Improved Mass Cytometry

Abstract

[0006] Embodiments of the present invention relate to replacing previous ICP-based ionization systems with new laser ionization systems, and provide improved mass spectrometer-based devices and methods for using them to analyze samples, particularly the use of mass spectrometry mass cytometry, imaging mass spectrometry, and imaging mass cytometry for the analysis of biological samples. Accordingly, embodiments of the present invention provide a device, e.g., a mass cytometer, that includes: (1) a sampler; (2) a laser ionization system that receives material removed from the sample by the sampler, the laser ionization system including an ionization system conduit and a pulsed laser adapted to ionize sample material passing through or exiting the ionization system conduit; and (3) a mass spectrometer that receives elemental ions from the ionization system and analyzes the elemental ions.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Application No. 62 / 862,849, filed June 18, 2019, the contents of which are incorporated herein by reference for all purposes.

[0002] (Technical field) The present embodiment relates to the analysis of samples using mass cytometry and / or elemental mass spectrometry. [Background technology]

[0003] The ability to analyze a single cell (or other particle, such as a single bead) is useful because it is possible to determine the properties of each element of a population individually. This analysis therefore provides greater insight than a single measurement that is a simple average of the properties of each element of the population.

[0004] Fluorescence-activated cell sorters (FACS) can measure the properties of cells or particles by scanning them as they pass through a laser beam. By labeling cells or particles with fluorescent dyes specific to cellular components, e.g., receptors on the cell surface and DNA in the cell nucleus, the amount of labeled components can be detected as fluorescence as the particle or cell crosses the excitation beam. Because the amount of emitted fluorescence is proportional to the amount of fluorescent probe bound to the cell / antigen, antibodies conjugated to fluorescent dyes are routinely used as reagents to qualitatively and quantitatively measure antigens on and within cells. Deficiencies in this approach relate to limitations and difficulties of cell staining methods as well as the spectral overlap of and fluorescent dyes. In other words, the emission of detected fluorescent dyes is not all at a specific wavelength, which means that when multiple labels are used, some of the detected emissions may be erroneously assigned to the incorrect label. This therefore limits the discriminatory power of the technique.

[0005] A technique that overcomes this problem is mass cytometry. It is similar to flow cytometry in that a label specifically binds to the material being analyzed. The label specifically targets an antigen on a cell or particle using a specific binding partner, such as an antibody. The label is a difference between flow cytometry and mass cytometry. In mass cytometry, one or more detectable labels are atoms of known specific mass, usually transition metals such as rare earth metals. Thus, when a detectable label atom is detected by MS, it can be inferred that the target of the specific binding partner is present in the sample being analyzed.

[0006] A related technique is imaging mass cytometry (IMC). Here, material is ablated from a biological sample, e.g. a tissue sample, rather than introducing cells in suspension into a mass cytometer as a suspension, and the ablated material is analyzed by a mass spectrometer. Similarly, elemental labels used to tag specific targets on the sample can be detected by MS and their presence in the ablated material can be inferred. By recording the location on the sample where ablation took place and the amount of elemental tag detected, it is possible to build up a picture of the distribution of targets in the sample. Similarly, as with mass cytometry of liquid samples, it is possible to detect many different elemental tags simultaneously using MS, which means performing highly multiplexed imaging.

[0007] Simultaneously, a map of the elements present in the tissue in its native state can also be recorded by imaging mass spectrometry, such as laser ablation ICP-MS of tissue sections. Summary of the Invention

[0008] For example, an instrument, such as a mass spectrometer, mass cytometer, imaging mass spectrometer, imaging mass cytometer, etc., typically includes three core components in embodiments.

[0009] The first is a sampler for introducing material from the sample to the other components of the device. After being taken into the device, the sample needs to be ionized before the atoms in the sample (including the detectable label atoms) can be detected by the mass analysis component (MS component; third component). Therefore, the device comprises a second component, which is an ionization system that ionizes the atoms to enable detection by the MS component based on mass / charge ratio. In this way, the sample is taken into the device, ionized by the ion source, and the ions of the sample are passed to the MS component. Although the MS component may detect many ions and may be adapted to do so in certain applications, most of these ions will be ions of the atoms that make up the sample. In some applications, such as the analysis of minerals in geological or archaeological applications, this may be sufficient.

[0010] In some cases, for example, when analyzing biological samples, the original elemental composition of the sample may not provide adequate information. This is because all proteins and nucleic acids are usually composed of the same main atoms, so that it is not possible to distinguish a particular protein from all other proteins, even if it is possible to distinguish between regions that contain proteins / nucleic acids and regions that do not contain such protein or nucleic acid material. However, certain characteristics of the sample can be determined by labeling the sample with atoms that are not present under normal conditions in the material being analyzed, or at least not present in large amounts (e.g., certain transition metal atoms such as rare earth metals, see labeling section below for details). As with IHC and FISH, detectable labels can be attached to specific targets on or in the sample (such as fixed cells or tissue samples on a slide) by using SBPs such as antibodies, nucleic acids or lectins, among others, that target molecules on or in the sample. As with detecting ions from atoms naturally present in the sample, a detector system is used to detect the ionized labels. Typically, when analyzing liquid samples, particles such as cells in solution are introduced into the ionization system one at a time, so that the resulting atoms detected by the MS component can be assigned to specific particles, allowing each analyzed portion of the sample to be characterized individually. However, in some cases where mass cytometry is performed on liquid samples, multiple particles in solution are intentionally ionized and detected simultaneously. If the sample is a solid biological sample such as tissue, then a plume of material is typically generated from the tissue sample using laser ablation for introduction into the ionization system and subsequent ion detection by the MS component. Here, each plume generated from the solid sample is analyzed plume by plume in a manner similar to the individual analysis of each particle in a liquid sample. Imaging of biological samples via LA-ICP-MS has previously been reported at cellular resolution.

[0011] Many current mass spectrometers, including mass cytometers, use an inductively coupled plasma (ICP) as the ionization system before the ionized material is introduced into the MS. The ICP is held in an ICP torch, as shown in FIG. 1. The mass analyzer can separate the one atomic mass unit ions generated by the ionization system with minimal interference between mass channels. Any type of mass analyzer can be used as the mass analyzer component of the systems described herein, for example, a time-of-flight (TOF) detector is useful.

[0012] The present invention relates to replacing conventional ICP-based ionization systems with new laser ionization systems and providing improved mass spectrometer-based devices and methods for analyzing samples therewith, in particular the use of mass spectrometry mass cytometry, imaging mass spectrometry, imaging mass cytometry for the analysis of biological samples. Accordingly, the present invention relates to: (1) A sampler; (2) a laser ionization system that receives material removed from the sample by the sampler, the laser ionization system comprising an ionization system conduit and a pulsed laser adapted to ionize sample material passing through or exiting the ionization system conduit; (3) a mass spectrometer that receives the elemental ions from the ionization system and analyzes the elemental ions; The present invention provides an apparatus, such as a mass cytometer, comprising:

[0013] Referring to FIG. 2, which illustrates one embodiment of the present apparatus, the apparatus includes a sampler 100 that provides a sample material for ionization by a laser ionization system 200. The sampler 100 is in communication with the laser ionization system 200, and the sample material passes from the sampler 100 to the laser ionization system 200 through a conduit. The sample material can be liquid and / or gas. The sample material is carried along the conduit, at least in part, by a carrier gas, such as a cloud of gaseous material in a carrier gas or droplets of liquid in a carrier gas. The laser ionization system 200 includes an ionization system conduit 210 and a laser 220. The material is ionized in the ionization system conduit 210 by laser light 221 from the laser 220. In some systems, the laser 220 is a pulsed laser, such as a femtosecond laser. After ionization, the ions are detected by a mass spectrometer 300. Many types of mass spectrometers are suitable for use with the present apparatus, such as, for example, a TOF detector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] (Sample introduction) The sampler of the device can take a variety of forms appropriate to the sample to be analyzed.

[0015] When the sample is liquid, then the sampler that introduces the sample to the laser ionization system can be a sample loop. The sample loop is used to store liquid manually injected into the mass cytometer by the user, and then a pump can drive the sample to the laser ionization system for ionization and subsequent analysis. The liquid sample usually contains particles such as cells (prokaryotic or eukaryotic) or viruses, which are introduced into the laser ionization system. The use of a loop is advantageous when the user wants to quickly introduce a sample into the mass cytometer.

[0016] In some mass cytometer embodiments of the present invention, an autosampler is used as the sampler. The autosampler automates the process of introducing samples into the mass cytometer system for subsequent analysis. The autosampler is a robotic component that introduces a precise volume (which may be user-defined) of sample into the system from one or more containers outside the system. Therefore, the autosampler allows multiple samples to be subjected to mass cytometry without the user having to monitor the system, and provides greater accuracy and reproducibility than manual sample introduction.

[0017] Autosamplers are commercially available, for example for gas or liquid chromatography analysis (such as those manufactured by Agilent or Thermo Scientific) and for flow cytometry (such as the HyperCyt® autosampler manufactured by Intellicyt or autosamplers manufactured by Life Technologies).

[0018] In some embodiments, the samples analyzed are solid samples, such as geological mineral samples, or biological samples on solid substrates (e.g., tissue sections or monolayers of cells or individual cells, such as when a cell suspension is dispensed onto a microscope slide, as described in more detail below). These samples are introduced into the laser ionization system by a laser ablation (LA) system. Thus, in some embodiments, the sampler is an LA system. The use of an LA system allows imaging of the sample. Different target molecules within the sample are labeled with different labeling atoms, and laser ablation is performed across multiple cells of the labeled tissue sample. The method allows the labeled target molecules to be localized to specific locations on the sample by linking the detected signals to the known locations of the laser ablations that generated those signals, building up an image of the sample.

[0019] (A component of imaging devices such as LA-mass cytometers) (Laser Ablation Sampling System) Briefly, the components of a laser ablation sampling system include a laser source that emits a beam of laser radiation that is directed at a sample. The sample is positioned on a stage within a chamber (sample chamber) of the laser ablation sampling system. The stage is typically a translation stage so that the sample can move relative to the beam of laser radiation, allowing different locations on the sample to be sampled for analysis. As described in more detail below, a gas flows through the sample chamber, and the gas flow carries away the plume of aerosolized material that is generated as the laser source ablates the sample for analysis and construction of an image of the sample based on its elemental composition (including labeled atoms, such as labeled atoms from an elemental tag). In another mode of operation, the laser system of the laser ablation sampling system can be used to desorb material from the sample, as described in more detail below.

[0020] Especially in the case of biological samples (cells, tissue sections, etc.), the samples are often heterogeneous (although heterogeneous samples are known in other fields of application of this disclosure, i.e. samples of a non-biological nature). A heterogeneous sample is one that contains regions composed of different substances, so that some regions of the sample can be ablated at a lower threshold fluence at a given wavelength than other regions. Factors that affect the ablation threshold are the absorption coefficient of the material and the mechanical strength of the material. In the case of biological tissue, the absorption coefficient will have a large effect, since it can vary by several orders of magnitude depending on the wavelength of the laser radiation. For example, in a biological sample, when using nanosecond laser pulses, regions containing proteinaceous materials absorb more readily in the 200-230 nm wavelength range, while regions containing mainly DNA absorb more readily in the 260-280 nm wavelength range.

[0021] It is possible to perform laser ablation with a fluence close to the ablation threshold of the sample material. When ablation is performed in this way, the formation of the aerosol can often be improved, which can consequently help to improve the quality of the data after analysis. To obtain the smallest craters and thus maximize the resolution of the images obtained, a Gaussian beam is often used. The cross section of a Gaussian beam records an energy density profile with a Gaussian distribution. In that case, the fluence of the beam varies depending on the distance from the center. As a result, the diameter of the ablation spot size is a function of (i) the Gaussian beam waist (1 / e 2 ) and (ii) the ratio of the applied fluence to the threshold fluence.

[0022] Therefore, to consistently remove a reproducible amount of material with each ablation laser pulse and maximize the quality of the imaging data, it is useful to maintain a consistent ablation diameter, which means adjusting the ratio of the energy delivered to the target by the laser pulse to the ablation threshold energy of the material being ablated. This requirement becomes problematic when ablating heterogeneous samples where the ablation threshold energy varies across the sample, such as biological tissues where the ratio of DNA to protein varies or geological samples where it varies depending on the specific composition of minerals in the region of the sample. To address this, multiple wavelengths of laser radiation can be focused onto the same ablation spot on the sample to more effectively ablate the sample based on the composition of the sample at that spot.

