Autofocus sample imaging apparatus and method

Abstract

This application describes a system and method for autofocus in imaging mass spectrometry. The application describes an improvement over current IMS and IMC devices and methods via an autofocus component that includes multiple apertures in the autofocus system, such as multiple apertures arranged in two dimensions. Because multiple apertures are used, the autofocus system provides redundancy in the event that a measurement of the focal spot on the sample from illumination radiation passed through one or more apertures fails, thereby reducing the number of failed autofocus attempts. [Selection diagram] Figure 6

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Patent Application No. 62 / 729,239, filed September 10, 2018, the entire contents of which are incorporated by reference for all purposes.

[0002] The present invention relates to imaging of samples following laser ablation using Imaging Mass Spectrometry (IMS) and imaging of biological samples by Imaging Mass Cytometry (IMC™). [Background technology]

[0003] Focus is an important parameter in many measurement techniques. Systems that can automatically focus radiation (autofocus systems) have been used for imaging biological materials and to enable laser radiation to ablate different locations on a sample.

[0004] One group of existing autofocus systems utilizes an aperture through which radiation is directed to one or more lenses and then directed to the sample and reflected towards a sensor. Several different relative positions of the aperture and the sample are tested, and the position with the greatest intensity is selected as the most focused. This type of autofocus system can fail for biological samples. This is a particular problem in systems that rely on removing sample material from the sample, for example by laser ablation. This is because failure to autofocus compromises both data quality and sample integrity when the laser is confocal with the autofocus system. Summary of the Invention [Problem to be solved by the invention]

[0005] It is an object of the present invention to provide further and improved devices and techniques for imaging of a sample. [Means for solving the problem]

[0006] Broadly speaking, the analytical apparatus disclosed herein comprises three broadly characterized systems for performing imaging elemental mass spectrometry.

[0007] The apparatus may include an autofocus system. The autofocus system may include an illumination source and an autofocus sensor. The apparatus may further include one or more of a sampling system, an optical microscope, and a movable sample stage. The sampling system and the autofocus system (and optionally also the optical microscope) may be confocal. The apparatus may include further systems as described herein.

[0008] The first system is an autofocus system, which positions the sample at the correct point in the sample chamber for optimal sampling by the laser of the sampling and ionization system.

[0009] The second system is a sampling and ionization system. This system includes a sample chamber, which is the component in which the sample is placed when it undergoes analysis. The sample chamber includes a stage that holds the sample (typically the sample, e.g., a tissue section, a monolayer of cells, or individual cells, is on a sample carrier, such as a microscope slide, and the slide is placed on the stage, such as when a cell suspension is dropped onto a microscope slide). A laser in the sampling and ionization system acts to remove material from the sample in the sample chamber, which is converted to ions as part of a process that causes removal of material from the sample or by a separate ionization system downstream of the sampling system (the removed material is referred to herein as sample material). To generate elemental ions, hard ionization techniques are used. The ionized material is then analyzed by a third system, which is a detector system. The detector system can take different forms depending on the particular properties of the ionized sample material to be determined, for example a mass detector in a mass spectrometry based analyzer device.

[0010] The present invention provides an improvement over current IMS and IMC devices and methods via an autofocus component that includes multiple apertures in an autofocus system, such as multiple apertures arranged in two dimensions. An illumination source of the autofocus system directs radiation through the multiple apertures to a sample to be ablated. Because multiple apertures are used (i.e., multiple apertures are available for focus analysis), the autofocus system provides redundancy in the event that a measurement of the focus of the sample from illumination radiation passing through one or more apertures fails (e.g., tissue with varying composition, non-uniform topology, or voids may not reflect illumination radiation at a particular location enough to detect it through a single aperture for autofocus), thereby reducing the number of failed autofocus attempts.

