 to 150 Hz. An ablation frequency exceeding 40 Hz enables imaging of a typical tissue sample to be achieved within a reasonable time. The frequency at which laser pulses can induce a spot on a sample (assuming complete ablation of the material at that spot) and still be individually resolved determines how quickly pixels of an image can be acquired. Thus, if the laser pulse duration required to ablate material at a single point means that fewer than 5 pulses can be induced per second on the sample, it will take more than 2 days to study an area of ​​1 mm × 1 mm ablated with a spot size of 1 μm. At a speed of 40 Hz, it will take approximately 6 to 7 hours, with further reductions in analysis time required to further increase the pulse frequency. At these frequencies, where it is desirable to resolve each ablated plume individually, the instrument must be able to analyze the ablated material quickly enough to avoid substantial signal overlap between successive ablations. It is desirable that the overlap between signals originating from successive plumes has an intensity of <10%, more preferably <5%, and ideally <2%.The time required for the analysis of the plume will depend on the washing time of the ablation chamber (see the ablation chamber section below), the time of the plume's passage of the sample material to and through the ionization system (optimization of transport to the ionization system is discussed above), and the time required to analyze the ionized material. Each laser pulse can be correlated with a pixel on a sample image constructed in succession, as described in more detail below.

[0124] ablation chamber

[0125] An ablation chamber (which may also be referred to herein as a sample chamber) with a short wash time (e.g., 100 ms or less) is advantageous for use with the apparatus and method of the present invention. Cells with long wash times will limit the rate at which images can be generated or lead to overlap between signals due to continuous sample spots (e.g., see Kindness et al. [(2003) Clin Chem 49:1916-23], where the signal duration exceeds 10 seconds). Therefore, 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 having a wash time of 100 ms or less are known in the art. For example, Gurevich & Hergenroder [(2007) J Anal. At. Spectrom., 22:1043-1050] discloses an ablation chamber with a wash time of less than 100 ms. An ablation chamber is [(2013) Anal. Chem. [85:10107-16] is disclosed (see also WO 2014 / 146724), and the resection chamber of said document has a flushing time of 30 ms or less, enabling high resection frequency (e.g., greater than 20 Hz) and consequently rapid analysis. Another such resection chamber is disclosed with reference to WO 2014 / 127034.The ablation chamber of this document comprises a sample capture cell configured to be operablely arranged in proximity to a target (the sample capture cell described herein is an example of a delivery conduit inlet modification that can be combined with a tapered and flow-sacrifice modification of a delivery conduit as described above), wherein the sample capture cell comprises: a capture cavity having an opening formed on the surface of the capture cell and configured to receive target material emitted or generated from the laser ablation site through the opening; and a guide wall exposed within the capture cavity and configured to guide a flow of a carrier gas (also referred to herein as capture gas) within the capture cavity from the inlet to the outlet so that at least a portion of the target material received within the capture cavity can be delivered to the outlet as a sample. The volume of the capture cavity within the ablation chamber of reference WO 2014 / 127034 is 1 cm³. 3 Less than 0.005 cm 3 It may be less. Sometimes the ablation chamber has a flushing time of 25 ms or less, for example, 20 ms or 10 ms or less. A sample cone of the delivery conduit, for example, an asymmetric sample cone, can also help reduce the flushing time of the ablation chamber and is an alternative to the capture cell discussed herein.

[0126] A conventional LA-ICP-MS system including a laser ablation chamber is illustrated in FIG. 4. The ablation chamber may include a movable sample stage for positioning a sample. A gas source may provide a capture gas that flows through the ablation chamber and carries the ablation plume into an injection tube (also referred to herein as an injector or transfer conduit) leading to the ICP torch. The injection tube may have a flexible tube between the ablation chamber and the ICP torch, or it may be rigid and / or straight. In a certain embodiment, the injector may be orthogonal to the sample and collinear with the gas flow illustrated in FIG. 2 or FIG. 3. The LA-ICP-MS system may include one or more additional gas sources for supplying the transfer gas and / or internal and external gases (also referred to as auxiliary gas and plasma gas). In a certain embodiment, the injection tube may receive a supplementary gas downstream of the laser ablation plume introduction section to the injection tube. One or more gas flows may include hydrogen. For example, the gas flow may include water vapor or alcohol vapor, as further described herein. Alternatively or additionally, the gas flow may originate from a pre-mixed compressed gas source containing hydrogen (e.g., hydrogen gas, ammonia, or methane), as further described herein.

[0127] In a given embodiment, the laser ablation chamber may include a capture gas (which enters the chamber and carries the ablation plume into the injection tube) and optionally may additionally include a delivery gas (which enters an injector upstream of the ablation plume). The injector may additionally include a replenishing gas (i.e., a gas that replenishes the injector downstream of where the ablation plume enters the injector). The ICP torch may include an internal gas (i.e., an auxiliary gas flowing into the internal tube of the ICP torch) and an external gas (i.e., a plasma gas flowing into the external tube of the ICP torch). Additional gas flows may be present. In a given embodiment, hydrogen (e.g., hydrogen gas or water vapor) may be introduced into one or more of the gas flows, as further described herein.

[0128] ionization system

[0129] Sample materials can be ionized by various techniques, for example, in plasma. The use of ICP is suitable for IMS and IMC analysis. ICP is a plasma source that is energized by an electric current generated by electromagnetic induction. Typically, the plasma source is based on argon gas. For example, the ionization system may include an ICP torch. IMC using ICP in the ionization system has been reported, for example, in the literature of Giesen et al. [(2014) Nature Methods. 11:417-422] and Wang et al. [(2013) Anal. Chem. 85:10107-16].

[0130] Therefore, 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 (for example, if the plume of the sample material is still in molecular form or even in the form of an aerosol of particulate matter), the ionization system acts to break down the material into elemental ions as part of the ionization process.

[0131] mass spectrometer

[0132] As previously mentioned, the third component of the device is a mass spectrometer. The mass spectrometer to be used in the present invention may be selected based on the operator's requirements or a specific application. Exemplary types of mass spectrometers include quadrupole, time-of-flight (TOF), magnetic sector, high-resolution, single or multiple collector-based mass spectrometers.

[0133] The time required to analyze ionized material will depend on the type of mass spectrometer / mass spectrometer used for the detection of ions. For example, an instrument using a Faraday cup may be too slow to analyze fast signals, but since not all analyses require rapid signal analysis, a person skilled in the art will be able to appropriately select a mass spectrometer or mass spectrometer. Overall, the desired analysis rate (and thus the frequency at which ablation plumes can be examined) and the degree of multiplexing (the number of atoms to be monitored simultaneously or quasi-simultaneously) will determine the type of mass spectrometer(s) to be used (or, conversely, the selection of the mass spectrometer will determine the rate and multiplexing that can be achieved).

[0134] Generally, time-of-flight mass spectrometers are used to record rapid transient events along with the expected transit duration from a rapid laser ablation setup.

[0135] TOF detectors can simultaneously register multiple masses in a single sample. Although TOF mass spectrometers are generally unpopular for atomic analysis due to the compromises required to handle the space charge effects of TOF accelerators and flight tubes, the effectiveness of the technique can be improved by using them only to detect a subset of the range. For example, in mass cytometry and imaging mass cytometry, the range can be selected so that only ions of labeled atoms used to label target molecules in biological samples are detected, while other atoms (e.g., atoms with an atomic mass of less than 80) are excluded. This generates a less dense ion beam enriched with masses in the 80–210 Dalton range, which can be manipulated and focused more efficiently, thereby facilitating TOF detection and utilizing the high spectral scan rate of TOF. Thus, rapid analysis can be achieved by combining the selection of uncommon labeled atoms in a sample with TOF detection, ideally by using, for example, higher mass transition elements that have masses greater than those seen in unlabeled samples. Further details regarding mass cytometry analysis can be found in the literature by Tanner et al. [Cancer Immunol Immunother (2013) 62:955-965] and U.S. Patent No. 7,479,630, and in the literature by Giesen et al. [(2014) Nature Methods. 11:417-422] regarding imaging mass cytometry.

[0136] Additional variants of the present invention to which the device in use and the aforementioned delivery conduit modification may be applied

[0137] The device of the present invention may be used for the analysis or imaging of biological samples that may be on a transparent substrate. In imaging embodiments, the laser may generally be operated as a continuous train of pulses or a burst of pulses directed to different locations on the sample, referred to as “spots of interest” or “ablation sites or zones.” The pulses may be directed to spots of a set pattern, such as a raster for two-dimensional imaging. Alternatively, multiple individual spots may be ablated at different locations (e.g., corresponding to individual cells). In some embodiments, the laser emits a burst of pulses that generates plumes from the same pixel (i.e., the same location on the target). The ablation plumes generated by individual pulses within the burst are expected to fuse into one plume and move within the device in a manner distinct from plumes generated from other pixels. To distinguish individual pixels, the duration between bursts (pixel call signals, which can be a single pulse or 100 pulses) is maintained beyond a specific limit determined by the time spread of the ion signal (at the detector) from the individual pixel.