[0023] (Laser) In general, the choice of wavelength and power of the laser used for ablation of the sample may follow the usual usage in cell analysis. The laser must have sufficient fluence to cause ablation to the desired depth without substantially ablating the sample carrier. Typically, it is 0.1-5 J / cm. 2 Laser fluence of, for example, 3-4 J / cm 2 or about 3.5 J / cm 2A laser fluence of 100 ns is suitable, and the laser is ideally capable of generating pulses with this fluence at a rate of 200 Hz or higher. In some instances, a single laser pulse from such a laser should be sufficient to ablate cellular material for analysis, such that the frequency of the laser pulse matches the frequency at which the ablation plume is generated. In general, to be a useful laser for imaging biological samples, the laser should generate pulses with durations of less than 100 ns (preferably less than 1 ns) that can be focused, for example, to a specific spot size as described herein.

[0024] For example, the frequency of ablation by the laser system may be within the range of 200 Hz to 100 MHz, 200 Hz to 10 MHz, 200 Hz to 1 MHz, 200 Hz to 100 kHz, 500 to 50 kHz, or 1 kHz to 10 kHz.

[0025] If it is desirable to resolve each ablation plume individually (which may not always be desirable when firing a burst of pulses at the sample, as shown below), the instrument must be able to analyze the ablated material at these frequencies quickly enough to avoid signal overlap between successive ablations. The overlap between signals resulting from successive plumes should be less than 10% intensity, more preferably less than 5%, and ideally less than 2% intensity. The time required to analyze the plume depends on the washout time of the sample chamber (see Sample Chamber section below), the transit time of the plume aerosol through the laser ionization system, and the time it takes to analyze the ionized material. Each laser pulse can be correlated to a pixel on the image of the sample that is subsequently constructed, as detailed below.

[0026] In some embodiments, the laser source is an ultrafast laser (1 ps (10 -12Femtosecond lasers have pulse durations of up to 1000 s (fs) or faster. Ultrafast pulse durations offer many advantages as they limit heat diffusion from the ablation region, thereby providing more precise and reliable ablation craters, as well as minimizing debris scattering from each ablation event. Femtosecond lasers are particularly useful in the systems and devices described herein.

[0027] In some examples, femtosecond lasers are used as laser sources. Femtosecond lasers are lasers that emit light pulses with durations less than 1 ps. To generate such short pulses, a technique called passive mode-locking is often used. Femtosecond lasers can be generated using various types of lasers. Typical durations of 30 fs to 30 ps can be achieved using passively mode-locked solid-state bulk lasers. Similarly, various diode-pumped lasers, for example based on neodymium or ytterbium-doped gain media, also operate in this region. Titanium sapphire lasers with advanced dispersion compensation are also suitable for pulse durations less than 10 fs, reaching up to about 5 fs in extreme cases. Pulse repetition rates are most often between 10 MHz and 500 MHz, but to obtain higher pulse energies, low repetition rate versions with repetition rates of several megahertz also exist (available from Lumentum (CA, USA), Radiantis (Spain), Coherent (CA, USA), etc.). This type of laser can be accompanied by an amplification system that increases the pulse energy.

[0028] There are many different types of ultrafast fiber lasers, but they are also mostly passively mode-locked and typically offer pulse durations of 50-500 fs and repetition rates of 10-100 MHz. Such lasers are commercially available from companies such as NKT Photonics (formerly Fianium, Denmark), Amplitude Systems (France), and Laser-Femto (California, USA). The pulse energy of this type of laser can be increased by amplifiers, often in the form of integrated fiber amplifiers.

[0029] Some mode-locked diode lasers can generate pulses of femtosecond duration. Considering the output of the laser directly, the pulse duration is typically on the order of a few hundred femtoseconds (e.g., available from Coherent, California, USA).

[0030] In some examples, picosecond lasers are used. Many types of lasers already described in the previous text can also be adapted to generate pulses with durations in the picosecond range. The most common light sources are actively or passively mode-locked solid-state bulk lasers, such as passively mode-locked Nd-doped YAG, glass or vanadium lasers. Similarly, picosecond mode-locked lasers and laser diodes are commercially available (e.g., NKT Photonics (Denmark), EKSPLA (Lithuania), etc.).

[0031] Alternatively, a continuous wave laser can be used that is externally modulated to produce sub-nanosecond duration pulses.

[0032] Typically, the laser beams used for ablation in the laser systems described herein have a spot size of 100 μm or less, i.e., at the sampling location, e.g., 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less, e.g., about 3 μm or less, about 2 μm or less, about 1 μm or less, etc. The distance referred to as the spot size corresponds to the longest internal dimension of the beam, e.g., the beam diameter in the case of a circular beam, the length of the diagonal between the opposing corners in the case of a square beam, the length of the longest diagonal in the case of a rectangle, etc. (As mentioned above, the diameter of a circular beam with a Gaussian distribution is the distance when the fluence is 1 / e of the peak fluence. 2(defined as the distance between points reduced by a factor of 100). Instead of a Gaussian beam, beam shaping and beam masking may be used to provide the desired ablation spot. For example, in some applications, a square ablation spot with a top-hat energy distribution (i.e., a beam with a nearly uniform fluence, as opposed to a Gaussian energy distribution) may be useful. This arrangement reduces the dependence of the ablation spot size on the ratio of the fluence at the peak of the Gaussian energy distribution to the threshold fluence. Ablation at a fluence close to the threshold fluence results in more reliable generation of ablation craters and controls debris generation. Thus, the laser system may include beam masking and / or beam shaping components, such as diffractive optical elements, arranged on the Gaussian beam to reshape the beam to generate a laser focal spot of uniform or nearly uniform fluence, e.g., varying less than ±25% across the beam, e.g., varying less than ±20%, less than ±15%, less than ±10%, or less than ±5%. In some cases, the laser beam has a square cross-sectional shape. In some cases, the beam has a top-hat energy distribution. As mentioned above, in the context of embodiments of the present invention, the distance referred to as the spot size is the longest internal dimension of the beam at the sampling location, i.e., the spot size is the lateral dimension of the beam, e.g., the spot size of a circular beam is the diameter. However, since the focal spot of an objective lens is a three-dimensional volume and the axial dimension of the focal spot size is generally longer than the lateral dimension, those skilled in the art will understand that in the context of embodiments of the present invention, the axial dimension of the focal spot may be longer than the distance referred to as the "spot size".

[0033] When analyzing biological samples, the spot size of the laser beam used to analyze individual cells depends on the size and spacing of the cells. For example, if the cells are closely packed together (such as in a tissue section), one or more laser sources of the laser system may have a spot size that is no larger than the cells. This size depends on the particular cells in the sample, but generally the diameter of the laser spot is less than 4 μm, e.g., about 3 μm or less, about 2 μm or less, about 1 μm or less. To analyze a given cell with subcellular resolution, the system uses a laser spot size that is no larger than the cells, and more specifically, a laser spot size that can ablate material with subcellular resolution. The smaller the spot size, the higher the resolution of the image. In some cases, for example, when the cells are scattered on the slide with spaces between the cells, single-cell analysis may be performed using a spot size larger than the size of the cells. In this case, the additional ablated area around the cell of interest does not contain additional cells, so a larger spot size may be used to achieve single-cell characteristics. Thus, the particular spot size used may be appropriately selected depending on the size of the cells to be analyzed. In a biological sample, the cells are almost never all the same size. Therefore, if imaging with subcellular resolution is desired, the ablation spot size should be smaller than the smallest cell if a constant spot size is maintained throughout the ablation procedure. Small spot sizes can be achieved using laser beam focusing. A laser spot diameter of 1 μm corresponds to a laser focus (i.e., the diameter at the focus of the laser beam) of 1 μm, but the laser focus can vary by + / - 20% or more due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and variations in the total energy of the laser relative to the threshold energy for ablation.

[0034] (Laser ablation focus) For the laser to maximize the efficiency of ablating material from the sample, the sample should be in a suitable position relative to the focus of the laser, e.g., at the focus. This is because the focus is where the laser beam has the smallest diameter and the most concentrated energy. This can be achieved in several ways. The first way is that the sample can be moved with the axis of the laser light directed at it (i.e., up and down the path of the laser light / towards and away from the laser source) to the desired point where the light has sufficient intensity to perform the desired ablation effect. Alternatively or in addition, a lens can be used to move the focus of the laser light, thus using the effective ability of the lens to ablate material at the location of the sample, e.g., by demagnification. One or more lenses are placed between the laser and the sample stage. A third way, which can be used alone or in combination with either or both of the two previous ways, can be to change the position of the laser.

[0035] To assist the user of the system in positioning the sample in the most suitable position for ablation of material, a camera may be directed at the stage holding the sample (described in detail below). Thus, the present disclosure provides a laser ablation sampling system with a camera directed at the sample stage. The image detected by the camera may be focused on the same point that the laser is focused on. This may be achieved by using the same objective lens for both laser ablation and optical imaging. The user may ensure that the laser ablation is most effective when the optical image is in focus by matching the two focal points. Precise movement of the stage to focus on the sample may be performed using piezo activators available from Physik Instrumente, Cedrat-technologies, Thorlabs and other suppliers.

[0036] (Sample chamber of laser ablation sampling system) When laser ablation is performed, the sample is placed in a sample chamber. The sample chamber includes a stage that holds the sample (usually the sample is on a sample carrier). When ablated, the material in the sample forms a plume, and the flow of gas in the sample chamber from the gas inlet to the gas outlet carries the plume of aerosolized material, including the labeling atoms, at the ablated location. The gas carries the material to an ionization system, which ionizes the material and allows detection by a detector. The atoms in the sample, including the labeling atoms, can be identified by the detector, so that their detection reveals the presence or absence of multiple targets in the plume and a determination of what targets were present at the ablated location on the sample. Thus, the sample chamber serves a dual role, not only to house the solid sample to be analyzed, but also as a starting point for moving the aerosolized material to the ionization and detection systems. This means that the flow of gas in the chamber can affect the spread of the plume of ablated material as it passes through the system. The washout time of the sample chamber is an indication of how far the ablated plume spreads. This number is a measurement of the time it takes for material ablated from the sample to be carried out of the sample chamber by the gas flowing through the chamber.

[0037] Thus, the spatial resolution of the signal generated from laser ablation (i.e., when ablation is used for imaging and not just removal, as described below) depends on factors including (i) the spot size of the laser when the signal is integrated over the entire area being ablated and the relationship between the speed at which the plumes are generated and the movement of the sample relative to the laser, and (ii) the speed at which the plumes can be analyzed relative to the rate at which they are generated, such that overlap of signals from successive plumes is avoided, as described above. Thus, when separate analysis of plumes is desired, allowing the plumes to be analyzed in as short a time as possible minimizes the chance of plume overlap (and thus allows plumes to be generated more frequently).

[0038] Therefore, sample chambers with short washout times (e.g., 100 ms or less) are advantageous for use in the devices and methods disclosed herein. Sample chambers with long washout times limit either the speed at which images can be generated or lead to overlap between signals generated from successive sample spots (e.g., Kindness et al. (2003; Clin Chem 49:1916-23) had signal durations of more than 10 seconds). Therefore, the aerosol washout time becomes a significant limiting factor for achieving high resolution without increasing total scan time. Sample chambers with washout times of 100 ms or less are known in the art. For example, Gurevich & Hergenroeder (2007; J. Anal. At. Spectrom. 22:1043-1050) disclose sample chambers with washout times of less than 100 ms. A sample chamber is disclosed in Wang et al. (2013; Anal. Chem. 85:10107-16) (see also WO 2014 / 146724) that has a washout time of 30 ms or less, allowing for high ablation frequencies (e.g., greater than 20 Hz) and therefore rapid analysis. Another such sample chamber is disclosed in WO 2014 / 127034. The sample chamber in WO 2014 / 127034 comprises a sample capture cell configured to be operatively positioned adjacent to a target, the sample capture cell including a capture cavity having an opening formed in a surface of the capture cell, the capture cavity configured to receive the target material released or generated from the laser ablation site through the opening, and a guide wall exposed in the capture cavity and configured to direct the flow of carrier gas in the capture cavity from the inlet to the outlet such that at least a portion of the target material received in the capture cavity is transferred to the outlet as a sample. The volume of the capture cavity in the sample chamber in WO 2014 / 127034 is 1 cm 3 Less than 0.005 cm3 In some cases, the sample chamber has a washout time of 25 ms or less. For example, the sample chamber has a washout time of 20 ms, 10 ms or less, 5 ms or less, 2 ms or less, 1 ms or less, or 500 μs or less, 200 μs or less, 100 μs or less, 50 μs or less, or 25 μs or less. For example, the sample chamber may have a washout time of 10 μs or more. Typically, the sample chamber has a washout time of 5 ms or less.

[0039] For completeness, in some cases, plumes from the sample may occur more frequently than the washout time of the sample chamber, causing smearing in the resulting images (e.g., when the highest possible resolution is not required for a particular analysis).

[0040] The sample chamber typically comprises a translation stage for holding the sample (and sample carrier) and for moving the sample relative to the beam of laser radiation. When using a mode of operation which requires irradiation of the sample with laser radiation via the sample carrier, the stage holding the sample carrier should also be transparent to the laser radiation used.