[0011] Thus, in operation, a sample is introduced into the device and how the sample is focused for a given relative position of the focus of the irradiating radiation and the sample is measured (e.g., by assigning a focus score). The relative position of the focus of the irradiating radiation and the sample is then changed and focus measurements are obtained for one or more changed positions. The positions can then be compared to establish a direction or position that increases the focus. The relative position can then be changed again and, optionally, further measurements can be obtained. This process can be repeated to further improve the focus score. Once a desired focus score is obtained, the sample can be sampled (e.g., by laser ablation) to produce an ionized material using a laser source (the sampling may produce a vapor / specific material, which is then ionized by an ionization system) and ions of the sample material are passed into a detector system. The detector system can detect many ions, but most of these ions will be ions of the atoms that necessarily form the sample. In some applications, this may be sufficient, for example for the analysis of minerals, such as geological or archaeological applications.

[0012] In some cases, for example when analyzing biological samples, the natural elemental composition of the sample may not be adequately informative. This is because all proteins and nucleic acids are generally composed of the same main atoms, so that while it is possible to distinguish areas containing proteins / nucleic acids from areas that do not contain such proteins or nucleic acid material, it is not possible to distinguish a particular protein from all other proteins. However, by labeling the sample with atoms that are not present or at least not present in significant amounts in the material to be analyzed under normal conditions (e.g., certain transition metal atoms such as rare earth metals; see the section on labeling below for further details), certain properties of the sample can be determined. As with IHC and FISH, detectable labels can be attached to specific targets on or in the sample (such as a given cell or a tissue sample on a slide) by using SBPs such as antibodies, nucleic acids or lectins, among others, that target molecules on or in the sample. To detect the ionized labels, a detector system is used, since it is adapted to detect ions from atoms that are necessarily present in the sample. By relating the detected signals to known locations of the sampling of the sample that gave rise to those signals, an image can be generated of the atoms present at each location, i.e., both the natural elemental composition and any labeled atoms. In embodiments where the sample's original elemental composition has been depleted prior to detection, the image may consist only of labeled atoms. This technique allows many labels to be analyzed in parallel (also called multiplexing), which is a great advantage in the analysis of biological samples.

[0013] The present invention provides an autofocus component that includes multiple apertures.

[0014] The present invention also provides an autofocus system for focusing on a sample, comprising: an illumination source for emitting radiation to illuminate the sample; an autofocus component of the present invention that allows radiation from an illumination source to pass through a plurality of apertures; an objective lens disposed in the path of the radiation to focus the radiation towards the sample; an autofocus sensor positioned to receive radiation reflected from the sample and positioned confocal with the autofocus component; An autofocus system comprising:

[0015] The present invention also provides an apparatus for analyzing a biological sample, comprising: Autofocus system and a sampling and ionization system for removing material from a sample and ionizing the material to form elemental ions, the sampling and ionization system comprising a laser source for sampling the sample; Equipped with The focus of the laser source of the sampling and ionization system is confocal with the autofocus component and autofocus sensor of the autofocus device, An apparatus is also provided.

[0016] In some embodiments, the sampling and ionization system comprises a sampling system and an ionization system, where the sampling system comprises a laser source and a sample stage, and the ionization system is adapted to receive material removed from the sample by the sampling system and ionize the material to form elemental ions.

[0017] Another aspect of the present invention is determining a focus score for a first position of the sample; moving the sample to a second location; determining a focus score for the second position; Comparing the focus scores to each other; Including, Determining the focus score includes illuminating the sample with radiation from an illumination source, where the illumination radiation is passed through an autofocus component comprising a plurality of apertures, and detecting the illumination radiation reflected from the sample, where moving the sample is a movement parallel to an axis along which the illumination radiation is directed to the sample (i.e., the movement is in the z-axis); An autofocus method is also provided.