[0138] According to the present invention, each separate sample plume can be clearly analyzed by a mass spectrometer. In one embodiment, the device is configured such that the diffusion of the plume in the ablation cell (ablation system) and the delivery conduit is smaller than the diffusion occurring in the ionization system and the mass spectrometer. In one embodiment, the plume can be clearly analyzed by delivering each ablated plume to the ionization system within a period within the cumulative transit time of the plume and by ion detection by the mass spectrometer. This can be achieved by capturing each sample plume through the gas flow and under the delivery configuration such that the ratio between the plume expansion during the delivery period (i.e., delivery of the ablation plume from the ablation site to the plasma) and the expansion during the ion transit period (i.e., delivery of ions from the plasma to the mass spectrometer) is 1 or less.

[0139] Generally, the sample particle size limit at which an ionization system (e.g., ICP) can effectively vaporize and ionize for analytical detection is approximately 10 μm or less. Particles generated by laser ablation on a 1 μm scale are less than 1 μm and are highly suitable as ICP ion sources. For discrete particle analysis (which can be performed, for example, using the CyTOF® instrument from Fluidigm Canada Inc.), the typical rate at which these particles can be ionized and analytically detected may be a function of the cumulative extension or diffusion of the sample's passage time within the plasma while the particles are vaporized and ionized, or a function of the extension or diffusion of the ion's passage time between ICP and detection by the mass spectrometer. Typically, the cumulative extension or diffusion may last for approximately 200 μs. Consequently, for spatially separated particles of 10 μm or less, the analysis of each distinct particle can be achieved by delivering each particle to the ionization system (e.g., ICP) for a period of approximately 200 μs. In some embodiments, particles are delivered to an ionization system (e.g., ICP) within less than 200 μs or less than 150 μs. Thus, in a sample introduction system in which imaging of a biological sample can be performed by laser ablation, the laser system may be configured to operate with a sufficiently focused laser pulse to ablate a sample region within about 1 μm, for example, within the application time of a femtosecond pulsed laser. In this configuration, the ablated plume formed by each laser pulse may typically contain sample particles having dimensions of about 1 μm or less. Under specific conditions as described herein, these particles may be captured and delivered to satisfy the delivery period as needed, and then each distinct plume may be effectively shaped and ionized by an ionization system.

[0140] Furthermore, while operating the laser with a series of continuous pulses, such as when rasterizing across a sample surface for two-dimensional imaging, the distinctiveness of each plume and the spatial separation between each subsequent plume can be maintained between the plume formation zone and the vaporization and ionization points at the ionization system ion source. For example, when a plume is transported through a conduit, particles within the plume may diffuse radially and expand outward before entering the ionization system (e.g., plasma in ICP). The diffusion of particles generated in the plume may depend on the diffusion coefficient, the velocity profile of the transport flow, and the distribution of particle density when the particles are formed and as they evolve while passing through the ionization system. For example, a femtosecond laser ablation spot size of 1 μm can generate a plume having an initial cross-sectional diameter of about 100 μm or less before further diffusion during passage. The degree of plume diffusion may also be a function of the dimensions of the ablated particles; Larger particles tend to have lower diffusion spread, but their higher momentum results in potential losses due to contact with the inner walls of the delivery conduit / injection tube. Therefore, it is desirable to minimize plume diffusion and / or deliver the plume to the ionization system within a sufficient time for vaporization and ionization before the degree of diffusion exhibits any troublesome effects.

[0141] Accordingly, in various embodiments, the use of a laser to ablate a 1 μm sample spot and efficiently transport a plume so that diffusion is maintained within the inner diameter of the delivery conduit / injection tube can be achieved by the exemplary arrangement described herein and illustrated in the attached drawings.

[0142] For a given laser ablation system and a given sample, the ablated plume expands after laser ablation until it reaches a characteristic volume referred to as the “sampling volume.” It is desirable to configure the system to minimize the sampling volume and increase the speed at which the gas flow transports the plume away from the sampling volume. The combination of a small sampling volume and a fast gas flow reduces the time for plume transport to diffuse into the delivery conduit / injector. The sampling volume can be described by the plume shell when the plume expansion rate of any of the dimensions drops substantially (about 10 times) below the speed of sound of the surrounding gas medium. Without limitation, an exemplary sampling volume is 10 -6 mm 3 to 10 mm 3 It can be in the range of. Often, the sampling volume is 0.001 mm 3 to 1 mm 3 It is a range. If a capture flow is present, it flows into at least a portion of the sampling volume and carries at least a portion of the plume into the delivery conduit / injector, whereby the capture flow can be carried by the delivery flow to an ionization system (e.g., ICP). The velocity of the capture flow upon entering the sampling volume is preferably substantial (e.g., >1 m / s, >10 m / s, >100 m / s, or >500 m / s). In some embodiments, the velocity of the capture flow upon entering the sampling volume can be estimated by measuring the velocity of the capture flow delivered into the delivery conduit / injector (e.g., through the delivery conduit / injector aperture). 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 discharged, the plume will continue to expand and spread, undesirably filling the entire resection cell.

[0143] In one embodiment, the present invention provides a laser ablation configuration in which a laser beam is guided toward 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.

[0144] In one embodiment, the present invention provides a laser ablation configuration for “through-target” ablation. In this configuration, a pulse of a laser beam is guided through a transparent target, and a sample plume (“ablated plume” or “plume”) is formed downstream of the beam into a delivery conduit / injector. Through-target illumination is advantageous for optimizing the extension of transit time due to the removal of optical elements (windows, objective lenses, etc.) from the straight path of the plume. In one embodiment, the present invention provides a laser ablation system comprising: (a) a laser capable of generating laser illumination; (b) a laser ablation cell (or laser ablation system) into which a transparent target can be introduced and a delivery conduit / injector having an opening into which an ablated plume can enter, wherein the laser illumination originates from a surface on one side of the transparent target, and the delivery conduit / injector opening is on the other side. Other features that may be included in the system are described throughout the entire disclosure, including examples.

[0145] In a given embodiment, a smaller ablation spot size (higher resolution) can be achieved with a high numerical aperture lens such as an immersion lens. Such an immersion lens can be configured for penetrating-target ablation (e.g., of thin samples such as tissue sections with a diameter of less than 500 nm).

[0146] Accordingly, in operating one device according to the present invention, a sample is inserted into the device and sampled, and an ionized substance is generated using a laser system comprising an optical device in which laser radiation is guided onto the sample through an immersion lens (the sampling may generate vapor / specific substance, which is subsequently ionized by an ionization system), and ions of the sample substance are transferred into a detector system.

[0147] The present invention overcomes the limitations of traditional IMC and IMS by utilizing an immersion medium. The immersion medium has a refractive index greater than 1.0 and is placed between the objective lens and the sample stage. In this way, the device of the present invention achieves an numerical aperture greater than 1.0, so that the spot size of the laser is less than 200 nm, less than 150 nm, or less than 100 nm. Accordingly, the present invention provides an imaging mass cell analysis device having a spatial resolution of 200 nm or more, 150 nm or more, or 100 nm or more.

[0148] Therefore, during operation, the sample stage secures the sample; typically, the sample rests on a sample carrier, and the same stage secures the sample carrier. Then, laser radiation is guided to the sample through the device's optics, objective lens, and immersion medium, where the radiation ablates material from the sample.

[0149] To achieve optimal focusing conditions for a laser, the immersion medium of the present invention has a refractive index greater than 1.00, e.g. 1.33 or higher, 1.50 or higher, 2.00 or higher, or 2.50 or higher.

[0150] Additionally, as further discussed herein, in order to reconstruct an image of a single layer of biological cell thickness (or less than the thickness), or to read thicker specimen layers layer by layer and generate a 3D image, the sample preferably has a thickness of 100 μm or less, e.g. 10 μm or less, 5 μm or less, 2 μm 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 immersion lens.

[0151] When a liquid immersion medium is used, the sample must be positioned on the opposite side of the sample carrier relative to the liquid medium so that the carrier gas can collect the ablated material (as shown in Fig. 3). Therefore, a through-sample carrier ablation technique must be applied here. This offers the additional advantage of a smaller achievable working distance for the ablated material collection hardware and eliminates the need to bend the transport conduit between the sample chamber and the detector. This also reduces transient time, thereby increasing the achievable ablation rate in spots per second.

[0152] Accordingly, the present invention provides an apparatus in which the solid immersion medium is a hemispherical solid immersion lens or a Weierstrass solid immersion lens. A biological sample may be mounted on the solid immersion material on the opposite side of the sample stage. The stage on which the sample is mounted may be made of a material having the same refractive index as the solid immersion lens, and the solid immersion lens may be thinned by an amount equal to the thickness of the substrate to maintain the focal position.