[0041] In this way, the sample may be located on the side of the sample carrier (e.g., a glass slide) that faces the laser radiation when the laser radiation is applied to the sample, such that the ablation plume is emitted and captured on the same side as the laser radiation is applied to the sample, or the sample may be located on the opposite side to the side on which the laser radiation is applied to the sample (and the ablation plume is emitted and captured on the opposite side to the laser light, i.e., the laser radiation passes through the sample carrier before reaching the sample).

[0042] One feature of the sample chamber that is particularly useful for ablating specific portions of various discrete regions of a sample is the large range of movement in which the sample can be moved in the x and y axes (i.e. horizontally) relative to the laser (the laser beam is irradiated on the sample in the z axis), where the x and y axes are perpendicular to each other. By moving the stage in the sample chamber and fixing the position of the laser in the laser ablation sampling system of the device, a more reliable and accurate relative position is obtained. Also, the larger the range of movement, the more distant the discrete ablation regions can be from each other. The sample is moved relative to the laser by moving the stage on which the sample is placed. Thus, the sample stage may have a range of movement in the sample chamber of at least 10 mm in the x and y axes. For example, it may have a range of movement in the sample chamber of 20 mm in the x and y axes, 30 mm in the x and y axes, 40 mm in the x and y axes, 50 mm in the x and y axes, e.g., 75 mm in the x and y axes. In some cases, the range of motion is such that the entire surface of a standard 25 mm x 75 mm microscope slide can be analyzed within the chamber. Of course, in addition to a wide range of motion, precise motion is necessary to make intracellular ablation feasible. Thus, the stage can be configured to move the sample in increments of less than 10 μm in the x and y axes, such as less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, less than 1 μm or less, less than 500 nm, less than 200 nm, less than 100 nm. For example, the stage can be configured to move the sample in increments of at least 50 nm. Precise stage movements can be performed in increments of about 1 μm, such as 1 μm ± 0.1 μm. Commercially available microscope stages, such as those available from Thorlabs, Prior Scientific, and Applied Scientific Instrumentation, can be used. Alternatively, a motorized stage can be constructed from components based on a positioner that provides the desired range of motion and suitable precision of motion, such as Smaract's SLC-24 positioner. The speed of movement of the sample stage can also affect the speed of analysis.Therefore, the sample stage has a movement speed of more than 1 mm / s, for example, 10 mm / s, 50 mm / s, 100 mm / s, etc.

[0043] Of course, when the sample stage within the sample chamber has a large range of movement, the sample chamber must be sized to adequately accommodate the movement of the stage. Thus, the sizing of the sample chamber depends on the size of the associated sample, which in turn determines the size of the movable sample stage. Exemplary sizes of the sample chamber are 10×10 cm, 15×15 cm, or 20×20 cm internal chambers. The depth of the chamber can be 3 cm, 4 cm, or 5 cm. A person skilled in the art will be able to select the appropriate dimensions following the teachings herein. The internal dimensions of the sample chamber for analyzing biological samples using a laser ablation sampler must be larger than the range of movement of the sample stage, e.g., at least 5 mm, at least 10 mm, etc. This is because if the walls of the chamber are too close to the edge of the stage, the flow of carrier gas passing through the chamber, which carries the ablated plume of material away from the sample and into the ionization system, can become turbulent. The turbulence disrupts the ablated plume, so that the plume of material begins to spread out after it has been ablated and transported to the ionization system of the device, instead of remaining as a dense cloud of ablated material. The broader peaks of ablated material adversely affect the data generated by the ionization and detection systems as they lead to interference due to peak overlap, ultimately resulting in data that is poorly resolved spatially unless the rate of ablation is slowed to a point where it is no longer of experimental interest.

[0044] As discussed above, the sample chamber includes a gas inlet and a gas outlet for introducing material into the ionization system, but may further contain ports that function as inlets or outlets for directing the flow of gas within the chamber and / or supplying a mixture of gases to the chamber, as determined by one of skill in the art to be appropriate for the particular ablation process being performed.

[0045] (camera) In addition to identifying the most effective location of the sample for laser ablation, the inclusion of a camera (e.g., a Charged Coupled Device Image Sensor-based (CCD) camera or an Active Pixel Sensor-based camera) or any other light detection means in the laser ablation sampling system allows for a variety of further analyses and techniques. The CCD is a means of detecting light and converting it into digital information to generate an image. Within a CCD image sensor, there is a series of capacitors that detect light, each capacitor representing a pixel on the determined image. These capacitors allow the incident light to be converted into an electric charge. The CCD is used to read this charge, and the recorded charge can be converted into an image. An APS (Active Pixel Sensor) is an image sensor that consists of an integrated circuit that contains an array of pixel sensors, each pixel containing a photodetector and an active amplifier, e.g., a CMOS sensor.

[0046] (Laser ablation) Generally, laser ablation is carried out in a manner as previously defined, for example, in Giesen et al., 2014 and WO2014169394, taking into account modifications herein (e.g., it is not necessary to use an ICP to ionize the sample material or to use a TOF MS detector). For example, the method may also be carried out with OES detection replacing mass spectrometry detection, as described below.

[0047] (Laser Ionization System) Conventional mass cytometry devices and many mass spectrometers use inductively coupled plasma (ICP) as a system for ionizing materials from a sample (i.e., the source of ions detected by MS). However, the ion transmission efficiency of ICP-MS is well below 1. That is, the number of ions detected from a material introduced into the ICP is much lower than would be expected if the material were fully ionized. The low transmission efficiency is related to the problem of space charge generated by ions of the carrier gas (usually argon). As explained below, the space charge effect can result in the repulsion of closely packed ions, or the space charge can lead to the formation of neutral species in the presence of particles with the opposite type of charge, resulting in them being undetectable by the mass spectrometer. Many of the ions in ICP are generated from the argon gas used to maintain the plasma at the torch tip (note that in the torch in Figure 1, an argon gas inlet labeled * has been added in addition to the flow that carries the material for ionization). Thus, an approach to generate elemental ions without a large amount of argon carrier ions and electrons generated from the carrier gas may improve the sensitivity of the instrument.

[0048] The inventors have determined that instead of using an ICP as the ionization system for mass cytometry and mass spectrometry as in standard mass cytometers, it is possible to use a laser ionization system to ionize samples. The inventors have determined that any suitable laser that is effective in converting sample material present in aerosol or vapor form into elemental ions can be used in the laser ionization system, provided that it does not generate excess ions from the carrier gas, and that picosecond and femtosecond lasers are useful for the laser ionization system of the present embodiment (femtosecond lasers are particularly useful). Laser ionization provides advantages because the energy in the laser light fragments, atomizes, and ionizes the sample material without adding significant amounts of carrier gas ions to the ion beam. This ionization system is compatible with both liquid and solid samples.

[0049] Applications in which this approach may be particularly advantageous include imaging of biological samples by imaging mass cytometry. In these applications, the biological sample is interrogated by a laser pulse (likely from a second laser in the setup), and a plume of ablated material is generated for each pixel of interest on the biological sample. The generated plume typically carries vapor and nanometer-scale aerosols from the interrogated area. According to embodiments of the invention, the vapor and aerosols may be directly ionized by providing laser irradiation of sufficient intensity to generate elemental ions from the plume material, as described in more detail below.

[0050] 2, a laser ionization system 200 generally comprises an ionization system conduit 210 and a laser system 220 arranged such that the laser system is directed at the ionization system conduit 210 to enable ionization of materials passing through the ionization system conduit by laser light 221 emitted from the laser 220. One end of the ionization system conduit communicates with a sampler 100 of the device, and an opposite end of the conduit communicates with a mass spectrometer 300 of the device. Thus, materials from the sampler are ionized by the laser light as they are conveyed to the mass spectrometer.

[0051] (Lasers used in laser ionization systems) Generally, lasers useful for ionization provide energy on the order of a few microjoules per pulse, but can be pulsed at high frequencies. The laser generates elemental ions from the sample material. The generated elemental ions can then be analyzed in a mass spectrometer within the device. The laser can be a picosecond laser or a femtosecond laser. In some embodiments, the laser is a femtosecond laser.

[0052] Femtosecond lasers can be solid-state lasers. Passively mode-locked solid-state bulk lasers can emit high-quality ultrashort pulses with typical durations of 30 fs to 30 ps. Examples of such lasers include diode-pumped lasers using neodymium-doped crystals or ytterbium-doped crystals, etc. Titanium:sapphire lasers can be used with pulse durations of less than 10 fs, down to about 5 fs in extreme cases (e.g., Octavius ​​Ti:Sapphire Lasers available from Thorlabs). Pulse repetition rates are in the range of 1 kHz to 500 MHz in most cases.

[0053] The femtosecond laser can be a fiber laser. Various types of ultrafast fiber lasers can also be passively modelocked, typically with pulse durations of 50-500 fs, repetition rates of 0.10-100 MHz, and average powers of a few milliwatts to a few watts (femtosecond fiber lasers are commercially available from Toptica, IMRA America, and Coherent, Inc.).

[0054] Femtosecond lasers can be semiconductor lasers. Some mode-locked diode lasers can generate pulses with femtosecond durations. Direct consideration of the laser's output typically produces pulse durations of at least a few hundred femtoseconds, although shorter pulse durations can be achieved by external pulse compression.

[0055] Vertical external cavity surface emitting lasers (VECSELs) can also be passively modelocked, which is interesting because it can provide a combination of particularly short pulse durations, high pulse repetition rates and potentially high average powers, but they are not suitable for high pulse energies.

[0056] Femtosecond lasers suitable for the systems and methods presented herein also include color center lasers and free electron lasers.

[0057] In some embodiments, the laser is adapted to generate pulses with nanosecond, picosecond or femtosecond scale pulse duration. For example, the laser can have a duration of 500 fs or less, such as 400 fs or less, 300 fs or less, 200 fs or less, 100 fs or less, 50 fs or less, 45 fs or less, 25 fs or less, 20 fs or less, or 10 fs or less. Femtosecond lasers are adapted to generate pulses with durations of less than 1 ps.

[0058] In some embodiments, the laser is adapted to have a pulse repetition rate of at least 100,000 Hz, e.g., at least 1 MHz, at least 2 MHz, at least 3 MHz, at least 4 MHz, at least 5 MHz, at least 10 MHz, at least 20 MHz, at least 50 MHz, at least 100 MHz, at least 200 MHz, at least 500 MHz, or 1 GHz or greater.

[0059] In some embodiments, the laser has a beam width (1 / e 2 ), e.g. beam width (1 / e 2 ). The focal point of the laser is where the energy of the beam is most concentrated, and therefore where maximum ionization is achieved.

[0060] In some embodiments, the laser is adapted to have a pulse energy of 1 nanojoule up to 50 millijoules. A laser for assisting in sputtering or ablation of a material can be adapted to have a pulse energy between 1 nanojoule and 100 microjoules, such as between 10 nanojoules and 100 microjoules, between 100 nanojoules and 10 microjoules, between 500 nanojoules and 5 microjoules, about 1 microjoule, about 2 microjoules, about 3 microjoules or about 4 microjoules. A laser for post-ionization can be adapted to have a pulse energy between 1 millijoule and 50 millijoules, such as between 5 millijoules and 40 millijoules, between 10 millijoules and 30 millijoules, between 20 millijoules and 35 millijoules, or about 25 millijoules or 35 millijoules.

[0061] In some embodiments, the laser is adapted to generate pulses having a pulse energy of about 1 millijoule, having a pulse repetition rate of at least 10 MHz, and having a duration of less than 100 fs, e.g., 50 fs or less, 45 fs or less, 25 fs or less, 20 fs or less, 10 fs or less, etc.

[0062] In addition to the benefit of reducing the number of ions from the carrier gas, laser ionization systems can be adapted to further increase ionization efficiency by avoiding space charge effects through other mechanisms. For example, laser ionization systems can be adapted to utilize multiple laser pulses of an ionizing laser to ionize the contents of a single plume or particle. When multiple pulses are used, the amount of ions generated in a single pulse is subsequently reduced.

[0063] As mentioned above, when a large amount of positive and negative charges are generated in a small volume, the motion of the formed ions is dominated by the local fields resulting from the space charges induced by the ions and electrons. If there are too many charged ions in a small volume, external magnetic fields, such as those from ion optics present in a mass spectrometer used to direct the resulting ions to a detector for detection, are not effective in separating the positive and negative charges, and such ion clouds eventually neutralize, reducing the ionization efficiency. For example, a 10 μm-scale ion cloud containing 10,000 elementary charges (diameter) will generate an electrostatic potential of about 3 V. A few eV is the energy that holds the electrons to the atoms, so it may be the energy level of the free electrons after ionization. As a result, an ion density of 10,000 ions in a 10-micrometer-scale volume is close to the limit where space charge behavior begins to dominate.