[0018] Another aspect of the present invention is determining a direction of the focal spot at position n of the sample; moving the sample in the direction of the focus to position (n+1); Including, Determining the direction of focus includes illuminating the sample with radiation from an illumination source, the illumination radiation being passed through an autofocus component having a plurality of apertures, at least two of the apertures of the autofocus component being offset in an axis along which the illumination radiation passes through the autofocus component, and detecting the illumination radiation reflected from the sample using the autofocus sensor, the step of moving the sample being a movement parallel to the axis along which the illumination radiation is directed to the sample (i.e., the movement is in the z-axis); An autofocus method is also provided.

[0019] Aspects of the invention also provide a method of mapping the topology of a surface comprising the steps of performing an autofocus method (e.g. the method comprises the autofocus method), moving the sample in the plane of the sample (i.e. in the X-axis and / or Y-axis) to a second position in the plane of the sample, and iteratively performing the method comprising the autofocus method of the invention to again record a best focus position at the second position in the plane of the sample.

[0020] The present invention provides a method for analyzing a sample, comprising the steps of: Implementing the method of the present invention to position a sample at a laser focus for laser ablation; laser ablation of a sample on a sample stage at a plurality of locations; subjecting the plume to ionization and mass spectrometry, where detection of atoms in the plume allows an image of the sample to be constructed, and optionally the plurality of locations is a plurality of known locations; Also provided is a method comprising: [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram of the illumination radiation path in a prior art autofocus system with a single aperture autofocus component that brings the sample into perfect focus. [Diagram 2] FIG. 1 is a schematic diagram of the illumination radiation path of light from an illumination source to an out-of-focus sample in a prior art autofocus system with a single aperture autofocus component. [Diagram 3] FIG. 1 is a schematic diagram of the illumination radiation path of light from an out-of-focus sample to an autofocus sensor in a prior art autofocus system with a single aperture autofocus component. [Figure 4] FIG. 1 is a schematic diagram of the illumination radiation path in a prior art autofocus system with a multi-aperture autofocus component that brings a sample into perfect focus. [Diagram 5] FIG. 2 is a schematic diagram of the illumination radiation path in a prior art autofocus system including the multiple aperture autofocus component of the present invention with an offset aperture that is perfectly in focus on the sample. [Figure 6] FIG. 1 is a schematic diagram of a process for bringing an autofocus component into a confocal position with an autofocus sensor during manufacturing of an autofocus system according to an embodiment of the present invention. [Figure 7]1 is a schematic diagram of a process for focusing laser radiation to a confocal position with autofocus components and an autofocus sensor during manufacture of an apparatus of the present invention comprising an autofocus system and a laser-based sampling and ionization system according to an embodiment of the present invention. [Figure 8] 1 is an image from an autofocus sensor in an autofocus system of an embodiment of the present invention detecting illumination radiation reflected from a nine-aperture autofocus component of the present invention, showing reflected radiation detected at nine expected locations (i.e., areas of interest). [Figure 9] 1 is an image from an autofocus sensor in an autofocus system of an embodiment of the present invention detecting illumination radiation reflected from a nine-stop autofocus component of the present invention, showing reflected radiation detected at some but not all of the expected locations (i.e., areas of interest). [Figure 10] FIG. 2 is a diagram of a hybrid autofocus component arrangement according to an embodiment of the present invention. [Figure 11] 1 is a plot of a sample Z-profile generated using the sample topology mapping method of an embodiment of the present invention. The plot is shaded to indicate the Z position at best focus, with a numerical range for each shade shown to the right of the graph. In this example, an increase in the X or Y coordinate corresponds to an increase in this Z position. [Figure 12] Schematic diagram of an autofocus setup according to an embodiment of the present invention. Two light sources are used whose light is guided through a small aperture focused on the focal plane of the objective lens. When the sample is out of focus, the camera observes the image from each LED to be not only blurred but also offset according to the illumination angle of each LED at the sample. [Figure 13]Top: LED images acquired at a defocus distance of +60 μm according to an embodiment of the invention. The two images are similarly blurred and offset along the illumination direction for each LED. Bottom: Calculated correlation function between the two images. A parabolic peak fit gives a peak location of -512.08 pixels, corresponding to a focus offset of +60 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In operation, radiation is emitted from the illumination source, passes through an aperture of an autofocus component that includes multiple apertures, and is directed to a sample on a stage. The illumination radiation is reflected by the sample. The reflected radiation is directed to an autofocus sensor that is confocal with the autofocus component. The laser of the laser-based sampling and ionization system, if present, is also confocal with the autofocus component and the autofocus sensor. The reflected radiation can be detected at several positions of the autofocus sensor, each position correlating to an aperture of the autofocus component through which the radiation passed. The reflected radiation detected at the sensor is analyzed (e.g., evaluating the intensity of the radiation at each position correlating to the aperture to calculate a focus score - the sum of the intensities from the positions corresponding to all the apertures). The relative position of the focal point of the illumination radiation to the sample is then changed, and further measurements of the focus by the autofocus sensor of the radiation reflected from the sample are obtained. The process is then repeated, adjusting the relative position of the focal point of the illumination radiation to the sample three times, measuring the focus a third time, and so on. This cycle can be repeated iteratively until a maximum focus is reached. The sample can then be sampled and ionized by a sampling and ionization system, including a laser, if present, for maximum efficiency by positioning the sample at the focal point of the laser, which is confocal with the autofocus sensor and autofocus aperture. Thus, various types of autofocus devices including autofocus components of the present invention with multiple apertures can be used in practicing the present disclosure, some of which are described in detail below.