[0153] In various embodiments, the sample of interest may be configured for laser ablation by using a sample formatted to be compatible with a transparent target. The sample may be placed on a transparent substrate, incorporated within a transparent substrate, or formed as a transparent target. Suitable laser-transparent substrates may 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 a maximum of 3 mm. In some embodiments, the substrate is crypted (see, for example, U.S. Patent Publication No. 2012 / 0061561 of Antonov, A. and Bandura, D. incorporated herein by reference). In such configurations, a pulse of the laser beam is guided through the transparent target, and a sample plume (“ablated plume” or “plume”) is formed downstream of the beam into a delivery conduit / injector.

[0154] A delivery conduit (i.e., an injection tube) may have an inlet configured to capture an ablated plume; for example, the inlet may be formed as a sample cone having a small opening or aperture. In this configuration, the sample cone may be located near the area or zone where the plume is formed. For example, the opening of the sample cone may be located 10 μm to 1000 μm from a transparent target, for example, about 100 μm away from a transparent target. Consequently, the ablated plume may be generated and formed at least partially within the extended area of ​​the cone. In some embodiments, the aperture of the gap (including the angle) and / or the diameter of the dimensions may be adjustable to allow optimization under various conditions. For example, for a plume having a cross-sectional diameter of 100 μm, the diameter of the aperture may be about 100 μm in size with sufficient spacing to prevent perturbation of the plume as it passes through.

[0155] In this configuration, the transfer conduit extends downstream of the sampling cone to accommodate the ablated plume, facilitating the movement of the plume and preserving the spatial distinctness of each subsequent plume as a function of the laser pulse. Thus, a gas flow is introduced to guide the plume through the aperture of the sampling cone to explicitly capture each plume (capture flow), and an additional gas flow may be introduced into the transfer conduit / injector to transfer each explicitly captured plume toward the ionization system (transfer flow or cover flow). Another function of the transfer flow or cover flow is to prevent particles generated from the plume from coming into contact with the walls of the transfer conduit / injector. The gas(s) may be, for example, argon, xenon, helium, nitrogen, or a mixture thereof, but are not limited thereto. In some embodiments, the gas is argon. The capture flow gas and the transfer flow gas may be the same or different.

[0156] Selecting or determining a gas flow rate suitable for the present invention is within the capability of those skilled in the art guided by this disclosure. The total flow through the transfer conduit is typically determined by the requirements of the ionization source (e.g., an ICP ionization source). The laser ablation setup needs to provide a flow that meets these requirements. For example, the transfer conduit may optionally have an inner diameter of 1 mm or less, with a cumulative gas flow rate of about 1 liter per minute (0.1 liters per minute capture flow + 0.9 liters per minute transfer flow). It is expected that transfer conduits of smaller or larger diameters, with correspondingly selected gas flow rates, can be applied to various presented geometries with similar expected results. Conditions for maintaining non-turbulent gas kinetics within the transfer conduit are desirable to preserve the distinctiveness of each separate ablated plume.

[0157] As described herein, given the specific configuration of the elements (e.g., gas inlet location, aperture, delivery conduit characteristics, and specific configuration of other elements), the capture and delivery flow rates are selected so that each ablated plume is delivered to the ionization system (e.g., ICP) within a period within the cumulative transit time of the plume between the detection of the plume by the mass spectrometer and the ionization system. This can be achieved by capturing each sample plume through the gas flow and under the delivery configuration such that the ratio between the plume expansion during the delivery period and the expansion during the ion transit period is 1 or less. This is a time expansion (or time diffusion) of the important transit signal. ICP-MS devices (e.g., CyTOF® ICP-TOF instrument, Fluidigm Canada Inc.) feature a unique signal expansion. In the case of laser ablation, the operation of injecting a single plume may or may not be faster compared to the time diffusion on the ICP-MS itself. Plume diffusion prior to ionization depends on the design of the laser ablation system, particularly the ablation chamber and the delivery conduit. It is desirable that the laser ablation system and the delivery conduit do not diffuse the original ablation plume beyond the intrinsic expansion of the rest of the device. This condition ensures that the spike in the detection signal generated by the ablation plume is as sharp (in terms of time) as can be for the selected device. If the plume's expansion is much longer than, for example, the expansion in an ICP-MS system, the laser ablation event caused by a single pulse will emerge much more broadly from the detector. However, if the expansion in the laser ablation section is smaller than the device's expansion, the device's expansion will be dominant over the total expansion. Therefore, the device's expansion can be measured using a calibration bead, followed by the total expansion caused by a single laser pulse, and these two numbers can be compared. If the expansion caused by laser ablation is smaller than the device's expansion, the total expansion will be less than twice the device's expansion.

[0158] Characteristic instrument time extensions can be measured experimentally, for example, using labeled cells or calibration beads. Whenever a single bead enters a mass cytometer (e.g., a CyTOF® ICP-TOF instrument), the bead undergoes vaporization and ionization within the plasma and then passes through the mass spectrometer until the signal reaches the detector. Transient events are detected and used to record information regarding a specific bead, such as the width of the transient signal (indicating the time elapsed from a single event) and the diffusion value starting from the ICP source and ending at the detector.

[0159] In some embodiments, the device is configured to allow a time spread of 10 to 1000 microseconds for a defined path between the ion detector of the mass spectrometer and the sample.

[0160] 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 generally at the lower end of the range (e.g., about 0.1 Lpm). Typical transfer flow rates are in the range of 0.1 to 1 Lpm. The optimal transfer flow rate can be determined experimentally, but is generally at the upper end of the range (e.g., about 0.9 Lpm). In some embodiments, the capture flow rate is lower than the transfer flow rate. In some cases, for example, when the capture flow rate is approximately 1 Lpm, the transfer flow rate may be 0. Often, the transfer flow rate is in the range of 0.4 to 1 Lpm (e.g., 0.4, 0.6, 0.8, or 1 Lpm).

[0161] This document is described with various embodiments, but is not intended to be limited to these embodiments. On the contrary, this document includes various alternatives, modifications, and equivalents as understood by those skilled in the art. For example, in the various embodiments illustrated in the drawings, the delivery conduit / injection tube is described with an inner diameter of 1 mm with a cumulative gas flow rate of approximately 1 liter / min (0.1 + 0.9 liters per minute). It is expected that delivery conduits / injectors of smaller or larger diameters with correspondingly selected gas flow rates can be applied to the various geometries presented with similar expected results. However, conditions for maintaining non-turbulent or near-non-turbulent gas kinetics within the injection tube may be desirable to preserve the distinctiveness of each separate excisional plume.

[0162] Additionally, in some cases of increased laser pulse rates, two or more ablated plumes can be clearly captured and delivered to an ionization system (e.g., ICP) within the diffused cumulative passage time as described above. For example, at a repetition rate of 10 kHz, a pulsed laser can generate two ablated plumes within 200 μs, which can then be delivered to an ICP for ionization. The ions generated from the two discrete plumes can be analyzed by a mass spectrometer as a single discrete packet of ions. Consequently, while the laser remains at the same ablation spot, or the laser's movement speed relative to the trace of the continuous spot is smaller than the repetition rate, the ablated plumes and subsequent ions can provide cumulative mass analysis at the same ablation spot or provide an average mass distribution along the trace, respectively. It should be noted that laser repetition rates as high as several MHz are used to generate a signal indicating the averaging of many laser pulses. The laser may also be fired in bursts to provide gaps in the data flow between individual sampling locations (or pixels).

[0163] It will be understood that the method and apparatus of the present invention may be used with any one of various types of samples, e.g., biological samples. In one approach, the sample is cellular material, e.g., tissue sections, cell monolayers, cell preparations, etc. The sample may be thinly sectioned biological tissue with a thickness of up to 100 μm, a tissue sample with a thickness of about millimeters, or an unsectioned tissue sample. In one embodiment, thin tissue sections (e.g., paraffin-embedded sections) may be used. For example, 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 cells selected from a cell population. For example, refer to U.S. Patent Publication No. 2012 / 0061561 of 2012 by Antonov, A. and Bandura, D. incorporated herein by reference.

[0164] By constructing image IMS and IMC, signals for multiple labeled atoms / element tags within a plume can be provided. Detecting a label in the plume reveals the presence of a cognate target at the excision site (or, correspondingly, the detachment site of the material slug). By generating a series of plumes at known spatial locations on the sample surface, the MS signal reveals the location of the label on the sample, and accordingly, the signal can be used to construct an image of the sample. Since it is possible to associate the location of the labeled atom with the location of the cognate target by labeling multiple targets with distinguishable labels, the present invention can construct complex images and achieve a level of multiplexing that far exceeds what is achievable using existing techniques. For example, Kylebank Software's GRAPHIS package can be used, but other packages such as TERAPLOT, ImageJ, and CellProfiler may also be used. It is known in the art to use MS data from technologies such as MALDI-MSI for imaging, and 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 the Matlab platform, and there are also instruments such as the 'Datacube Explorer' program for rapidly exploring and visualizing both 2D and 3D MSI datasets at full spatial and spectral resolution.