[0064] In addition to using more pulses to ionize the contents of a single plume or particle, the inventors have also identified further techniques and modifications to address this issue by controlling the amount of material ionized by the pulses of the ionizing laser. These techniques can be used alone or in combination with each other, and optionally using multiple pulses of the ionizing laser to ionize the contents of a single plume or particle. This results in fewer ions being produced, and the ion optics can perform charge separation before charge recombination and neutralization.

[0065] Then, if the sampler is a laser ablation device, the amount of material per plume can be controlled by ablating only a small amount of material to generate each plume for ionization by the laser ionization system. There are two main ways to do this. First, ablate a small spot of material from the sample, e.g., less than 4 μm, e.g., less than 3 μm, less than 2 μm, less than 1 μm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, or less than 100 nm, to generate each plume. Typically, the spot size is less than 1 μm. Second, control the depth of the ablation spot by appropriate settings of the ablation laser, e.g., adjusting the power per pulse. The resulting crater depth can be less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 100 nm, less than 50 nm, or less than 20 nm (i.e., each laser pulse ablates less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 100 nm, or less than 50 nm of material from the sample within the spot diameter of the laser pulse). If the total sample depth is greater than the sample depth ablated per laser pulse, multiple pulses can be applied to the same location of the sample to completely ablate that location of the sample.

[0066] A plume generated from a 1 × 1 μm spot and ablated to a depth of 100 nm is approximately 10 10 In this case, 10 10 Each atom is 10 4 Contains 10 atoms 6 In this case, each part can be ionized by direct laser ionization, such as femtosecond laser ionization based on the tunneling effect or the multiphoton effect. Such ionization can achieve ionization efficiencies close to 100%, so that the ionization efficiency can be as high as 10 4 atoms to 10 4ions can be generated. This number of ions is within the boundaries of the space charge, ensuring reasonable efficiency in separating positive and negative particles. For an ablation spot of 300 nm × 300 nm and a sample thickness of 100 nm, the amount of material ablated is 10 times larger and the number of ionizing laser pulses required to ionize the ablation plume is 10 times larger. 5 A further advantage is provided by reducing the amount of material ablated since the laser energy required for ablation is reduced to 0.05 keV.

[0067] When this setup was used for imaging mass cytometry or imaging mass spectrometry, one pulse of the ablation laser to generate a plume from the sample was followed by 10 pulses of the ionization laser to ionize the ablation plume in the hope of achieving nearly complete ionization of the sample. 5 The smaller the diameter of the ablation spot and / or the shallower the ablation depth, the fewer the number of pulses from the laser of the laser ionization system required to ablate the ablated plume. Thus, in some embodiments, the laser of the laser ionization system is 4 More than double, 5×10 4 Over 10 times 5 More than double, 5×10 5 Over 10 times 6 More than double or 10 6 More than 10 times the repetition rate of the laser ablation system 3 The pulse generator is configured to generate pulses at a repetition rate greater than twice the normal repetition rate.

[0068] If less ionization, for example 10% ionization, is acceptable, the corresponding number of pulses of the ionization laser may be reduced. For example, when analyzing biological samples, if two or more copies of an elemental tag are used to mark a target molecule, a reduced degree of ionization may be acceptable. For example, if 10 or more copies of an elemental tag are covalently attached to an antibody that binds to a target antigen, 10% ionization may be acceptable because at least one elemental tag is ionized and detected.

[0069] (Ionization system conduit) To maximize ionization, the plume generated by ablation may be expanded so that it can be ionized by a series of pulses from an ionizing laser. Several parameters may be employed to increase the volume of the plume. Thus, in one embodiment of the apparatus, the sampler comprises a laser ablation system configured to generate a plume, and the apparatus includes an arrangement to increase the volume of the plume prior to laser ionization.

[0070] One parameter is the pressure in the ablation chamber of the ablation system. When ablating a sample, the plume volume of the resulting ablation plume when it stops expanding is inversely proportional to the pressure in the ablation chamber (from which the ionization system conduit carries the ablation plume). Thus, the ablation chamber can be maintained at a pressure lower than atmospheric pressure. For example, operating at 0.5 atm instead of 1 atm increases the gas volume of the ablation plume by a factor of two before the plume enters the ionization system conduit. The ablation chamber can be operated at less than 0.5 atm, for example, less than 0.1 atm or less than 0.05 atm.

[0071] The second parameter is the residence time of the sample in the ionization system conduit between the ablation chamber and the point where ionization occurs. As the ablation plume is transported along the conduit from the ablation chamber, it diffuses, with the degree of diffusion being proportional to the time the plume spends in the gas flow (the plume's residence time). Thus, the longer the conduit, the more diffuse and thus the wider the plume that is suitable for efficient ionization (when a more diffuse plume is ionized, fewer ions are produced per unit volume, and therefore the charge neutralization effect is less likely to be affected). Similarly, slowing the flow of gas in the conduit increases the residence time and promotes greater diffusion of the ablation plume. This can be controlled by the volume of gas introduced into the mass cytometer or mass spectrometer system per unit time and the diameter of the conduit between the ablation chamber and the ionization system. Variation of these parameters to appropriately control diffusion can be easily performed by one skilled in the art.

[0072] In some embodiments, reshaping of the plume of ionized sample material can be done similarly to or instead of relying on diffusion to reduce space charge effects. The length of time it takes to analyze material from an event (e.g., an individual ablation plume) depends on how diffuse the plume of material is, and therefore how diffuse the resulting ion cloud is; a more diffuse cloud will take longer to analyze.

[0073] Because the size of the volume that can be ionized by a femtosecond laser is typically on the micrometer scale (typically smaller than the plume generated by ablation of, e.g., a 1 μm spot), the gaseous sample material can be reshaped from the spherical shape generated by diffusion into an elongated thread shape that is only a few micrometers in diameter in order to maximize the efficiency of ionization.

[0074] A simple approach to reshape the plume of sample material is to utilize an ionization system conduit that is tapered towards the output direction. This is shown in Figure 3A-D, where a plume of sample material 400 is shown traveling through a carrier gas flowing along the ionization system conduit 210. As the cloud passes through the taper 420 of the ionization system conduit 210, its width narrows but its length increases. For example, the inner diameter of the ionization system conduit may be x μm at the input end of the conduit, but may taper down to x / 10 μm near the output end (e.g., 300 μm at the input end and 30 μm at the output end). The taper may occur along the length of the ionization system conduit, or it may only taper part of the conduit (i.e., the conduit has the same inner diameter for a first portion of its length, followed by a second portion where the inner diameter decreases). If the plume in the ionization system conduit expands to a diameter of 100 μm in the first section, the plume maintains approximately the same diameter ratio as it passes through the tapered section. Thus, if the diameter at the beginning of the taper is 10 times that at the end of the taper, the diameter of the plume passing through the tapered section is 10 μm. At the same time, in this example, the length of the plume increases from 100 μm to 10 mm, since the volume of the plume is preserved. Thus, in some embodiments, the ionization system conduit includes a tapered section, and the inner diameter of the conduit decreases by more than two times throughout the taper, such as by more than two times, more than three times, more than four times, more than five times, more than ten times, more than twenty times, or more than fifty times. The inner diameter here is the measurement of the longest cross section through the conduit. For example, if the conduit is circular, the inner diameter is simply the diameter of the circle, but if the conduit is rectangular, it is the diameter.

[0075] In some embodiments, the flow of the carrier gas in the tapered section is near sonic or even supersonic. This provides the highest flow velocity to elongate the plume. This also reduces the residence time in the tapered section, thus minimizing undesirable diffusion broadening that can lead to loss of ions in this part of the device. In some embodiments, the ionizing laser is positioned to ionize the sample at this narrowest point (where the gas is fastest in the taper). The higher flow velocity allows for a higher repetition rate of the laser used to ionize the sample material without hitting the portion of the sample ionized by the previous pulse. By doing so, the plume can be divided into smaller parts suitable for minimizing space charge effects that lead to post-ionization neutralization as described above.

[0076] (Maximizing the efficiency of laser light usage) By appropriate design of the laser optics and ionization system conduit, it is possible to direct the laser of the laser ionization system so that it reflects through the sample material passing through and / or exiting the ionization system conduit, such that the laser light can be focused multiple times into the sample material, thereby resulting in the most efficient use of the laser light for ionizing the sample passing through the conduit and / or the sample material exiting the conduit.

[0077] Thus, in some embodiments, the laser ionization system further comprises a reflector arrangement for making multiple passes of the laser light generated by the laser through the sample. In some cases, the reflector arrangement comprises one or more reflectors shaped to provide multiple focal points within the ionization conduit, and in some cases arranged to provide multiple focal points at the exit of the ionization conduit (usually where the sample material has not expanded from the narrow diameter shape into which it was forced by the ionization system conduit). This is illustrated in Figures 4 and 5. In Figure 4, the laser light 221 is shown reflected within the ionization conduit 210 according to a taper 420 by reflectors arranged such that multiple focal points are formed 430 along the conduit.

[0078] To maximize the use of the laser light, the laser light may be introduced into a multi-pass cell. A typical multi-pass cell utilizes a convex reflector 500 arranged so that the light is reflected multiple times through the same space in the ionization conduit 210 to ionize the sample material 400 passing along the conduit. In doing so, the laser light may be focused to multiple focal points within the ionization conduit (FIG. 5A) or at the exit of the tapered end of the conduit between the conduit and the skimmer or sampler cone of the MS 520 (FIG. 5B), so that ionization of the sample material with the same laser light may be performed multiple times, maximizing absorption by the sample to produce elemental ions for downstream analysis by the MS. Thus, in some embodiments, the reflector arrangement comprises at least one convex reflector. As shown diagrammatically in FIG. 5, multiple convex reflectors may be arranged along the length of the ionization conduit to increase the number of focal points. Although no focal points are shown in FIG. 5, similar to FIG. 4, the reflectors are arranged to focus the light at the center of the conduit where the amount of material to be ionized is greatest.

[0079] In some embodiments, the laser light is launched into a tapered section of a conduit that acts as a waveguide, such that the energy density reaches a suitable level for ionization only in the tapered section. This is shown in Figure 6, where the laser light 211 leaves the laser 210 and, as the taper narrows, is reflected off the inner surface of the taper towards the narrow end of the taper until it reaches a density of laser light that can ionize the sample material 610.

[0080] Referring to FIG. 7, in some embodiments, the tapered conduit has a divergence 710 after narrowing. The laser light 221 is launched from the diverging side towards the end of the taper 420 (the ionizing laser is positioned to irradiate the laser light from the diverging side of the taper). The angle of the divergence can be adjusted to the numerical aperture of the objective lens used to focus the laser light. A wider angle allows the use of lenses with a higher numerical aperture and helps to minimize the diameter of the laser focus. Ions created by ionization travel along with the gas flow. The ions can be deflected by the ion optics to change their trajectory from the direction of the gas flow so that they do not collide with the objective lens. In some embodiments, the objective lens can have a hole in the center to allow the ions to pass through.

[0081] (Ionization of liquid samples using a multi-laser system) If the sample is liquid, particles such as cells or viruses are broken down into smaller particles by a first laser upstream of the ionizing laser. The ionizing laser can then ionize the particles to produce elemental ions as described above. The laser can act to break down the cells while they are in the liquid. Alternatively, the cells can be fragmented in the gas phase. Here, the liquid sample can be atomized to produce gas phase cells, which are broken down by the first laser into fragments that can be ionized by the ionizing laser. Another method of fragmenting in the gas phase is to introduce the cells into a gas stream using a drop-on-demand device (http: / / www.microfab.com / dispensing-devices, http: / / www.polypico.com / wp-content / uploads / 2015 / 06 / Poly-Pico_Brochure_-2015.pdf) or a device based on an inkjet printer head. In some embodiments, two or more lasers are used to break down the cells into fragments that can be ionized by an ionizing laser, such as two, three, four, or five or more lasers. For example, a first laser may break the sample into fragments of a first diameter, and a second laser located downstream of the first laser may break the fragments of the first diameter into fragments of even smaller diameters. The same principle applies when there are more than two lasers, with each laser breaking the fragments into smaller fragments. The pre-ionized fragments are in a cloud of material that can be manipulated in the same way as the plume described above, especially when discussing tapered conduits using the laser ablation embodiment as an example. All the features described above in relation to this device may be applied to the analysis of single cells or particles in liquid samples, for example mass cytometers.

[0082] (Laser Ablation with Laser Ionization) As described above, when the sampler is a laser ablation system, the sampler is configured to generate a plume of sample material and the laser ionization system is configured to ionize it. In some embodiments, the laser ionization system is configured to generate multiple laser pulses to ionize a single plume. In some embodiments, the laser ionization system is configured to generate multiple laser pulses to ionize a single plume. 4 More than double, 5×10 4 Over 10 times 5 More than double, 5×10 5 Over 10 times 6 More than 10 times the repetition rate of the laser ablation system 3 The pulse generator is configured to generate pulses at a repetition rate greater than twice the normal repetition rate.