[0023] Mass detection-based analyzers The autofocus system and method of the present application may provide one or more advantages, such as no pre-processing required, rapid autofocus, low-cost solution to autofocus, feature creation, focal map generation, and / or improved accuracy. In certain embodiments, the autofocus system and method can perform autofocus for laser ablation-based mass spectrometry. The autofocus system and method can also perform focus for an optical microscope (e.g., an inspection system) used to guide laser ablation-based mass spectrometry. The sample may be biological tissue with different surface topologies. In certain embodiments, the sample may be stained with mass-tagged SBP. Unlike conventional fluorescence microscopy, imaging mass cytometry does not simultaneously acquire images over a wide field of view. Rather, individual spots (e.g., pixels) are removed from the sample by radiation (e.g., laser ablation) and sent to a mass spectrometer. To acquire images with single-cell resolution or better, the individual spots are small (e.g., within the range of about one micron in size). Therefore, it is important to precisely focus the radiation on the sample. Variations in the tilt of the sample (or support slide), variations in the control of the sample stage, system drift, and / or variations in the topology of the sample can lead to variations in the relationship between the focal point and the sample surface, which can adversely affect sampling and sampling consistency.

[0024] An apparatus of the present application may include an autofocus system according to any of the embodiments described herein. The autofocus system may include an illumination source and an autofocus sensor. As discussed further herein, the apparatus may include further components.

[0025] In certain embodiments, the apparatus further comprises a sampling system, such as a laser ablation system. The sampling system and autofocus system may be confocal. The apparatus may further comprise an optical microscope for inspection of the sample, for example to identify regions of interest. The apparatus may further comprise a movable sample stage.

[0026] The illumination source of the autofocus system may be, for example, a laser diode with appropriate beam-shaping optics, or a bright LED with a specific lens system and an aperture or set of apertures. The detector may be, for example, the same camera used for inspection (the distinction between autofocus light and illumination light can be achieved in real time by LED pulsing, optical filtering, or non-real time by performing autofocus at the beginning or end of each line), a line sensor oriented along the expected translation direction, or a position-sensitive photodiode. In certain embodiments, the illumination source of the autofocus system may be shared with the optical microscope. Alternatively, the illumination source of the autofocus system may be separate from the illumination source of the optical microscope.

[0027] An autofocus system may provide an illumination scheme in which a strip or other pattern of light is incident on the slide at an angle, and then use some detector (camera or other position sensitive device) to detect the XY position of the light pattern on the slide surface. Due to the angle between the incident light and the slide normal, the displacement of the returned light compared to some calibrated best focus position depends linearly on the amount of defocus, so that the best focus position can be found directly (no full scan is required).