[0165] Sample

[0166] The present invention provides a method for imaging samples. All types of samples may be analyzed by a method including alloys, geological samples, and archaeological samples. Biological samples may also be analyzed. These samples may contain multiple cells, and multiple of these cells may undergo IMS and / or IMC to provide images of these cells in the sample. Generally, the present invention is now studied by immunohistochemistry (IHC) technology, but it may be used for the analysis of tissue samples using labels suitable for detection by IMC.

[0167] Any suitable tissue sample may be analyzed. For example, the tissue may be epithelial tissue, muscle tissue, nerve tissue, etc., and combinations thereof. For diagnostic or prognostic purposes, the tissue may be derived from a tumor. In some embodiments, the sample may be derived from known tissue, but it may not be known whether the sample contains tumor cells. Imaging may facilitate diagnosis by revealing the presence of targets indicating the presence of a tumor. The tissue sample may include breast cancer tissue, for example, human breast cancer tissue or human mammary gland epithelial cells (HMLE). The tissue sample may include formalin-fixed paraffin-embedded (FFPE) tissue, frozen tissue, or tissue embedded in a suitable resin. The tissue may be obtained from any living multicellular organism, but will generally be human tissue.

[0168] Tissue samples are generally sections with a thickness in the range of, for example, 2 to 10 μm, e.g., 4 to 6 μm. Thinner tissue sections with a thickness of less than 2 μm, e.g., less than 1 μm, less than 500 nm, less than 250 nm, or even less than 100 nm, may also be analyzed. Although thinner tissue samples generate lower signals due to the reduction in the volume of the sample excised by the later pulse, thinner sections allow for the generation of more sections from the tissue sample, which offers advantages in terms of 3-D imaging by imaging multiple sections. However, thinner sections (e.g., sections with a resolution lower than that of laser ablation) may allow for easier resection through a slide (e.g., the entire depth of the tissue section can be resected at the laser ablation spot). Techniques for preparing such sections, including dehydration steps such as embedding, are well known in the field of IHC using microtomes. Therefore, the tissue can be chemically fixed, and then sections can be prepared in the desired plane. Freeze cutting or laser capture microdissection may also be used to prepare tissue samples. The sample can be permeable, for example, so that reagents can label intracellular targets (see above).

[0169] The size of the tissue sample to be analyzed will be similar to current IHC methods, but the maximum size will be determined by the laser ablation device, specifically by the size of the sample that can fit into the ablation chamber. While a maximum size of 5 mm x 5 mm is common, smaller samples (e.g., 1 mm x 1 mm) are also useful (these dimensions refer to the size of the section, not its thickness).

[0170] labeling of tissue samples

[0171] In some embodiments, as described above, the apparatus and method of the present invention detect atoms added to a sample (i.e., not normally present). These atoms are referred to as labeled atoms (thus, labeled atoms represent elemental tags). The sample is a biological sample, generally comprising cells, and the labeled atoms are used to label target molecules within the cell or on the cell surface. In some embodiments, simultaneous detection of two or more labeled atoms, e.g., at least 3, 4, 5, 10, 20, 30, 32, 40, 50, or even 100 different labeled atoms is possible, enabling multiplexed label detection. By labeling different targets with different labeled atoms, it is possible to determine the presence of multiple targets on a single cell.

[0172] The labeled atoms that can be used with the present invention include any species that can be detected by MS and are substantially absent in the unlabeled sample. Thus, for example, 12C atoms are unsuitable as labeled atoms because they are naturally abundant, whereas 11C can theoretically be used because it is an artificial isotope that does not occur naturally. However, in a preferred embodiment, the labeled atoms are transition metals, such as rare earth metals (including 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 and neodymium has 7 stable isotopes, all of which are available in enriched form. The 15 lanthanide elements provide at least 37 isotopes having masses that are not double-inherent. Examples of elements suitable for use as labeling atoms include lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), and yttrium (Y). In addition to rare earth metals, other metal atoms, such as gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), and bismuth (Bi), are suitable for detection by MS. The use of radioisotopes is undesirable because they are less convenient to handle and are unstable; for example, Pm is not a preferred labeling atom among the lanthanides.

[0173] To facilitate TOF analysis (see above), it is helpful to use labeled atoms with atomic weights within the range of 80 to 250, e.g., the range of 80 to 210, or the range of 100 to 200. This range includes all lanthanides, but excludes Sc and Y. The 100 to 200 range allows for theoretical 101-plex analysis by using different labeled atoms, while enabling the present invention to utilize the high spectral scan rate of TOF MS. As previously mentioned, by selecting a labeled atom with mass in the window above what is seen in unlabeled samples (e.g., within the 100 to 200 range), rapid analysis at a biologically significant level can be provided using TOF detection.

[0174] Labeling a sample generally requires that a labeled atom be attached to one member of a specific bonding pair (sbp). By coming into contact with the sample, this labeled sbp interacts with the other member of the sbp (the target sbp member) (if present) to localize the labeled atom to the target molecule within the sample. Then, the method of the present invention detects the presence of the labeled atom on the particle when the particle is analyzed by a mass cytometer. Rare earth metals and other labeled atoms can be attached to the sbp member by known techniques. For example, the literature by Bruckner et al. [(2013) Anal. Chem. [86:585-91] describes attaching lanthanide atoms to oligonucleotide probes for MS detection, Gao & Yu's literature [(2007) Biosensesor Bioelectronics 22:933-40] describes the use of ruthenium to label oligonucleotides, and Fluidigm Canada sells MaxPar™ metal labeling kits that can be used to conjugate more than 30 different labeling atoms to proteins (including antibodies).

[0175] Multiple various label atoms can be attached to a single sbp member, and greater sensitivity can be achieved when more label atoms are attached to any sbp member. For example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more than 100 label atoms can be attached 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. For example, DTPA is about 10 -6 It binds to 3+ lanthanide ions with a dissociation constant of M [Tanner et al. Cancer Immunol Immunother (2013) 62:955-965]. These polymers can be terminated at thiol-reactive groups (e.g., maleimides) that can be used to attach to sbp members. For example, thiol-reactive groups can bind to the Fc region of the antibody. Other functional groups may also be used for the conjugation of these polymers, such as amine-reactive groups like N-hydroxysuccinimide esters, or groups reactive to carboxylates or glycosylation of the antibody. Any number of polymers may be bound 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. The use of metal nanoparticles may also be employed to increase the number of atoms in the label.

[0176] As described above, a labeled atom is attached to an sbp member, and this labeled sbp member comes into contact with a sample, wherein the labeled sbp member can locate a target sbp member (if present) and form a labeled sbp. The labeled sbp member may comprise any chemical structure suitable for detection according to the present invention after being attached to the labeled atom.

[0177] Generally, the method of the present invention may be based on any sbp already known to be used to determine the presence of a target molecule in a sample (e.g., as used in IHC or fluorescence in situ hybridization (FISH)) or fluorescence-based flow cytometry, but the sbp member in contact with the sample has a labeled atom detectable by MS. Accordingly, the present invention can be easily implemented using available flow cytometry reagents by modifying a FISH probe having a label detectable by MS, for example, by modifying a previously used label.

[0178] The sbp may comprise any one of the following: a nucleic acid divalent; an antibody / antigen complex; a receptor / ligand pair; or an aptamer / target pair. Thus, a labeling atom may be attached to a nucleic acid probe, and then, to form, for example, a DNA / DNA divalent, a DNA / RNA divalent, or an RNA / RNA divalent, this nucleic acid probe may be brought into contact with a sample so that the probe can hybridize to the complementary nucleic acid(s) within it. Similarly, a labeling atom may be attached to an antibody and then brought into contact with a sample so that it can bind to an antigen. A labeling atom may be attached to a ligand and then brought into contact with a sample to bind to its receptor. A labeling atom may be attached to an aptamer ligand and then brought into contact with a sample to bind to its target. Thus, the labeled sbp members can be used to detect various target molecules in a sample, including DNA sequences, RNA sequences, proteins, sugars, lipids, or metabolites.

[0179] In a typical embodiment, the labeled sbp member is an antibody. Labeling the antibody can be achieved by conjugating one or more labeled atom-binding molecules to the antibody, for example, using the MaxPar™ conjugation kit as described above. The target molecule of the antibody is referred to as its antigen and may be a protein, carbohydrate, nucleic acid, etc. Antibodies that recognize cellular proteins useful for mass cytometry are already widely available for IHC use, and by using labeled atoms instead of current labeling techniques (e.g., fluorescence), these known antibodies can be easily configured for use in the method of the present invention, while having the advantage of increasing multiplexing capability. The antibody used with the present invention can recognize targets on the cell surface or targets within the cell. Antibodies can recognize various targets, for example, they can specifically recognize individual proteins, recognize multiple related proteins that share a common epitope, or recognize specific post-translational modifications to proteins (for example, this allows distinguishing between tyrosine and phospho-tyrosine on the protein of interest, distinguishing between lysine and acetyl-lysine, and detecting ubiquitination). After binding to the target, the presence of the target in the sample can be revealed by detecting the labeled atom(s) conjugated to the antibody.