[0083] In some embodiments, the apparatus includes a single laser, which is a laser of a laser ionization system adapted to ablate a solid sample to generate a plume, and ionizes the plume of material. In this arrangement, the repetition rate of the laser pulse in the laser ionization system must often be several orders of magnitude greater than the repetition rate of the laser in the ablation chamber to generate a plume of ionized material. For this reason, when the same laser is used for ablation and ionization, the apparatus further includes a pulse picker (available from Del Mar photonics, KMLabs, EKSMA Optics) configured to control the laser pulses directed to the ablation chamber. In some embodiments, the apparatus includes a laser pulse power control module programmed to control the power of the laser pulses for ablation and ionization. In some embodiments, the apparatus includes two optical attenuators (commercially available from Thorlabs, Newport, etc.), one for the ablation laser pulses and one for the ionization laser pulses, to independently control the power of each type of pulse.

[0084] (Mass Detector System) Exemplary types of mass detector systems include quadrupole, time-of-flight (TOF), magnetic sector, high-resolution, single or multi-collector based mass spectrometers. In particular, magnetic sector instruments are suitable for high speed recording of 1 megapixel / second or higher.

[0085] The time it takes to analyze ionized material depends on the type of mass spectrometer used to detect the ions. For example, instruments that use Faraday cups are generally too slow to analyze rapid signals. Overall, the desired imaging speed, resolution and degree of multiplexing will determine the type of mass spectrometer to be used (or conversely, the choice of mass spectrometer will determine the speed, resolution and multiplexing that can be achieved).

[0086] Mass spectrometry instruments that detect ions at only one mass-to-charge ratio (m / Q, commonly referred to as m / z in MS) at a time, such as point ion detectors, provide poor imaging detection results. First, the time required to switch between mass-to-charge ratios can limit the speed at which multiple signals can be determined, and second, when ions are in low abundance, signals can be missed when the instrument is focused on other mass-to-charge ratios. Therefore, it is preferable to use a technique that detects ions with different m / Q values ​​substantially simultaneously.

[0087] (Detector type) (Quadrupole detector) A quadrupole mass spectrometer comprises four parallel rods with a detector at one end. Alternating RF potentials and fixed DC offset potentials are applied between one pair of rods and the other pair, so that each pair of rods (opposite each other) has an opposite alternating potential with respect to the rods of the other pair. The ionized sample passes through the center of the rods in a direction parallel to the rods towards the detector. The applied potentials affect the ion trajectories so that only ions of a certain mass-to-charge ratio have a stable trajectory and reach the detector. Ions of other mass-to-charge ratios will collide with the rods.

[0088] (Magnetic Sector Detector) In magnetic sector mass spectrometry, the ionized sample passes through a curved flight tube towards an ion detector. A magnetic field applied across the flight tube deflects the ions from their path. The amount of deflection of each ion is based on the mass-to-charge ratio of each ion, so that only some ions strike the detector while others are deflected away from it. Multi-collector sector field instruments use an array of detectors to detect ions of various masses. Some instruments, such as the ThermoScientific Neptune Plus and Nu Plasma II, combine a magnetic sector with an electrostatic sector to provide a dual-focusing magnetic sector instrument that analyzes ions by kinetic energy in addition to mass-to-charge ratio. Multi-detectors with Mattauch-Herzog geometry may be used (e.g., SPECTRO MS, which can simultaneously record all elements from lithium to uranium in a single measurement using a semiconductor direct charge detector). These instruments may measure multiple m / Q signals substantially simultaneously. Their sensitivity may be increased by including electronic multipliers in the detector. However, array sector instruments are effective at detecting increasing signals but are less effective at detecting decreasing signal levels, making them less suitable, especially for situations where labels are present in highly variable concentrations.

[0089] (Time-of-Flight (TOF) detector) A time-of-flight mass spectrometer comprises a sample inlet, an acceleration chamber with a strong electric field applied, and an ion detector. A packet of ionized sample molecules is introduced into the acceleration chamber through the sample inlet. Initially, each ionized sample molecule has the same kinetic energy, but as the ionized sample molecules accelerate through the acceleration chamber, they are separated by their mass, with lighter ionized sample molecules moving faster than heavier ions. The detector detects all the arriving ions. The time it takes each particle to reach the detector depends on the mass-to-charge ratio of the particle.

[0090] Thus, a TOF detector may semi-simultaneously register multiple masses in a single sample. In theory, TOF techniques are not ideally suited for ICP ion sources due to their space charge characteristics, but in practice, TOF instruments may analyze ICP ion aerosols quickly and sensitively enough to enable single cell imaging. TOF mass analyzers are not common in atomic analysis, typically due to the compromises required to deal with space charge effects in the TOF accelerator and flight tube. On the other hand, tissue imaging according to the present disclosure may be effective by detecting only labeled atoms, so that other atoms (e.g., atoms with atomic masses less than 100) may be filtered out. This results in a less dense ion beam enriched in masses in the 100-250 Dalton range (for example) that may be more efficiently manipulated and focused. This facilitates TOF detection and allows the high spectral scanning speed of TOF to be utilized. Thus, high speed imaging may be achieved by combining TOF detection with the selection of labeled atoms that are rare in the sample and ideally have masses above those found in unlabeled samples. Thus, the use of a narrow window of label masses means that TOF detection can be used for efficient imaging.

[0091] Suitable TOF instruments are commercially available from Tofwerk, GBC Scientific Equipment (e.g., Optimass 9500 ICP-TOFMS), and Fluidigm Canada (e.g., CyTOF™, CyTOF™2, etc.). These CyTOF™ instruments are more sensitive than the Tofwerk and GBC instruments and have known applications in mass cytometry, as they can rapidly and sensitively detect ions in the mass range of rare earth metals (especially in the m / Q range of 100-200) (see Bandura et al. (2009; Anal. Chem. 81:6813-22)). Thus, they are preferred instruments for use in the present disclosure and can be used for imaging in instrument settings already known in the art, such as Bendall et al. (2011; Science 332,687-696) and Bodenmiller et al. (2012; Nat. Biotechnol. 30:858-867). These mass spectrometers can detect multiple markers semi-simultaneously at high mass spectral acquisition frequencies in the time required for high frequency laser ablation or sample desorption. They can measure the abundance of labeled atoms with detection limits of as low as 100 per cell, allowing for sensitive construction of images of tissue samples. Because of these features, mass cytometry can be used to meet the demands of sensitivity and multiplexing in imaging tissues with subcellular resolution. By combining mass cytometry instruments with high resolution laser ablation sampling systems and fast transit low dispersion sample chambers, it is now possible to construct highly multiplexed images of tissue samples in practical turnaround times.

[0092] The TOF can be coupled with a mass corrector. In the majority of ionization events, an electron is knocked out of the atom, M +Ions are produced. Due to the mode of operation of the TOF MS, in some cases there is some bleeding (or crosstalk) of ions of one mass (M) into channels for adjacent masses (M±1), especially when multiple ions of mass M enter the detector (i.e., if the instrument was equipped with an ion deflector, the ion counts will be high, but not so high that the ion deflector located between the sampling ionization system and the MS will prevent ions from entering the MS). + The arrival times of ions at the detector follow a probability distribution around a mean value (known for each of the M ions), so that + When the number of ions is large, some of them are usually M-1 + or M+1 + ions arrive at a time associated with the M channel. However, because each ion has a known distribution curve upon entering the TOF MS, it is possible to determine the overlap of the M channel with the M±1 channel based on the peak of the M channel (by comparison with known peak shapes). This calculation is particularly suited to TOF MS, since the peaks of the ions detected in TOF MS are asymmetric. Thus, the readings of the M-1, M, and M+1 channels can be corrected to properly assign all of the detected ions to the M channel. Such corrections are particularly useful in correcting imaging data due to the nature of the large number of ion packets generated by ionization systems such as those encompassing laser ablation (or desorption as described below) as a technique for removing material from samples as described herein. Programs and methods for improving data quality by deconvoluting data from TOF MS are described in WO 2011 / 098834, U.S. Pat. No. 8,723,108, and WO 2014 / 091243.

[0093] (Building an image) The above device can provide signals of multiple atoms in a packet of ionized sample material removed from the sample. Detection of the atom in the packet of sample material reveals its presence at the location of ablation, either because the atom is naturally present in the sample or because the atom is localized to that location by a labeling reagent. By generating a series of packets of ionized sample material from known spatial locations on the sample surface, the detector signal reveals the location of the atom on the sample, which can be used to construct an image of the sample. By labeling multiple targets with distinguishable labels, it is possible to correlate the location of the labeled atom with the location of the same target, so that this method can reach a level of multiplexing far beyond conventional techniques such as fluorescence microscopy to construct complex images.

[0094] Assembling signals into images can be achieved using known techniques and software packages using computers. For example, Kylebank Software's GRAPHIS package can be used, and other packages such as TERAPLOT can also be used. Imaging with MS data from techniques such as MALDI-MSI is known in the art, for example, Robichaud et al. (2013; J Am Soc Mass Spectrom 24 5:718-21) discloses the "MSiReader" interface for displaying and analyzing MS imaging files on a Matlab platform, and Klinkert et al. (2014; Int J Mass Spectrom http: / / dx.doi.org / 10.1016 / j.ijms.2013.12.012) discloses two software instruments, such as the "Datacube Explorer" program, for rapid data exploration and visualization of both 2D and 3D MSI data sets at full spatial and spectral resolution.

[0095] The images obtained by the methods disclosed herein can be further analyzed in the same manner as analyzing the results of IHC, for example. For example, the images can be used to clarify cell subpopulations within a sample, providing information useful for clinical diagnosis. Similarly, SPADE analysis can be used to extract cell hierarchies from the high-dimensional cytometry data provided by the methods disclosed herein (Qiu et al. (2011; Nat. Biotechnol. 29:886-91)).

[0096] (sample) Certain aspects of the present disclosure provide methods for imaging biological samples. Such samples may contain a plurality of cells that may be subjected to imaging mass cytometry (IMC) to provide images of these cells within the sample. Embodiments of the present invention may be used to analyze tissue samples currently studied by immunohistochemistry (IHC) techniques with labeled atoms suitable for detection by mass spectrometry (MS) or optical emission spectroscopy (OES). Additionally, embodiments of the present invention provide various techniques for preparing tissue samples, providing better resolution than IMC and IMS techniques using samples prepared by traditional methods. In particular, embodiments of the present invention provide techniques for preparing samples suitable for imaging by electron microscopy, techniques for preparing ultra-thin samples, and combinations thereof. These methods are further described herein.

[0097] Any suitable tissue sample may be used in the methods described herein. For example, the tissue may include tissue from one or more of epithelium, muscle, nerve, skin, intestine, pancreas, kidney, brain, liver, blood (e.g., blood smear), bone marrow, cheek swipe, cervical swipe, or any other tissue. The biological sample may be an immortalized cell line or primary cells obtained from a living organism. For diagnostic, prognostic, or experimental (e.g., drug development) purposes, the tissue may be from a tumor. In some embodiments, the sample may be from a known tissue, but it may be unknown whether the sample contains tumor cells. Imaging may reveal the presence of targets that indicate the presence of a tumor, facilitating diagnosis. Tissue from a tumor may contain immune cells that are also characterized by the method of interest, providing insight into the biology of the tumor. The tissue sample may comprise formalin-fixed paraffin-embedded (FFPE) tissue. The tissue may be obtained from any living multicellular organism, such as a mammal, an animal research model (e.g., of a particular disease, such as an immunodeficient rodent with a human tumor xenograft), or a human patient.

[0098] The tissue sample may be a section having a thickness in the range of, for example, 2-10 μm, for example 4-6 μm. Techniques for preparing such sections are known in the field of IHC and include dehydration steps, fixation, embedding, permeabilization, sectioning, etc., for example with a microtome. Thus, the tissue may be chemically fixed and then sections may be prepared in the desired plane. Also, frozen sectioning or laser capture microdissection may be used to prepare the tissue sample. The sample may be permeabilized (see above), for example to allow the uptake of reagents for labeling of intracellular targets.

[0099] The size of the tissue sample analyzed will be similar to current IHC methods, although the maximum size will be dictated by the size of the sample that can fit into the laser ablation device, specifically the sample chamber. Sizes up to 5 mm x 5 mm are common, although smaller samples (e.g., 1 mm x 1 mm) are also useful (these dimensions refer to the size of the section, not the thickness of the section).