[0028] Autofocus System An autofocus system typically includes a series of components, such as an autofocus component with multiple apertures, an illumination source, and an autofocus sensor. Depending on the arrangement of these components, various optical components may be included as needed.

[0029] The autofocus system described herein may be used in the methods of the invention described herein that employ an autofocus step. Thus, the invention provides the use of the autofocus system of the invention to autofocus an apparatus (e.g., an imaging mass cytometer of an imaging mass spectrometer).

[0030] The apparatus may be configured to perform autofocus (e.g., autofocus correction) by moving the sample stage and / or adjusting optical elements. In certain embodiments, the apparatus may be configured to perform autofocus by moving the sample stage in response to a readout from an autofocus component. In certain embodiments, the apparatus is not configured to perform autofocus by adjusting optical elements, for example when only the z position of the sample stage is moved. Thus, the optical elements may not be able to move to achieve focus.

[0031] The apparatus may perform autofocus that is sample independent, e.g., that is not dependent on the characteristics or contrast provided by the sample. The autofocus system may perform autofocus during a sample run, e.g., when the sample stage positions the sample at different X,Y coordinates.

[0032] In certain embodiments, the autofocus system provides fast autofocus (e.g., fast autofocus correction). For example, the autofocus system can provide autofocus feedback at a rate of 100 Hz or more, 200 Hz or more, 500 Hz or more, 1 kHz or more, 2 kHz or more, 5 kHz or more, or 10 kHz or more.

[0033] In certain embodiments, the autofocus may be implemented as a closed loop, such as a PID loop, where the autofocus provides instantaneous adjustments to the actuator. In certain embodiments, the PID controller continuously calculates an error value as the difference between a desired set point and a measured process variable, and applies a correction based on proportional, integral, and derivative terms.

[0034] In certain embodiments, autofocus may be performed at a hardware level without processing autofocus data at a software level. Hardware-based autofocus may significantly reduce cycle times for autofocus.

[0035] Autofocus correction may be performed by adjusting optical elements (e.g., the distance between optical elements), via a positioner that adjusts the focus in the Z direction, and / or by adjusting the sample stage in the Z direction. In certain embodiments, autofocus correction may be performed by adjusting the z position of the sample stage.

[0036] In certain embodiments, the autofocus system may project a focus map across X, Y, or XY coordinates (e.g., create a focus map that guides autofocus correction across a range of coordinates). The focus map may make or be used to make adjustments to the focus of the laser ablation optics across multiple coordinates without having to perform autofocus at each coordinate. In some cases, the coordinates may be sample coordinates (e.g., coordinates across a tissue sample). The sample stage may be moved to position the sample at the new coordinates. Alternatively or additionally, the laser may be scanned continuously across the coordinates by one or more positioners. The positioners may be mirror-based positioners (such as galvanometer mirrors, polygon scanners, MEMS mirrors, piezoelectric device mirrors, etc.) or solid-state positioners (such as AODs and EODs). The focus map may provide best focus (e.g., in the z direction) across multiple coordinates (e.g., at multiple coordinates) or the optics may be adjusted to obtain best focus from an initial position. Thus, the focal map may be based on a relative (e.g., starting) or objective reference system. In certain embodiments, the sample stage may be moved in the z direction while the laser is scanned along the focal line in the X,Y plane.

[0037] In certain embodiments, autofocus may enable selective sampling or removal of surface material at higher z positions compared to the surrounding material, such that a uniform sample surface is obtained for subsequent investigation.

[0038] The device may be further configured to provide an optical image during the sample run. For example, the device may include an image sensor, such as a CCD or CMOS, that provides an optical image of the sample. The device may include an optical microscope (of which the image sensor is a part), such as a bright field / wide field microscope or a fluorescence microscope. The optical microscope may be used to inspect the sample, for example to identify regions of interest in the sample by laser ablation.