[0180] The labeled sbp member will generally interact directly with the target sbp member in the sample. However, in some embodiments, the labeled sbp member may interact indirectly with the target sbp member; for example, a primary antibody may bind to the target sbp member, and then a labeled secondary antibody may bind to the primary antibody in a sandwich assay manner. However, generally, the present invention relies on direct interaction because direct interaction is more easily achievable and allows for higher multiplexing. However, in both cases, the sample is brought into contact with the sbp member capable of binding to the target sbp member in the sample, and the label attached to the target sbp member is detected in a later step.

[0181] One feature of the present invention is the ability to detect a number of different target sbp members in a sample (e.g., 10 or more, even 100 or more), e.g., the ability to detect a number of different proteins and / or a number of different nucleic acid sequences in a sample. To allow for the different detection of these target sbp members, each of these sbp members must have a different labeling atom so that their signals can be distinguished by MS. For example, if 10 different proteins are detected, 10 different antibodies (each specific to a different target protein) may be used, each of which has a unique labeling so that signals from the different antibodies can be distinguished. In some embodiments, for example, it is preferable to use a number of different antibodies for a single target that recognizes different epitopes on the same protein.

[0182] When two or more labeled antibodies are used, it is desirable that the antibodies have similar affinities for each of these antigens, as this helps ensure that the relationship between the amount of detected labeled atoms and the abundance of the target antigen becomes more consistent across different sbp (especially at high scanning frequencies).

[0183] When the target sbp member is located within a cell, it is typically necessary to permeate the cell membrane before or during contact between the sample and the label. For example, if the target is a DNA sequence but the labeled sbp member cannot permeate the membrane of a living cell, the sample cell can be fixed and permeated. Then, the labeled sbp member can enter the cell and form an sbp together with the target sbp member.

[0184] Generally, the method of the present invention detects at least one intracellular target and at least one cell surface target. However, in some embodiments, the present invention may be used to detect multiple cell surface targets while ignoring the intracellular target. Overall, the selection of targets will be determined by the information preferred in the method.

[0185] The labeling of a sample does not rely entirely on sbp. In some cases, classical dyes can be used to highlight desired features on the tissue. In many cases, the dyes used in microscopy contain elements that are rare in the natural cellular state. Therefore, during the process of staining the tissue, the tissue is enriched with specific elements that are readable by the apparatus and method described herein.

[0186] Accordingly, in some embodiments, the analysis method described above includes the step of labeling a sample with at least one labeled atom. Then, such atom can be detected using the method described above.

[0187] Signal enhancement

[0188] Aspects of the present application include enhancing the signal by adding hydrogen to the LA-ICP-MS as further described herein. The LA-ICP-MS system may have one or more gas flows as illustrated in FIG. 4, such as a capture gas flow, a transfer gas flow, an internal (auxiliary) gas flow into an internal torch tube, and / or an external (plasma) gas flow into an external torch tube. For reference, the gas source illustrated in FIG. 4 may be configured differently so that a pre-mixed or humidified gas is supplied to any one or more of the capture gas flow, the transfer gas flow, the internal gas flow, and / or the external gas flow. In a certain embodiment, the LA-ICP-MS system may include three gas sources, such as a separate gas source for the transfer flow, in comparison to the internal and external gas flows. Alternatively or additionally, the laser ablation system may include an injector orthogonal to the sample, as illustrated in FIG. 2 and 3, for example. In a certain embodiment, only one gas flow (e.g., capture gas) may flow through the injector. In a given embodiment, the injector includes a supplementary gas flow downstream of the laser ablation plume.

[0189] Aspects of the present application include apparatus and workflow for imaging mass spectrometry (IMS) that improve sample acquisition speed, signal sensitivity, and / or signal stability. Imaging mass cell analysis (IMC) is the detection of mass tags at cellular resolution or subcellular spatial resolution by imaging mass spectrometry. IMC systems and methods may include any one of the aspects described in the present application. In a certain aspect, mass cell analysis may include laser ablation (LA)-induced coupled plasma (ICP) mass spectrometry (MS). Using non-endogenous elements, such as heavy metal mass tags, enables detection of endogenous elements. These endogenous elements, such as carbon, oxygen, and nitrogen, and light metals, such as calcium, may be depleted by a mass spectrometer, for example, by a high-pass mass filter (e.g., RF quadrupole). In a certain aspect, argon dimers (byproducts of argon-based ICP) may also be depleted, for example, by a high-pass mass filter with a cutoff of at least 80 amu.

[0190] Heavy metal mass tags may contain more than 80 amu of heavy metal, for example, as further described herein. In a given embodiment, individual mass tags may contain a plurality of label atoms enriched with heavy metal isotopes. These label atoms may be present on the polymer, for example, by being bound to a chelating pendant group on the polymer, which is then conjugated to a specific binding partner (SBP) that binds to a specific target, such as an antibody that binds to a specific protein target. For example, Maxpar tags supplied by Fluidigm each comprise a polymer loaded with a plurality of label atoms consisting of a single isotope, such as a lanthanide isotope, and may be conjugated to an antibody that binds to a specific protein expressed by a cell. Detection of the isotope by ICP-MS indicates the presence of the corresponding target (e.g., protein).

[0191] Using enriched isotopes as labeling atoms can increase the number of different targets that can be detected compared to those detectable with elemental mass tags containing a natural mixture of isotopes. However, using more than 20, 30, or 40 isotope mass tags often results in isotopes of 16 amu being included among them, so an oxide of one mass tag can interfere with the detection of another mass tag containing an isotope with a mass exceeding 16 amu. Oxides, such as lanthanides, used as mass tags are byproducts of ICP-based atomization and ionization. In certain embodiments, a method or system for increasing sensitivity or signal stability can be implemented in a manner that avoids the formation of excess oxides.

[0192] As such, in contrast to other forms of IMS such as MALDI, oxidation can be a unique problem for atomic IMC, as detecting the atomic mass of a metal isotope of 16 amu from another mass tag is susceptible to oxide spillover. This consideration is further complicated by the demand for sensitive and stable detection at each laser-absorbed spot (pixel). Each ablation creates a small (e.g., micron or sub-micron in size) ablation crater where only a few given mass tags may exist. Additionally, IMC relies on the ability to accurately compare the expression of different targets across samples, such as tissue sections (e.g., measured as signals in different mass channels corresponding to different masses of the isotope mass tag).

[0193] In a specific embodiment, the IMC system may be used for suspension mass cell analysis, such as when the ICP torch of the system can be coupled to a nebulization chamber for introducing whole cells instead of a laser ablation source. As such, the ICP torch can nebulize and ionize whole cells (e.g., cells with a diameter of at least 15 μm or at least 20 μm). As such, the plasma generated by the ICP torch may not be specifically designed for the efficient ionization and / or nebulization of the laser ablation plume (e.g., may have a longer path length than required for the laser ablation plume). Alternatively, designing the LA-ICP-MS system to have a short transient may involve reducing the path length of the plasma, which may result in inefficient ionization and / or nebulization (unless modified by introducing hydrogen as described herein, for example). The inventors discovered that humidity levels affect the sensitivity of the IMC system and can lead to signal drift if, for example, the humidity of the system tends to decrease relative to the sample run (e.g., if there is an initial humidity level due to air in the ablation chamber before operation). Indeed, as further discussed herein, both water vapor and hydrogen gas have been found to increase signal sensitivity and further improve signal stability. As such, the efficiency of ionization and / or atomization (e.g., measured by increased sensitivity in one or more mass channels 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 may be water vapor or alcohol (e.g., ethanol) vapor. Alternatively or additionally, suitable hydrogen-containing molecules may be hydrogen gas, methane, or ammonium, provided as a premix with, for example, helium or argon.

[0194] In a given embodiment, a portion of the LA-ICP-MS system may be open to the atmosphere, so that air may be present in the laser ablation chamber, fluid, and / or ICP torch. This air may ultimately be purged or consumed by the operation of the ICP-MS system. However, changes in humidity and / or oxygen levels caused by this air may affect the efficiency of the ICP plasma, such as ionization efficiency and / or oxide formation. In a given embodiment, humidity may be controlled throughout the entire sample run as described herein (e.g., so that the mass signal increases and / or stabilizes but oxidation is minimized). Alternatively or additionally, hydrogen may be pre-mixed with a gas (e.g., helium or argon) to increase sensitivity and / or signal stability.

[0195] The inventors have discovered that the addition of water or hydrogen during LA-ICP-MS analysis of heavy metal mass tags can improve sensitivity and regulate signal stability. In certain embodiments, one or more hydrogen-containing gases, such as hydrogen gas, water vapor, methane, and / or ammonia, may be introduced during LA-ICP-MS. In certain embodiments, the gas (e.g., hydrogen gas, methane, or ammonia) may be supplied from a pressurized gas source in a state pre-mixed with an ICP gas, such as helium or argon. In certain embodiments, water vapor may be mixed with a gas such as argon gas.