[0100] In addition to being useful for imaging tissue samples, the present disclosure may also be used to image cell samples such as monolayers of adherent cells or cells immobilized on solid surfaces (as in traditional immunocytochemistry). These embodiments are particularly useful for the analysis of adherent cells that cannot be easily solubilized for cell suspension mass cytometry. Thus, the present disclosure is not only useful for improving current immunohistochemistry analyses, but may also be used to improve immunocytochemistry.

[0101] (Ultra-thin sample) As mentioned above, conventional IMC and IMS techniques use tissue samples that are a few micrometers thick.

[0102] Thus, an embodiment of the present invention provides a method of preparing a biological sample for analysis, comprising: labeling the sample with a labeling atom (the labeling atom is further described herein) and dividing the sample into thin sections, whereby the sample is divided into sections with a thickness of less than 10 micrometers or less, such as 1 micrometer or less, or 100 nm or less, or 50 nm or less, or 30 nm or less, if desired. An embodiment of the present invention also provides a method of preparing a biological sample for analysis, comprising: labeling the sample with a labeling atom (the labeling atom is further described herein) and dividing the sample into sections with a thickness of less than 10 micrometers or less, such as 1 micrometer or less, or 100 nm or less, or 50 nm or less, or 30 nm or less, if desired. An automated microtome, such as an ATUMtome available from RM Boeckeler, may be used to divide the sample into sections with a thickness according to the methods described herein. Ultra-thin sections can contribute to a smaller plume of material ablated from the sample, which can contribute to improved efficiency of ionization in the ionization conduit.

[0103] Samples prepared by the above methods may be used for any of the IMC and IMS techniques described herein, however, samples prepared by the above methods are particularly suited to analysis by laser ionization.

[0104] (Sample Carrier) In certain embodiments, the sample may be immobilized on a solid support (i.e., a sample carrier) and positioned for imaging mass spectrometry. The solid support may be optically transparent, for example, made of glass or plastic.

[0105] In some cases, the sample carrier includes features that serve as reference points for use with the devices and methods described herein, e.g., to calculate the relative position of a feature / region of interest that is ablated or desorbed and analyzed. The reference points may be optically resolvable or mass spectrometrically resolvable.

[0106] (Target element) Imaging mass spectrometry may be of interest in the distribution of one or more target elements (i.e., elements or elemental isotopes). In certain aspects, the target element is a labeled atom as described herein. The labeled atom may be added directly to the sample alone or may be covalently attached to or in a biologically active molecule. In certain embodiments, the labeled atom (e.g., a metal tag) may be attached to a member of a specific binding pair (SBP), such as an antibody (conjugated to its cognate antigen), an aptamer, or an oligonucleotide for hybridizing to a DNA or RNA target, as described in detail below. The labeled atom may be attached to the SBP in any manner known in the art. In certain aspects, the labeled atom is a metal element, such as a lanthanide or transition element, or another metal tag as described herein. The metal element may have a mass of more than 60 amu, more than 80 amu, more than 100 amu, or more than 120 amu. The mass spectrometers described herein can deplete elemental ions less than the mass of the metallic element, such that abundant lighter elements do not create space charge effects and / or overwhelm the mass detector.

[0107] Labeling of tissue samples The present disclosure produces images of samples labeled with labeled atoms, e.g., multiple different labeled atoms, where the labeled atoms are detected by an apparatus capable of sampling a specific region of the sample, preferably an area within a cell (therefore, the labeled atoms represent elemental tags). Reference to multiple different atoms means that multiple atomic species are used to label the sample. These atomic species can be identified using a mass detector (e.g., they have different m / Q ratios), such that the presence of two different labeled atoms in the plume results in two different MS signals. An optical spectrometer can also be used to distinguish atomic species (e.g., different atoms have different emission spectra), such that the presence of two different labeled atoms in the plume results in two different emission spectral signals.

[0108] (Mass tagged reagents) As used herein, the mass tagged reagent comprises several components. The first is an SBP. The second is a mass tag. The mass tag and the SBP are linked by a linker formed at least in part by the conjugation of the mass tag and the SBP. The bond between the SBP and the mass tag may also comprise a spacer. The mass tag and the SBP may be conjugated together by various reaction chemistries. Exemplary conjugation reaction chemistries include thiol maleimide, NHS ester and amine or click chemistry reactants (preferably Cu(I)-free chemistries), such as strained alkynes and azides, strained alkynes and nitrones, and strained alkenes and tetrazines.

[0109] (Mass tag) Mass tags used in embodiments of the present invention may take a variety of forms. Typically, a tag comprises at least one labeling atom, as described later in this specification.

[0110] Thus, in its simplest form, a mass tag may comprise a metal chelating moiety, which is a metal chelating group having a metal label atom coordinated within a ligand. In some instances, it may be sufficient to detect a single metal atom per mass tag. However, in other instances, it may be desirable for each mass tag to contain two or more label atoms. This may be achieved in a number of ways, as described below.

[0111] The first means for generating mass tags that may contain two or more labeling atoms is the use of polymers with metal chelating ligands attached to two or more subunits of the polymer. The number of metal chelating groups capable of binding at least one metal atom in the polymer may be about 1 to 10,000, for example, 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500 or 500 to 1,000. At least one metal atom may be bound to at least one of the metal chelating groups. The polymer may have a degree of polymerization of about 1 to 10,000, for example, 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500 or 500 to 1,000. Thus, a polymer-based mass tag may comprise from about 1 to 10,000 label atoms, such as, for example, 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500 or 500 to 1,000.

[0112] Labeling of Samples for Mass Cytometry Applications In some embodiments, as described above, the devices and methods detect atoms that are added to the sample (i.e., not normally present). Such atoms are called labeling atoms. In some embodiments, simultaneous detection of multiple labeling atoms is possible, for example, multiple labeling detection of at least 3, 4, 5, 10, 20, 30, 32, 40, 50, or 100 different labeling atoms. Labeling atoms can also be used in a combinatorial manner, further increasing the number of distinguishable labels. By labeling different targets with different labeling atoms, it is possible to determine the presence of multiple targets on a single cell.

[0113] Labeled atoms that may be used in this embodiment include species that are detectable by MS or OES and that are substantially absent in unlabeled tissue samples. For example, 12 C atoms are abundant in nature and therefore unsuitable as labeling atoms. 11 C is an artificial isotope that does not occur naturally, so theoretically it could be used for MS. The labeled atom is often a metal. However, in a preferred embodiment, the labeled atom is a transition metal, such as a rare earth metal (15 lanthanides plus scandium and yttrium). These 17 elements (which can be distinguished by OES and MS) provide many different isotopes that can be easily distinguished (by MS). These various elements are available in enriched isotopic forms. For example, samarium has 6 stable isotopes and neodymium has 7 stable isotopes, all of which are available in enriched forms. The 15 lanthanide elements provide at least 37 isotopes with unique, non-overlapping masses. Examples of elements suitable for use as labeling atoms include lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), yttrium (Y), etc. In addition to rare earth metals, other metal atoms are suitable for detection, such as gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), bismuth (Bi), etc. The use of radioisotopes is not preferred, since they are more inconvenient to handle and unstable, for example Pm is not a preferred labeling atom among the lanthanides.

[0114] To facilitate time-of-flight (TOF) analysis (as described herein), it is useful to use labeled atoms with atomic masses in the range of 80-250, e.g., in the range of 80-210 or in the range of 100-200. This range includes all of the lanthanides but excludes Sc and Y. The 100-200 range allows for theoretical 101-plex analysis using a variety of labeled atoms while taking advantage of the high spectral scan rates of TOF MS. As previously mentioned, by selecting labeled atoms whose masses are within a window above those seen in unlabeled samples (e.g., in the range of 100-200), TOF detection may be used to provide rapid imaging at biologically significant levels.

[0115] Depending on the mass tags used (and the number of label atoms per mass tag) and the number of mass tags attached to each SBP, various numbers of label atoms may be attached to a single SBP element. Higher sensitivity may be achieved as more label atoms are attached to the SBP element. For example, more than 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 label atoms may be attached to the SBP element, such as up to 10,000, such as 5-100, 10-250, 250-5,000, 500-2,500 or 500-1,000 label atoms. As mentioned above, monodisperse polymers containing multiple monomer units, each containing a chelating agent such as diethylenetriaminepentaacetic acid (DTPA) or DOTA, may be used. DTPA, for example, chelates 3+ lanthanide ions at approximately 10 -6The polymers are bonded with a dissociation constant of M. These polymers may terminate with thiols that can be used to attach to the SBP via reaction with maleimides that attach click chemistry reactants along the lines of the above description. Other functional groups can also be used to attach these polymers, such as amine-reactive groups such as N-hydroxysuccinimide esters, carboxyl groups or groups reactive to glycosylation of antibodies. Any number of polymers can be attached to each SBP. 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 can also be used to increase the number of atoms in the label, as described above.

[0116] In some embodiments, all the labeled atoms of the mass tag are the same atomic mass. Alternatively, the mass tags may comprise labeled atoms of different atomic masses. Thus, in some examples, a labeled sample may be labeled with a series of mass-tagged SBPs, each of which comprises only a single type of labeled atom (each SBP is conjugated to its cognate target, so that each type of mass tag is localized to a specific location on the sample, e.g., an antigen). Alternatively, in some examples, a labeled sample may be labeled with a series of mass-tagged SBPs, each of which comprises a mixture of labeled atoms. In some examples, the mass-tagged SBPs used to label a sample may comprise a mixture of those with mass tags of a single labeled atom and a mixture of labeled atoms in those mass tags.

[0117] (Spacer) As mentioned above, in some cases, the SBP is attached to the mass tag via a linker with a spacer. There can be a spacer between the SBP and the click chemistry reagent (e.g., between the SBP and the strained cycloalkyne (or azide); strained cycloalkene (or tetrazine); etc.). There can be a spacer between the mass tag and the click chemistry reagent (e.g., between the mass tag and the azide (or strained cycloalkyne); tetrazine (or strained cycloalkene); etc.). In some examples, there can be a spacer between both the SNP and the click chemistry reagent and between the click chemistry reagent and the mass tag.

[0118] The spacer can be a polyethylene glycol (PEG) spacer, a poly(N-vinylpyrrolide) (PVP) spacer, a polyglycerol (PG) spacer, a poly(N-(2-hydroxypropyl)methacrylamide) spacer, or a polyoxazoline (POZ, such as polymethyloxazoline, polyethyloxazoline, or polypropyleneoxazoline, etc.) or a C5-C20 acyclic alkyl spacer. For example, the spacer can be a PEG spacer having 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more EG (ethylene glycol) units. The PEG linker can have 3 to 12 EG units, 4 to 10 EG units, or can have 4, 5, 6, 7, 8, 9, or 10 EG units. The linker can include cystamine or a derivative thereof, can include one or more disulfide groups, or can be other suitable linkers known to those of skill in the art.

[0119] Spacers can be beneficial to minimize steric effects on the SBP to which the mass tag is attached, and hydrophilic spacers, such as PEG-based spacers, can act to improve the solubility of the mass-tagged SBP and can serve to prevent aggregation.

[0120] (SBP) Mass cytometry, including imaging mass cytometry, is based on the principle of specific binding between members of a specific binding pair. A mass tag binds to a member of a specific binding pair, which localizes the mass tag to the target / analyte, which is the other member of the pair. However, specific binding does not require binding to only one molecular species to the exclusion of other molecular species. Rather, it defines that the binding is not non-specific, i.e., not a random interaction. Thus, an example of an SBP that binds to multiple targets is an antibody that recognizes an epitope common to multiple different proteins. Here, the binding is specific and mediated by the CDR of the antibody, but multiple different proteins are detected by the antibody. The common epitope can be naturally occurring or an artificial tag, such as a FLAG tag. Similarly, in the case of nucleic acids, a nucleic acid of a defined sequence may not exclusively bind to a completely complementary sequence, but as one skilled in the art will appreciate, the use of hybridization conditions of different stringency can introduce various heterogeneous tolerances of mismatched pairs. Nevertheless, this hybridization is not non-specific since it is driven by homology between the SBP nucleic acid and the target analyte. Similarly, a ligand may specifically bind to multiple receptors, e.g., TNFα binds to both TNFR1 and TNFR2.

[0121] SBPs may comprise either a nucleic acid duplex, an antibody / antigen complex, a receptor / ligand pair, or an aptamer / target pair. Thus, a labeled atom may be attached to a nucleic acid probe that contacts a tissue sample, such that the probe hybridizes with a complementary nucleic acid in the tissue, forming, for example, a DNA / DNA duplex, a DNA / RNA duplex, or an RNA / RNA duplex. Similarly, a labeled atom may be attached to an antibody, which may then contact a tissue sample, which may then bind to its antigen. A labeled atom may be attached to a ligand, which may then contact a tissue sample, which may then bind to its receptor. A labeled atom may be attached to an aptamer ligand, which may then contact a tissue sample, which may then bind to its target. In this way, labeled SBP elements may be used to detect a variety of targets in a sample, including DNA sequences, RNA sequences, proteins, sugars, lipids, or metabolites.