[0039] The autofocus system may comprise at least one aperture. An illumination source (such as from an LED) impinging on the aperture may provide a feature that is reflected from the sample (or sample support) to the autofocus sensor. In certain embodiments, the autofocus system comprises multiple apertures, such as three or more apertures arranged in two dimensions.

[0040] The autofocus system may provide multiple features (i.e., a spot of any shape) impinging on the autofocus sensor. The autofocus system may provide multiple features using multiple apertures, alternating illumination sources (such as two or more LEDs or laser diodes), and / or diffractive beam splitters.

[0041] Autofocus components with multiple apertures The present invention provides an autofocus component comprising a plurality of apertures, the apertures allowing radiation to pass through the apertures, and generally the material of the autofocus component does not otherwise allow radiation to pass through it.

[0042] Previous approaches to autofocus are characterized by a reliance on a single aperture, which results in unproductive autofocus attempts when the illumination radiation passing through the single aperture is blocked or otherwise impeded (illumination radiation from the aperture does not properly reach the sensor). Thus, erroneous intensity (or lack of intensity) is detected. For example, a point on a sample where illumination radiation is directed through a single aperture may not be reflective because, for example, no biological material is present there (as can happen when voids are introduced into a tissue section as a result of a preparation procedure or as a result of random distribution of cells on a slide of a cell smear).

[0043] By providing multiple apertures, the autofocus component introduces redundancy into the autofocus system, whereby failure of the illumination radiation to reflect off the sample can be compensated for by the reflected illumination radiation detected from the remaining apertures. Thus, the autofocus system is made more robust, which reduces the time spent on failed autofocus attempts and improves efficiency. Increasing the number of apertures improves the robustness of the autofocus by making additional apertures available in case other apertures fail. Thus, in some cases, the autofocus component comprises at least 2 apertures, e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 16, or at least 25 apertures.

[0044] In some cases, the apertures of the autofocus components are arranged in a regular shape, which allows for efficient coverage of the area of ​​the sample. Thus, in some cases, the apertures of the autofocus components are arranged at the vertices of a regular polygon, such as three vertices of a triangle, four vertices of a square or rectangle, five vertices of a pentagon, six vertices of a hexagon, seven vertices of a heptagon, eight vertices of an octagon, nine vertices of a nonagon, or ten vertices of a decagon.

[0045] When the apertures of the autofocus components are arranged in a regular shape, the vertices of the regular shape can be arranged at a fixed distance from the center of gravity of the polygon. Such polygons with a short distance provide higher redundancy over a small area of ​​the sample. Conversely, polygons with a large distance allow for more rapid topological analysis over a larger area of ​​the sample, as described in more detail below. In some cases, the distance from the center of gravity of the polygon to each vertex is less than 10 μm, 15 μm, 20 μm, 25 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 750 μm, or 1 mm. In some cases, the distance from the centroid of the polygon to each vertex is greater than 10 μm, 15 μm, 20 μm, 25 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 750 μm or 1 mm.

[0046] In the preferred case where the apertures of the autofocus component are arranged in a regular shape, the component may comprise further apertures in addition to the vertex apertures described above. In one such example, there is a further aperture at the center of gravity of the polygon. This center of gravity aperture is generally arranged to deliver the irradiating radiation to the point on the sample where ablation of the sample occurs, for example by a laser ablation sampling system as discussed herein. Thus, if multiple apertures (few center of gravity apertures) allow measurement of the focus around the sampling position, the center of gravity aperture contributes to the focus calculation by readings from the exact ablation point. In another such example, the component comprises further apertures at the midpoints of the sides of the polygon defined by the vertex apertures. These peripheral apertures enhance the efficient coverage of the sample area by providing additional positions that can be used for the focus calculation.