[0196] Generally, hydrogen gas can be supplied to the ICP source at a flow rate sufficient to provide signal enhancement and / or stability.

[0197] Signal enhancement may be for one or more mass channels, e.g., a metal isotope channel of a mass tag used to label a biological sample analyzed by the device. In a given embodiment, the metal isotope comprises a lanthanide isotope. The signal enhancement may be at least 20%, at least 30%, at least 50%, at least 80%, or at least 100% compared to a signal without hydrogen gas. In a given embodiment, the signal enhancement is an average signal enhancement over a range of mass channels in which a signal (e.g., from a metal isotope mass tag) is detected. In a given embodiment, the average signal enhancement for a lanthanide isotope measured by at least 10 counts is at least 20%, at least 30%, or at least 50%. The signal enhancement, also referred to as sensitivity enhancement, may be measured as an increase in the count per laser ablation plume (e.g., average number of counts) when analyzing labeled atoms of a sample, e.g., or when analyzing an elemental standard containing a known number of detectable atoms.

[0198] In a given embodiment, the amount of hydrogen gas flows into the ICP source in a strong amount for a change in the hydrogen gas flow such that a 20% or 50% change in amount results in a change of less than 10% of the signal, for example, a change of less than 5% of the signal (for example, in the case of a standard, labeled atom, some labeled atom, or all labeled atom detected when exceeding 10 counts).

[0199] Apparatus and method for introducing hydrogen-containing molecules (e.g., into a pre-mixed gas or vapor) to improve signal sensitivity and / or stability are described herein. In a given embodiment, the introduction of hydrogen improves signal stability such that external humidity has a signal sensitivity change of less than 10% or less than 5% over a range (e.g., 0 to 2000 microbar or 0 to 4000 microbar).

[0200] Hydrogen gas for signal enhancement

[0201] In a given embodiment, an apparatus, method, and / or a pre-mixed compressed gas source for introducing hydrogen gas into an ICP torch to improve signal sensitivity and / or signal stability may be provided.

[0202] In a given embodiment, the device comprises one or more of the following: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to transport an ablation plume generated from the sample by the laser ablation source to the ICP torch; and a pre-mixed compressed gas source comprising hydrogen gas as a mixture with at least one of helium and argon.

[0203] The hydrogen gas in the pre-mixed compressed gas source may be 0.1 volume% to 5 volume%, for example, 1 volume% to 4 volume%.

[0204] The pre-mixed compressed gas source may be at least 50 volume% helium or at least 50 volume% argon. The pre-mixed compressed gas source may supply gas to an ablation chamber including a sample stage. The pre-mixed gas source may supply capture gas carrying the ablation plume into the injector. Alternatively or additionally, the pre-mixed gas source may supply a transfer gas (i.e., transfer gas entering the injector upstream of the ablation plume), a replenishment gas (i.e., replenishment gas in the injector downstream of where the ablation plume enters the injector), and / or an internal gas (i.e., auxiliary gas flowing into the internal tube of the ICP torch).

[0205] For example, when the capture gas raises the ablation plume into the delivery gas of the injector, an additional gas source may supply the delivery gas to the injector. The delivery gas may contain argon (e.g., at least 50% argon). The capture gas may contain 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.

[0206] In a specific embodiment, for example, when only the capture gas enters the injector, there may be no separate capture and delivery gas. In a specific embodiment, the injector is configured to guide the laser ablation plume to the vertical ICP torch, for example, when the device includes a vertically oriented ICP torch.

[0207] The device may further include an additional gas source supplying at least one of a delivery gas, a supplement gas, an internal (auxiliary) gas, and / or an external (plasma) gas. The additional gas source may be compressed gas or liquid dewar, and may be at least 50 volume% argon, and the pre-mixed gas source may include at least 50 volume% helium. In contrast, the pre-mixed gas source may not be liquid dewar, as different gases may vaporize (and thus be depleted) at different rates during operation.

[0208] If the device includes a supplementary flow to the injector downstream of where the laser ablation plume enters the injector, the supplementary flow may additionally be optionally downstream of the sacrifice flow.

[0209] In a given embodiment, a pre-mixed compressed gas supply source supplies at least one of a capture gas, a transfer gas, and an internal torch gas.

[0210] The above device may be configured to provide a hydrogen gas flow of 0.001 L / min to 0.1 L / min, for example, a hydrogen gas flow of 0.001 L / min to 0.02 L / min into the ICP torch. Alternatively or additionally, the hydrogen gas may be 0.002% to 1% of the total gas flow into the ICP torch, for example, 0.01% to 0.1% of the total gas flow into the ICP torch. The total gas flow into the ICP torch may be 5 to 30 L / min, for example, 10 to 25 L / min. For example, U.S. Patent No. US8633416, incorporated by reference, reports a gas flow of 5 L / min. In a given embodiment, the hydrogen gas flow into the ICP torch may vary by more than 20%, for example, more than 50%, without reducing the sensitivity (for example, to one, some, or all labeled atoms) by more than 5%. In a given embodiment, the amount of hydrogen gas flow into the ICP torch during sample analysis may vary by more than 10%, more than 20%, or more than 50%.

[0211] As further described herein, the device may further include a mass spectrometer configured to detect ionized atoms generated by an ICP torch. In a given embodiment, the mass spectrometer includes a high-pass filter configured to remove ions having a mass of at least 80 amu or less.

[0212] An ICP torch is configured to atomize and ionize whole cells in a cell suspension mode where the ICP torch is separated from the laser ablation source. For example, the ICP torch can be separated from the laser ablation source and is sufficient to atomize and ionize whole cells introduced in a suspension state into the atomization chamber upstream of the ICP. Characteristics of the ICP torch, such as size and geometry, may be suitable for the atomization and ionization of whole cells. Due to these design considerations, the ICP torch may be less efficient at atomizing and / or ionizing material into a laser ablation plume, e.g., a plume generated by a laser with a spot size of less than 2 microns, e.g., a spot size of less than 1 micron.

[0213] Any one of the aforementioned embodiments may further include a humidification system configured to humidify the gas flow (e.g., as further described herein).

[0214] The method of the present application may include the step of analyzing a sample by LA-ICP-MS using an apparatus of any one of the aforementioned embodiments. The sample may be a biological sample, such as a tissue section, as further described herein. The sample may include a label atom, such as a label atom combined with an SBP that binds to a target within the sample, as further described herein. Thus, the analysis method may further include the step of labeling the sample with a label atom before analyzing the sample by LA-ICP-MS. In a given embodiment, the label atom does not have an oxide overflow of more than 3%.

[0215] In a given embodiment, hydrogen gas provides at least 20% increased sensitivity for at least some labeled atoms, such as labeled atoms having an average ionic coefficient of at least 10 per ablation plume, or at least 50% increased sensitivity for at least some labeled atoms.

[0216] In a given embodiment, the average sensitivity to a labeled atom (or elemental standard) during any given 5-minute period may not change by more than 10% over at least 1 hour of analyzing the sample.

[0217] Although hydrogen gas has been described above, other hydrogen-containing gases may be used instead of or in addition to hydrogen gas in any of the above embodiments. For example, methane or ammonia may be used instead of or in addition to hydrogen gas.

[0218] Pre-mixed gas source and its use

[0219] An aspect of the present application comprises a pre-mixed compressed gas source for an inductively coupled plasma device. The pre-mixed gas source may comprise at least 50 volume% of helium or argon (e.g., at least 70 volume%, at least 90 volume%) and may further comprise at least 0.1 volume% of a gas, wherein the gas comprises elemental hydrogen. The gas may be, for example, hydrogen gas, ammonia, and / or methane.

[0220] In a given embodiment, the gas comprises hydrogen gas. The pre-mixed gas source may contain 0.1 volume% to 5 volume%, e.g., 1 volume% to 4 volume% of hydrogen gas. The pre-mixed gas source may contain hydrogen gas below its flash point (e.g., less than about 4%). The pre-mixed gas source may be a compressed gas cylinder for use in LA-ICP-MS.

[0221] An embodiment includes an LA-ICP-MS system comprising the aforementioned pre-mixed compressed gas source. In a given embodiment, the mass spectrometer may instead be an elemental analyzer, such as an optical emission spectrometer.

[0222] Gas humidification for signal enhancement

[0223] An aspect of the present invention includes a gas humidification device and a method for LA-ICP-MS.

[0224] In a given embodiment, the device comprises one or more of the following: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) source; an injector configured to transport an ablation plume generated from the sample by the laser ablation source to an ICP torch; and a humidification system configured to humidify the gas flow.

[0225] The gas flow may include one or more of a transfer gas flow, a capture gas flow, a supplementary gas flow, and an auxiliary gas flow. In a given embodiment, the gas flow is a transfer gas flow.