[0122] As such, the mass-tagged SBP can be a protein or peptide, or a polynucleotide or oligonucleotide.

[0123] Examples of protein SBPs include antibodies or antigen-binding fragments thereof, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, antibody fusion proteins, scFvs, antibody mimics, avidin, streptavidin, neutravidin, biotin or combinations thereof, where optionally the antibody mimics include nanobodies, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, DARPins, finomers, Kunitz domain peptides, monobodies or any combination thereof, receptors such as receptor-Fc fusions, ligands such as ligand-Fc fusions, lectins, e.g., agglutinins such as wheat germ agglutinin.

[0124] The peptide may be a linear peptide or may be a cyclic, such as a bicyclic peptide. One example of a peptide that may be used is phalloidin.

[0125] Polynucleotide or oligonucleotide generally refers to a single- or double-stranded polymer of nucleotides containing deoxyribonucleotides or ribonucleotides linked by 3'-5' phosphodiester bonds, as well as polynucleotide analogs. Nucleic acid molecules include, but are not limited to, DNA, RNA, and cDNA. Polynucleotide analogs may have backbones other than the standard phosphodiester bonds found in natural polynucleotides, and may optionally have one or more modified sugar moieties other than ribose or deoxyribose. Polynucleotide analogs contain bases that are capable of hydrogen bonding to standard polynucleotide bases by Watson-Crick base pairing, where the analog backbone presents the bases in a manner that allows such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in the standard polynucleotide. Examples of polynucleotide analogs include, but are not limited to, xenonucleic acid (XNA), bridged nucleic acid (BNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), yPNA, morpholino polynucleotide, locked nucleic acid (LNA), threose nucleic acid (TNA), 2'-0-methyl polynucleotide, 2'-0-alkylribosyl substituted polynucleotide, phosphorothioate polynucleotide, and boronophosphate polynucleotide. Polynucleotide analogs can have purine or pyrimidine analogs, including, for example, 7-deazapurine analogs, 8-halopurine analogs, 5-halopyrimidine analogs, or universal base analogs that can pair with any base, including hypoxanthine, nitroazole, isocarbostyril analogs, azolecarboxamide, and aromatic triazole analogs, or base analogs with additional functionality, such as a biotin moiety for affinity binding.

[0126] (antibody SBP element) In a typical embodiment, the labeled SBP element is an antibody. Labeling of the antibody can be achieved through conjugation of one or more label-atom binding molecules to the antibody, for example, by attaching a mass tag using NHS-amine chemistry, sulfhydryl-maleimide chemistry, or click chemistry (strained alkyne and azide, strained alkyne and nitrone, strained alkene and tetrazine, etc.). Antibodies that recognize cellular proteins useful for imaging are already widely available for IHC, and these known antibodies can be easily adapted to the methods disclosed herein by using label atoms instead of current labeling techniques (e.g., fluorescence), but with the advantage of increased multiplexing capabilities. Antibodies can recognize cell surface targets or intracellular targets. Antibodies can recognize a variety of targets. For example, they can specifically recognize individual proteins, recognize multiple related proteins that share a common epitope, and recognize specific post-translational modifications on proteins (e.g., to distinguish tyrosine from phosphotyrosine, to distinguish lysine from acetyl-lysine, to detect ubiquitination on a target protein). After binding to the target, the label atom attached to the antibody can be detected to reveal the location of the target in the sample.

[0127] The labeled SBP element usually interacts directly with the target SBP element in the sample. However, in some embodiments, it is possible for the labeled SBP element to interact indirectly with the target SBP element. For example, a primary antibody can bind to the target SBP element, and then a labeled secondary antibody can bind to the primary antibody in a sandwich assay method. However, usually, this method relies on direct interaction, since it can be more easily realized and allows for higher multiplexing. However, in both cases, the sample is contacted with an SBP element that can be bound to the target SBP element in the sample, which detects the label bound to the target SBP element at a later stage.

[0128] (Changes in nucleic acid SBP and labeling method) RNA is another biomolecule that can be detected by the method and apparatus disclosed herein in a specific sensitive and, if necessary, quantitative manner. RNA can be detected by using SBP elements labeled with element tags that specifically bind to RNA in the same manner as described above for the analysis of protein (e.g., complementary sequence polynucleotides or oligonucleotides as described above, including complementary sequence lock nucleic acid (LNA) molecules, complementary sequence peptide nucleic acid (PNA) molecules, complementary sequence plasmid DNA, complementary sequence amplified DNA, complementary sequence RNA fragments, and complementary sequence genomic DNA fragments). RNA includes not only mature mRNA, but also RNA processing intermediates and nascent pre-mRNA transcripts.

[0129] In some embodiments, the methods described herein are used to detect both RNA and protein.

[0130] To detect RNA, cells in a biological sample described herein may be prepared for analysis of RNA and protein content using the methods and devices described herein. In certain aspects, cells are fixed and permeabilized prior to the hybridization step. Cells may be prepared in a fixed and / or permeabilized state. Cells may be fixed by a cross-linking fixative such as formaldehyde, glutaraldehyde, etc. Alternatively or in addition, cells may be fixed using a precipitating fixative such as ethanol, methanol, or acetone. Cells may be permeabilized by detergents such as polyethylene glycol (e.g., Triton X-100), polyoxyethylene (20) sorbitan monolaurate (Tween-20), saponin (amphiphilic glycosidic groups), or chemicals such as methanol or acetone. In some cases, fixation and permeabilization may be performed with the same reagent or set of reagents. Fixation and permeabilization techniques are described by Jamur et al. in "Permeabilization of Cell Membranes" (Methods Mol. Biol., 2010).

[0131] Detection of target nucleic acids in cells, or "in situ hybridization" (ISH), has been performed using fluorescently tagged oligonucleotide probes. As described herein, mass-tagged oligonucleotides can be combined with ionization and mass spectrometry to detect target nucleic acids in cells. In-situ hybridization methods are known in the art (see Zenobi et al. "Single-Cell Metabolomics: Analytical and Biological Perspectives," Science vol. 342, no. 6163, 2013). Hybridization protocols are also described in U.S. Patent No. 5,225,326, U.S. Patent Application Publication No. 2010 / 0092972 and U.S. Patent Application Publication No. 2013 / 0164750, which are incorporated herein by reference.

[0132] Before hybridization, cells in suspension or cells immobilized on solid support can be immobilized and permeabilized as described above.Permeabilization can allow cells to retain target nucleic acid while allowing target hybridization nucleotides, amplification oligonucleotides and / or mass tagged oligonucleotides to enter the cells.After any hybridization step, for example, after hybridization of target hybridization oligonucleotides to nucleic acid target, after hybridization of amplification oligonucleotides and / or after hybridization of mass tagged oligonucleotides, cells can be washed.

[0133] Cells can be in suspension in all or most of the steps of the method to facilitate handling.However, the method is also applicable to cells in solid tissue samples (e.g., tissue sections) and / or cells immobilized on solid supports (e.g., slides or other surfaces).Thus, in some cases, cells may be in suspension in the sample and during the hybridization step.In other cases, cells are immobilized on solid supports during hybridization.

[0134] Target nucleic acids include any nucleic acid present in sufficient numbers in a cell of a subject to be detected by the method. Target nucleic acids can be RNAs present in multiple copies in a cell. For example, 10 or more, 20 or more, 50 or more, 100 or more, 200 or more, 500 or more, or 1,000 or more copies of a target RNA can be present in a cell. Target RNAs can be messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (IncRNAs), or other types of RNA known in the art. Target RNAs can be 20 nucleotides or more, 30 nucleotides or more, 40 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 200 nucleotides or more, 500 nucleotides or more, 1,000 nucleotides or more, 20-1,000 nucleotides, 20-500 nucleotides in length, 40-200 nucleotides in length, etc.

[0135] In certain embodiments, mass-tagged oligonucleotides may be hybridized directly to the target nucleic acid sequence, however, it may be possible to improve specificity and / or signal amplification by hybridizing additional oligonucleotides.

[0136] In certain embodiments, two or more target hybridization oligonucleotides may hybridize to proximal regions on the target nucleic acid, together providing sites for hybridization of additional oligonucleotides in a hybridization scheme.

[0137] In certain embodiments, mass tagged oligonucleotides can be directly hybridized to two or more target hybridization oligonucleotides.In other embodiments, one or more amplification oligonucleotides can be added simultaneously or sequentially to hybridize two or more target hybridization oligonucleotides and provide multiple hybridization sites to which mass tagged oligonucleotides can be bound.One or more amplification oligonucleotides can be provided as multimers that can hybridize to two or more target hybridization oligonucleotides with or without mass tagged oligonucleotides.

[0138] The use of two or more target hybridization oligonucleotides improves specificity, while the use of amplification oligonucleotides increases the signal. Two target hybridization oligonucleotides are hybridized to a target RNA in a cell. The two target hybridization oligonucleotides together provide a hybridization site to which an amplification oligonucleotide can bind. Hybridization of the amplification oligonucleotide and / or subsequent washing can be performed at a temperature that allows hybridization to two adjacent target hybridization oligonucleotides, but above the melting temperature of hybridization of the amplification oligonucleotide to just one target hybridization oligonucleotide. The first amplification oligonucleotide provides multiple hybridization sites to which the second amplification oligonucleotide binds to form a branched pattern. The mass-tagged oligonucleotide can bind to multiple hybridization sites provided by the second amplification oligonucleotide. These amplification oligonucleotides (with or without mass-tagged oligonucleotides) are both referred to herein as "multimers." Thus, the term "amplification oligonucleotide" includes oligonucleotides that provide multiple copies of the same binding site to additional oligonucleotides that can be annealed. By increasing the number of binding sites for other oligonucleotides, the final number of labels that can be found on the target increases. In this way, multiple labeled oligonucleotides are indirectly hybridized to a single target RNA. This allows the detection of low copy numbers of RNA by increasing the number of detectable atoms of the elements used per RNA.

[0139] One particular method for carrying out this amplification involves using the RNAscope® method from Advanced cell diagnostics, as described in detail below. Yet another method is the use of an adapted QuantiGene® FlowRNA method (Affymetrix eBioscience). This assay is based on an oligonucleotide pair probe design with branched DNA (bDNA) signal amplification. More than 4,000 probes are listed in the catalog, and custom sets can be requested at no extra cost. In line with the previous sentence, this method works by hybridizing a target hybridization oligonucleotide to a target, then forming a branched structure with a first amplification oligonucleotide (referred to as a preamplification oligonucleotide in the QuantiGene® method) and stemming to a plurality of second amplification oligonucleotides that can anneal (referred to simply as amplification oligonucleotides in the QuantiGene® method). A plurality of mass-tagged oligonucleotides can then be attached.

[0140] Another means of amplifying RNA signals relies on rolling circle amplification means (RCA). There are various ways in which this amplification system can be introduced into the amplification process. In a first example, a first nucleic acid is used as a hybridization nucleic acid, and the first nucleic acid is circular. The first nucleic acid can be single-stranded or double-stranded. The first nucleic acid comprises a sequence complementary to the target RNA. After the first nucleic acid hybridizes to the target RNA, a primer complementary to the first nucleic acid is hybridized to the first nucleic acid and used for primer extension, usually using a polymerase and a nucleic acid that are exogenously added to the sample. However, in some cases, the first nucleic acid may already have a primer bound when it is added to the sample. As a result of the first nucleic acid being circular, once the extension of the primer has completed a full round of replication, the polymerase can displace the primer and the extension continues (i.e., without 5'→3' exonuclease activity), generating further bound and linked copies of the complement of the first nucleic acid, thereby amplifying the nucleic acid sequence. Thus, the oligonucleotide with element tag (such as RNA or DNA, or LNA or PNA as described above) can be hybridized to the linked copy of the complement of the first nucleic acid.The degree of amplification of the RNA signal can therefore be controlled by the length of time allocated to amplifying the circular nucleic acid.

[0141] In another application of RCA, the first, e.g., oligonucleotide, hybridized to the target RNA can be linear rather than circular, with a first portion having a sequence complementary to the target and a second portion selected by the user. A circular RCA template with a sequence homologous to the second portion can then be hybridized to this first oligonucleotide, and RCA amplification is performed as described above. By using a first, e.g., oligonucleotide, with a target-specific portion and a user-selected portion, the user-selected portion can be selected to be common among a variety of different probes. This is reagent-efficient, since the same subsequent amplification reagents can be used in a series of reactions that detect different targets. However, as will be appreciated by those skilled in the art, when this strategy is employed, in order to individually detect a specific RNA in a multiplex reaction, each first nucleic acid hybridized to the target RNA must have a unique second sequence, and as a result, each circular nucleic acid should contain a unique sequence that can be hybridized by a labeled oligonucleotide. In this way, the signal from each target RNA can be specifically amplified and detected.