[0226] In a given embodiment, for example, when the gas flow is a transfer gas, the gas flow contains at least 50% argon (e.g., at least 70% or at least 90% argon). Alternatively, for example, when the gas flow is a capture gas, the gas flow may contain at least 50% helium.

[0227] In a specific embodiment, the humidification system has an adjustable range including at least 500 μm (microbar) to 5000 uB, e.g. 1000 uB to 4000 uB or 1500 uB to 3000 uB. This can produce a humidifying gas with better stability than 20%, e.g. better stability than 20%. In a specific embodiment, the humidity is not controlled within a range of less than 3%, e.g. less than 5%, because such control may not be necessary for signal stability.

[0228] The humidification system may include a water diffusion tube. In a certain embodiment, the humidification system controls the temperature of the diffusion tube. Alternatively or additionally, the humidification system includes an adjustable variable splitter for diverting a gas flow (e.g., argon gas flow) around the water diffusion tube. The variable splitter may be controlled by a controller coupled to a humidity sensor. In a certain embodiment, the controller and the humidity sensor are configured together to maintain a humidity level by diverting the gas flow around the diffusion tube. Alternatively to the diffusion tube, the humidification system may include a water pump configured to inject water directly into the gas flow.

[0229] The method may include the step of analyzing a sample by LA-ICP-MS using any suitable chest system, such as a device comprising a humidification system of one of the aforementioned embodiments.

[0230] The sample may be a biological sample, such as a tissue section, as further described herein. The sample may contain a labeled atom, such as a labeled atom combined with an SBP that binds to a target within the sample, as further described herein. As such, the analytical method may further include the step of labeling the sample with a labeled atom before analyzing the sample by LA-ICP-MS. In a given embodiment, the labeled atom does not have an oxide overflow exceeding 3%. For example, high humidity may result in an oxide overflow exceeding 3%. As such, humidity and / or plasma temperature may be maintained at a level that allows for sensitivity improvement as described below, but may be sufficiently low to avoid an oxide overflow exceeding 3% for any labeled atom (e.g., average oxide overflow over any 5 minutes of a sample run). For example, the temperature may be controlled by adjusting the plasma temperature through a supplementary gas flow. If the temperature is not properly controlled, even a small amount of humidity may lead to very high oxides.

[0231] In a specific embodiment, humidification provides at least a 20% increase in sensitivity for at least some labeled atoms, such as labeled atoms having an average ionic coefficient 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 a specific embodiment, the average sensitivity for a labeled atom (or elemental standard) over any given 5-minute period may not change by more than 10% over at least 1 hour of analyzing the sample. In a specific embodiment, humidity may change by more than 20%, e.g., more than 50%, without reducing the sensitivity (e.g., for one, some, or all labeled atoms) by more than 5%.

[0232] Although gas humidification (using water vapor) has been described above, another hydrogen-containing molecule may be introduced into the gas stream as vapor as an alternative to or in addition to water. For example, an alcohol such as ethanol may be introduced into the gas stream by any mode, such as the diffusion tube described above for water vapor.

[0233] Conventional laser ablation-guided coupled plasma devices use a combination of gas flows to transport ablated material from the ablation point to the plasma. Sensitivity is affected by the amount of moisture in the gas flows. Adding water to the gas flows via atomized water mist disrupts the flow and, in imaging applications, will negatively impact transient signals and pixel rates. Humidifying the gas flows before they enter the machine does not cause these problems. Significant temperature stabilization is required to ensure normally stable humidity. By measuring the humidity of the exhaust gas flow, feedback can be provided to allow the humidifier to regulate humidity. The humidifier itself is a gas flow passing through a diffusion tube. Since the flow passing through the tube is saturated and exits the tube, the inventors use a variable gas splitter to alter the flow passing through the diffusion tube, thereby changing the moisture in the gas flow after it is recombined. Changing the gas flow is the feedback mechanism.

[0234] Hydrogen supply device and method for signal enhancement

[0235] An aspect of the present application includes an apparatus and a method for introducing a hydrogen-containing molecule into an ICP torch of an LA-ICP-MS. The hydrogen-containing molecule may be a gas such as hydrogen gas, ammonia, or methane. Alternatively or additionally, a hydrogen-containing molecule such as water or an alcohol (e.g., ethanol) may be introduced into the gas stream as a vapor.

[0236] An embodiment comprises a device comprising one or more of the following: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to transport an ablation plume generated from the sample by the laser ablation source to the ICP torch; and a pre-mixed compressed gas source comprising a hydrogen-containing gas as a mixture with at least one of helium and argon. In a given embodiment, the hydrogen-containing gas is methane, ammonia, or hydrogen gas.

[0237] An embodiment comprises a device comprising one or more of the following: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) source; and an injector configured to transport an ablation plume generated from the sample by the laser ablation source to an ICP torch. The device may be configured to supply a vapor containing a hydrogen gas flow. The vapor may include water vapor or alcohol vapor (e.g., ethanol).

[0238] An embodiment comprises an apparatus comprising one or more of the following: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch coupled to a mass spectrometer; and an injector configured to transport an ablation plume generated from the sample by the laser ablation source to the ICP torch. The apparatus may be configured to supply hydrogen-containing molecules to the plasma of the ICP torch during operation for laser ablation ICP mass spectrometry (e.g., as described elsewhere in this invention), but may not supply hydrogen-containing molecules to the plasma of the ICP torch during cell suspension mode. In cell suspension mode, the ICP torch may be separated from the laser ablation source and may be sufficient to ablate and ionize the entire cell introduced in suspension into the ablation chamber upstream of the ICP.

[0239] experiment

[0240] The inventors discovered that signal stability and signal enhancement for lanthanide-labeled atoms or other labeled atoms in biological or inorganic samples (e.g., elemental standards) can be achieved with small amounts of hydrogen. The hydrogen can be hydrogen gas (below the flammability limit), and such stability and enhancement can be robust against differences in the amount of hydrogen gas (e.g., due to changes in gas flow during operation to maintain a constant plasma). Alternatively, hydrogen can be introduced as water vapor, and signal stability and enhancement can likewise be robust against a range of humidity.

[0241] The inventors observed that the sensitivity of the Fluidigm Hyperion Imaging System (IMC) decreased during operation and recovered when turned off. They determined that this was because a small amount of moisture diffused into the system, accumulated during downtime, and gradually dried out during operation. This led to recommendations to reduce not only the method of moisture diffusion into the system but also the need to humidify the gas flow within the system. Upon investigation, the inventors discovered that the amount of hydrogen in the system was a critical factor. Initially, the inventors dismissed the idea of ​​mixing pure hydrogen into the injection gas, deeming it too expensive due to concerns regarding handling excess gas and safety reasons. Instead, they first developed an argon flow humidifier setup, which could reliably supply H2O at the required levels. However, image analysis by biological experts revealed concerns regarding images of mass channels contaminated by oxide formation. In this case, the oxygen originated from water molecules within the argon flow. The inventors then decided to seek a method to obtain the benefits of pure hydrogen mixed into the injector flow without adding oxygen, in a configuration that eliminates flammability and safety issues. The inventors realized that the pre-mixed gas could be diluted to a non-flammable level by existing gas handling components, and that providing a sufficient concentration of hydrogen could increase sensitivity and stabilize the signal.

[0242] Initially, the inventors discovered that the signal is sensitive to humidity and can vary depending on external humidity (e.g., the air within the space of the LA-ICP-MS instrument). As illustrated in Fig. 5, the delivery gas humidity range of 0 to 4000 uB accounts for about 30% variation in the sensitivity of the lutetium-containing elemental standard, measured as the average number of lutetium per ablation shot across the humidity range. However, the inventors also found that the signal is stable and high within the humidity range (about 2000 to 3000 uB). The system used was similar to that illustrated in Fig. 4.

[0243] The inventors also discovered that while adding water can significantly increase oxide levels, oxides can be maintained at a specific level by controlling the flow rate of the delivery gas, as opposed to simply adjusting to maximum sensitivity. The results of sample adjustments below demonstrate that the oxide ratio can be limited to < 3% (or less, e.g., < 2% or < 1%) while achieving significant improvements in sensitivity after humidifier use:

[0244]

[0245] One additional point to note is that Ce140 to Gd156 generally have the worst oxide overflow among all mass channels, and therefore, limiting oxide overflow in those channels to < 3% during adjustment may result in much less in other channels. In a given embodiment, oxide overflow changes by less than 1% point, e.g., less than 0.5% point or less than 0.25% point, during a sample run (e.g., for periods exceeding 5 minutes, 10 minutes, or 30 minutes of the sample run).

[0246] Additionally, the inventors confirmed that adding hydrogen (H2) gas (as a gas source in which 3% hydrogen is pre-mixed with helium) is strongly correlated with increased signal / ion image brightness. Signal enhancement for the two approaches is illustrated in Fig. 9. Specifically, the delivery gas humidification (left) shows an improved signal for the common marker, and the pre-mixed hydrogen-helium capture gas (right) also shows an improved signal for the common marker.

[0247] The inventors also confirmed that very faint channels experience less sensitivity improvement compared to bright channels, which is likely due to the background. As such, sensitivity improvement is mainly observed for channels detected at an average of 10 or more ion counts per ablation shot.

[0248] A comparison of the compromises between these two approaches is below.

[0249]

[0250] The inventors attempted multiple humidification systems as illustrated in Fig. 6 and discovered that directly injecting water with a water pump (top of Fig. 6) caused some stability issues related to wetting and droplet formation at the ends of the capillaries. It was found that flowing gas through the diffusion tube using temperature and / or humidity feedback to regulate the diversion of gas flow around the diffusion tube (bottom of Fig. 6) provided better humidification stability. When operated overnight at a constant rate while stabilizing the temperature, a significantly stable signal is provided despite humidity fluctuations of + / -10% (see Fig. 7). However, removing temperature stabilization and using feedback on the gas flow instead allows for better humidity control. Humidity is controlled by varying the gas flow through the diffusion tube in response to humidity detected by a sensor downstream of the tube. Controlling the gas flow through the diffusion tube provides more consistent humidity and a good response time to changes.

[0251] Conventional laser ablation-guided coupled plasma devices use a combination of gas flows to transport ablated material from the ablation site to the plasma. Sensitivity is affected by the amount of hydrogen in the gas flow.

[0252] The inventors used various amounts of hydrogen gas to determine the appropriate amount of hydrogen required for the LA-ICP-MS system, similar to what is illustrated in Fig. 4. They used a mixed gas ratio for the helium-hydrogen gas flow, which would provide an appropriate amount of hydrogen over the typical helium flow range. This allows for conversion to a hydrogen mixed gas system without any other changes to the hardware or software, other than removing pure helium and replacing it with mixed hydrogen-helium gas. The total number of cylinders / regulators / mass flow controllers remains the same. Unlike the humidifier system, the mixed gas system does not add oxygen, and the oxide ratio detected by the MS system was not affected.

[0253] In an alternative setup, hydrogen is pre-mixed into argon (e.g., specifically as a gas source for the delivery gas and / or capture gas). In some LA-ICP-MS systems, argon can be used as a capture gas for the ablation plume. In addition to LA-ICP-MS, in mass cytometry instruments, neon may be considered for some applications, but it can be prohibitively expensive. In another alternative setup, hydrogen pre-mixed into argon is added to the delivery gas. The delivery gas is part of the total injection flow; it is mixed with the ablation capture gas that carries the plume. When an H2 / argon premix is ​​added to the delivery gas flow, the user can change the ratio between pure argon and the H2 / Ar premix, controlling the total flow of H2 into the injector while independently adjusting the optimal argon flow. That is, the flow of the premix controls the mass flow of H2, and the combined flow of the premix and the remaining argon controls the total argon flow of the delivery gas. The total argon flow at the injector outlet needs to be carefully adjusted to achieve optimal plasma temperature and maximum sensitivity. Then, the H2 level provides a second dimension for sensitivity improvement. The proposed H2 / argon premix contains low-concentration hydrogen and is below the flammability limit. This setup has the disadvantage of requiring additional cylinders for the premix gas and an independent additional mass flow controller for the premix flow. However, the advantage of this setup is the ability to independently regulate and adjust the hydrogen portion of the total flow for a given instrument and plasma conditions. For example, this arrangement is considered as Plan B for a deuterium project using an H2 / helium premix, which is Plan A. The H2 / helium mix is ​​generally a simple, low-cost, and limit-free solution without independent H2 flow control.

Claims

Claim 1 A method comprising analyzing a sample by LA-ICP-MS using a device, wherein the device comprises: a sample stage configured to move the sample in at least two directions; a laser ablation source configured to ablate the sample mounted on the sample stage; an inductively coupled plasma (ICP) torch; an injector configured to transport an ablation plume generated from the sample by the laser ablation source to the ICP torch; and a pre-mixed compressed gas source comprising hydrogen gas as a mixture with at least one of helium and argon, wherein the average sensitivity to a labeled atom during any given 5 minutes over at least 1 hour of analyzing the sample does not change by more than 10%. Claim 2 A method according to claim 1, wherein the hydrogen gas in the pre-mixed compressed gas source exceeds 0.1% and is below the flammability limit of hydrogen. Claim 3 A method according to claim 1, wherein the hydrogen gas is 1 volume% to 4 volume%. Claim 4 A method according to claim 1, wherein the pre-mixed compressed gas supply comprises at least 50 volume% of helium. Claim 5 A method according to claim 1, wherein the pre-mixed compressed gas supply comprises at least 50 volume% of argon. Claim 6 A method according to claim 1, wherein a pre-mixed compressed gas supply source supplies gas to a cutting chamber including a sample stage. Claim 7 A method according to claim 1, wherein a pre-mixed gas supply source supplies a capture gas that transports the ablation plume into the injector. Claim 8 A method according to claim 7, wherein an additional gas supply source supplies a delivery gas to an injector, and the capture gas raises a cut plume into the delivery gas of the injector. Claim 9 A method according to claim 8, wherein the transfer gas comprises argon and the capture gas comprises a mixture of helium and hydrogen gases. Claim 10 A method according to paragraph 7 in which, in addition to the capture gas supplied from a pre-mixed gas source, there is no separate capture gas and a delivery gas. Claim 11 A method according to claim 1, wherein the injector guides the laser ablation plume into a vertical ICP torch. Claim 12 A method according to claim 1, further comprising an additional gas supply source that supplies the delivery gas to the injector. Claim 13 A method according to claim 12, wherein the additional gas source comprises at least 50 volume% argon and the pre-mixed gas source comprises at least 50 volume% helium. Claim 14 A method according to claim 1, wherein the flow of pre-mixed compressed gas is configured to introduce a make-up flow into the injector downstream of where the laser ablation plume enters the injector. Claim 15 In paragraph 14, the supplementary flow is introduced downstream of the sacrifice flow. Claim 16 In paragraph 15, the device is a method that does not include a sacrifice flow. Claim 17 A method according to claim 1, wherein a pre-mixed compressed gas supply provides at least one of a capture gas, a delivery gas, and an internal torch gas. Claim 18 The method of claim 1, configured to supply a hydrogen gas flow of 0.001 L / min to 0.1 L / min into an ICP torch. Claim 19 A method according to claim 18, configured to supply a hydrogen gas flow of 0.001 L / min to 0.02 L / min into an ICP torch. Claim 20 The method of claim 1, further configured to supply hydrogen gas of 0.002% to 1% of the total gas flow into the ICP torch. Claim 21 A method according to claim 20, wherein the device is configured to supply hydrogen gas of 0.01% to 0.1% of the total gas flow into an ICP torch. Claim 22 A method according to claim 20, wherein the total gas flow into the ICP torch is 5 L / min to 30 L / min. Claim 23 The method of claim 1, further comprising a mass spectrometer configured to detect ionized atoms generated by an ICP torch. Claim 24 In paragraph 23, the method comprises a mass spectrometer comprising a high-pass filter configured to remove ions having a mass of at least 80 amu or less. Claim 25 A method according to claim 1, wherein the ICP torch is configured to atomize and ionize whole cells in a cell suspension mode in which the ICP torch is separated from the laser ablation source. Claim 26 A method according to claim 1, further comprising a humidification system configured to humidify the gas flow. Claim 27 In paragraph 26, the humidified gas flow is a transfer gas flow. Claim 28 In paragraph 1, the method wherein the sample is a biological sample. Claim 29 In paragraph 28, the method wherein the sample comprises a labeled atom. Claim 30 A method according to claim 29, further comprising labeling a sample with a labeled reactor before analyzing the sample by LA-ICP-MS. Claim 31 In paragraph 30, a method wherein the average oxide spillover of the labeled atoms additionally does not exceed 3%. Claim 32 A method according to claim 1, wherein the hydrogen gas provides at least a 20% increase in sensitivity for at least some labeled atoms. Claim 33 In paragraph 32, the method wherein the hydrogen gas provides at least a 50% increase in sensitivity for at least some labeled atoms. Claim 34 A device comprising: a sample stage configured to move a sample in at least two directions; a laser ablation source configured to ablate a sample mounted on the sample stage; an inductively coupled plasma (ICP) torch coupled to a mass spectrometer; and an injector configured to transport an ablation plume generated from a sample by the laser ablation source to the ICP torch, wherein the ICP torch is configured to atomize and ionize whole cells in a cell suspension mode in which the ICP torch is separated from the laser ablation source; and wherein the device is configured to supply hydrogen-containing molecules to the plasma of the ICP torch during operation for laser ablation ICP mass spectrometry, but not to supply hydrogen-containing molecules to the plasma of the ICP torch during the cell suspension mode. 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