[0142] Other configurations for effecting RCA analysis will be known to those skilled in the art. In some instances, the first, e.g., oligonucleotide, may be fixed (e.g., with formaldehyde) after hybridization to prevent the first, e.g., oligonucleotide, from dissociating from the target during subsequent amplification and hybridization steps.

[0143] Additionally, hybridization chain reaction (HCR) can be used to amplify RNA signals (see, e.g., Choi et al., 2010, Nat. Biotech, 28:1208-1210). As Choi explains, HCR amplifiers consist of two nucleic acid hairpin species that do not polymerize in the absence of an initiator. Each HCR hairpin consists of an input domain with an exposed single-stranded end and an output domain with a single-stranded end hidden within the folded hairpin. Hybridizing an initiator to the input domain of one of the two hairpins opens the hairpin, exposing the output domain. Hybridizing this (previously hidden) output domain to the input domain of a second hairpin opens the hairpin, revealing an output domain with the same sequence as the initiator. Regeneration of the initiator sequence provides the basis for a chain reaction of alternating polymerization steps of the first and second hairpins, forming a nicked double-stranded "polymer." In the application of the methods and devices described herein, either or both of the first and second hairpins can be labeled with element tags. Because the amplification procedure relies on the output domain of a specific sequence, various discrete amplification reactions using sets of individual hairpins can be carried out independently in the same process. This amplification therefore also allows for the amplification of multiple RNA species in multiplex analysis. As Choi points out, HCR is an isothermally triggered self-assembly process. Therefore, the hairpins penetrate the sample before undergoing in situ self-assembly, suggesting the possibility of deep penetration into the sample and high signal-to-background ratio.

[0144] Hybridization may include contacting cells with one or more oligonucleotides, such as target hybridization oligonucleotides, amplification oligonucleotides and / or mass-tagged oligonucleotides, and providing conditions that allow hybridization to occur. Hybridization may be performed in a buffer, such as saline-sodium citrate (SCC) buffer, phosphate-buffered saline (PBS), saline-sodium phosphate-EDTA (SSPE) buffer, TNT buffer (with Tris HCl, sodium chloride and Tween 20) or any other suitable buffer. Hybridization may be performed at a temperature that is about equal to or lower than the melting temperature of hybridization of one or more oligonucleotides.

[0145] Specificity can be improved by performing one or more washes after hybridization to remove unbound oligonucleotides. Increasing the stringency of the wash can improve specificity but reduce the overall signal. The stringency of the wash can be increased by increasing or decreasing the concentration of the wash buffer, by increasing the temperature, and / or by increasing the time of the wash. RNAse inhibitors can be used in any or all hybridization incubations and subsequent washes.

[0146] A first set of hybridization probes, including one or more target hybridizing oligonucleotides, amplification oligonucleotides and / or mass tagged oligonucleotides, can be used to label a first target nucleic acid. Additional sets of hybridization probes can be used to label additional target nucleic acids. Each set of hybridization probes can be specific to a different target nucleic acid. Additional sets of hybridization probes can be designed to reduce or prevent hybridization between oligonucleotides of different sets, and can be hybridized and washed. In addition, each set of mass tagged oligonucleotides can provide a unique signal. In this way, multiple sets of oligonucleotides can be used to detect 2, 3, 5, 10, 15, 20 or more different nucleic acid targets.

[0147] In some cases, the different nucleic acids to be detected are splice variants of a single gene. Mass tagged oligonucleotides can be designed to hybridize (directly or indirectly via other oligonucleotides, as described below) within the sequence of an exon to detect all transcripts containing that exon, or to cross splice junctions to detect specific variants (e.g., if a gene has three exons and two splice variants, exons 1-2-3 and exons 1-3, the two can be distinguished; i.e., variant 1-2-3 can be specifically detected by hybridizing to exon 2, and variant 1-3 can be specifically detected by hybridizing across the exon 1-3 junction).

[0148] (Single cell analysis) The disclosed method includes laser ablation of a plurality of cells within a sample, such that the plume from the plurality of cells is analyzed and the contents are mapped to specific locations within the sample to provide an image. In most cases, the user of the method will need to localize the signal to a specific cell within the sample, rather than the entire sample. To do so, the boundaries of the cells within the sample (e.g., cell membranes, and possibly cell walls) may be defined.

[0149] The definition of cell boundaries can be performed in various ways. For example, the sample can be surveyed using conventional techniques that can define cell boundaries, such as microscopy as described above. Thus, when performing these methods, an analysis system equipped with a camera as described above is particularly useful. The method of the present disclosure can then be used to create an image of the sample, which can be overlaid on the previous result, thereby localizing the detected signal to a specific cell. Indeed, as described above, in some cases, laser ablation can be directed only to a subset of cells in the sample that have been determined to be of interest using a microscope-based technique.

[0150] However, as part of the imaging method of the present disclosure, it is possible to define the cell boundary to avoid the need to use multiple techniques. Such boundary definition strategies are well known in IHC and immunocytochemistry, and these approaches can be adapted by using detectable labels. For example, the method may include labeling target molecules known to be located at cell boundaries, and signals from these labels can be used to define the boundary. Suitable target molecules include abundant or ubiquitous markers of cell boundaries, such as elements of adhesion complexes (such as β-catenin and E-cadherin). In some embodiments, two or more membrane proteins may be labeled to define the boundary.

[0151] Thus, in addition to defining the boundaries of the cell by including appropriate labels, it is also possible to define specific organelles. For example, antigens such as histones (e.g. H3) can be used to identify the nucleus, and it is also possible to label mitochondrial-specific antigens, cytoskeleton-specific antigens, Golgi-specific antigens, ribosome-specific antigens, etc. This allows the method of the present disclosure to analyze the ultrastructure of the cell.

[0152] The signal that defines the boundary of a cell (or organelle) can be evaluated visually or analyzed by computer using image processing. Such techniques are known to those skilled in the art as other imaging techniques, such as the segmentation scheme that uses spatial filtering to determine cell boundaries from fluorescent images described by Arce et al. (2013; Scientific Reports 3, article 2266), the algorithm that determines boundaries from bright-field microscopy images disclosed by Ali et al. (2011; Mach Vis Appl 23:607-21), the CellSeT method that extracts cell shape from confocal microscopy images disclosed by Pound et al. (2012; The Plant Cell 24:1353-61), and the CellSegm Matlab toolbox for fluorescent microscopy images disclosed by Hodneland et al. (2013; Source Code for Biology and Medicine 8:16). The method useful in this disclosure uses watershed transformation and Gaussian blurring. These imaging techniques may be used alone or may be used followed by visual confirmation.

[0153] Once cell boundaries are clearly defined, it becomes possible to assign signals from specific target molecules to individual cells, and by calibrating the method with quantitative standards, it is also possible to quantify the amount of target analyte within individual cells.

[0154] (Reference particle) As described herein, reference particles of known elemental or isotopic composition can be added to a sample (or sample carrier) for use as a reference during detection of target element ions in the sample. In certain embodiments, the reference particles comprise metal elements or isotopes, such as transition metals or lanthanides. For example, the reference particles can comprise elements or isotopes with masses of more than 60 amu, more than 80 amu, more than 100 amu, or more than 120 amu.

[0155] Target elements, such as label atoms, can be normalized within a sample run based on elemental ions detected from individual reference particles. For example, a subject method can include switching between detecting elemental ions from individual reference particles and detecting only target elemental ions.

[0156] The term "comprising" encompasses "including" and "consisting of," for example, a composition "comprising" X may consist only of X, or may include additional items, such as X+Y.

[0157] The term "about" in relation to a numerical value x is arbitrary and means, for example, x±10%.

[0158] The word "substantially" does not exclude "completely", for example a composition that is "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of an embodiment of the invention. [Brief description of the drawings]

[0159] [Figure 1] FIG. 1 is a diagram of an ICP torch. [Diagram 2] FIG. 1 is a diagram of a mass cytometer according to an embodiment of the present invention. [Diagram 3]Illustration of plume reshaping by passage through a tapered conduit. Here, an initial spherical cloud of material decreases in diameter as it passes through a narrowing conduit. Because the flow is usually fastest in the center of the conduit, the cloud elongates as it is carried along the conduit by the gas flow. By properly designing the tapered conduit and gas flow, a long vapor stream with a very small diameter can be produced from the previously spherical cloud. [Figure 4] FIG. 1 is a diagram of a reflector arrangement. [Diagram 5] 5A and 5B are diagrams of reflector arrangements using a convex reflector, showing reflection through a conduit and reflection at a tapered end of the conduit, respectively. [Figure 6] FIG. 1 is a diagram of laser ionization where the laser light is directed at a wide section of a conduit acting as a waveguide, and the energy density only reaches a level suitable for ionization in the tapered section. [Figure 7] FIG. 1 is a diagram of laser ionization using an embodiment of an ionization conduit with a divergence.

Claims

1. An apparatus comprising: (a) a sampler; (b) a laser ionization system that receives material removed from the sample by the sampler, the laser ionization system comprising an ionization system conduit and a pulsed laser adapted to ionize sample material passing through or exiting the ionization system conduit, the ionization system conduit comprising a tapered section therealong along which an inner diameter of the ionization system conduit decreases; (c) a mass spectrometer that receives elemental ions from the laser ionization system and analyzes the elemental ions; An apparatus comprising:

2. The apparatus of claim 1 , wherein the pulsed laser of the laser ionization system is a picosecond laser or a femtosecond laser.

3. 3. The apparatus of claim 2, wherein the pulsed laser of the laser ionization system is a solid-state laser, a fiber laser, a semiconductor laser, or a VECSEL.

4. 4. The apparatus of claim 2 or claim 3, wherein the pulsed laser of the laser ionization system is adapted to generate pulses having a duration of less than 1 ps.

5. 5. The apparatus of claim 4, wherein the pulsed laser of the laser ionization system is adapted to generate pulses of duration less than or equal to 500 fs.

6. 6. The apparatus of claim 5, wherein the pulsed laser of the laser ionization system is adapted to generate laser pulses at a repetition rate of at least 100,000 Hz.

7. 7. The apparatus of claim 1, wherein the pulsed laser of the laser ionization system is adapted to have a beam width of 100 μm or less.

8. 8. The apparatus of claim 1, wherein the reduction in inner diameter of the ionization system conduit is about two times or more.

9. 9. The apparatus of claim 1, wherein the tapered section is adapted such that gas flow at a narrow end of the tapered section is supersonic.

10. 10. The apparatus of claim 1, wherein the ionization system conduit comprises a divergence downstream of the tapered section, and the pulsed laser is positioned to irradiate laser light from the diverging side of the tapered section.

11. 11. The apparatus of claim 1, wherein the laser ionization system further comprises a reflector arrangement for causing laser radiation generated by the pulsed laser to pass multiple times through the ionization system conduit or a path of material exiting the ionization system conduit.

12. The apparatus of claim 11 , wherein the reflector arrangement comprises one or more reflectors shaped to provide multiple focal points within or at the exit of the ionization system conduit.

13. 13. The apparatus of claim 1, wherein the sampler is a laser ablation system.

14. 14. The apparatus of claim 13, wherein a laser of the laser ablation system is adapted to ablate a sample at a repetition rate of about 10 Hz or greater.

15. 15. The apparatus of claim 13 or 14, wherein the sampler is configured to generate a plume of sample material, and the laser ionization system is configured to generate multiple laser pulses to ionize a single plume.

16. The apparatus of claim 15 , wherein the pulsed laser of the laser ionization system is configured to generate pulses at a repetition rate greater than 10 3 times the repetition rate of the laser ablation system.

17. 17. The apparatus of claim 13, wherein a laser of the laser ablation system is configured to ablate the sample with a spot size diameter of less than 4 μm.

18. 18. The apparatus of any one of claims 13 to 17, wherein the laser of the laser ablation system is configured to ablate the sample to a depth of 500 nm or less per laser pulse.

19. 19. The apparatus of any one of claims 1 to 18, which is a mass cytometer.

20. A method for performing mass cytometry using an apparatus described in any one of claims 1 to 18 or a mass cytometer described in claim 19, comprising the steps of obtaining a sample material from the sampler, ionizing the sample material obtained from the sampler with the pulsed laser to generate sample ions, and detecting the sample ions by mass spectrometry.

21. A method for analyzing a sample using an apparatus as described in any one of claims 1 to 18 or a mass cytometer as described in claim 19, comprising the steps of obtaining a plurality of portions of sample material from the sampler, separately ionizing each portion of the sample material obtained from the sampler with the pulsed laser to produce sample ions, and detecting the sample ions by mass spectrometry, wherein each portion of material is ionized by a plurality of laser pulses.

22. 20. A method of performing mass cytometry using an apparatus according to any one of claims 1 to 18 or a mass cytometer according to claim 19.

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