[0047] In another example, the apertures are arranged in a regular grid, which gives the same efficient coverage of the sample area but may be preferred for a particular shape and / or topology of the sample. The apertures are arranged in a regularly spaced grid, such as a 2x2 grid, a 3x3 grid, a 4x4 grid, a 5x5 grid, a 6x6 grid, a 7x7 grid, an 8x8 grid, a 9x9 grid, a 10x10 grid, or a grid greater than 10x10.

[0048] When the apertures of the autofocus component are arranged in a regular grid, adjacent grid apertures may be spaced apart from the center point of the aperture by a certain inter-aperture distance. Such a grid with a short distance provides greater redundancy over a small area of ​​the sample. Conversely, a grid with a long distance allows for more rapid topological analysis over a larger area of ​​the sample, as described in more detail below. The inter-aperture distance between adjacent grid apertures, measured from the center point of the aperture, may be 10 μm, 15 μm, 20 μm, 25 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm, 250 μm, 500 μm, 750 μm, 1 mm. In some cases, the apertures are polygonal, such as triangular or square.

[0049] In some cases, the shape of the aperture is selected to maximize the robustness of the focus score measurement. In some cases, the aperture is circular and has a diameter of 1 μm, 2.5 μm, 5 μm, 7.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 50 μm, 75 μm, or 100 μm or less. In some cases, the aperture is circular and has a diameter of 1 μm, 2.5 μm, 5 μm, 7.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 50 μm, 75 μm, or 100 μm or more.

[0050] For some method applications described herein involving calculation of an optimal focus position based on collection of illumination radiation focused through multiple apertures in a single focal plane, an autofocus component with this aperture placement is required, and thus in some embodiments the autofocus component is planar (so that all apertures are in the same focal plane).

[0051] In some embodiments, the autofocus component comprises apertures that are offset relative to each other in the axis of the illumination radiation. That is, the autofocus component comprises one or more steps, or for that matter, two or more aperture planes. See, for example, feature 502 in FIG. 5. In some embodiments, the autofocus component comprises two aperture planes. In some embodiments, the autofocus component comprises three aperture planes. In some embodiments, the autofocus component comprises four or more aperture planes. In some embodiments, each aperture plane comprises two or more apertures, for example, three apertures per aperture plane. In some cases with two aperture planes, the planes are offset equally on either side of the plane that is confocal with the detector. Thus, in use, the autofocus component indicates perfect focus when both aperture planes are equally defocused. When one level of aperture is more defocused than the other level of aperture, the direction of the focal plane from the sample is indicated. In an embodiment with three aperture planes, one aperture plane may be confocal with the autofocus detector, and two more planes are offset equally on either side of the aperture plane that is confocal with the detector. Thus, in use, the autofocus component will indicate perfect focus when the first aperture plane is in focus and both further aperture planes are equally defocused.

[0052] In some embodiments, the autofocus component acts as a hybrid mask for the illumination source. Such a hybrid autofocus component comprises opaque areas where multiple apertures are located to block the illumination radiation, but otherwise comprises areas that allow the illumination radiation to pass through to the sample (either by using a radiation transparent material or by simply leaving the voids of the autofocus component relatively large). The size of the areas is different from the apertures of the autofocus component. Thus, by using such an autofocus component, autofocus can be performed along the description herein, but at the same time the illumination radiation is allowed to go to the sample so that it can be visually inspected by the autofocus sensor (when the autofocus sensor is a camera or device with equivalent functionality). In some embodiments, the apertures of the hybrid autofocus component are offset relative to each other in the axis of the illumination radiation. Thus, in some embodiments, the hybrid autofocus component comprises an offset aperture, such as an offset surface of the aperture, as described in any embodiment of the previous paragraph. Using such a hybrid autofocus component allows for "live" autofocus - i.e., maintaining the best focus when the sample is moved in a direction perpendicular to the optical axis (i.e., during ablation of the sample for "live" autofocus). This autofocus component and method allows for simultaneous ablation, sample presentation, and focus tracking.

[0053] The present invention provides for the use of such autofocus components in the methods described herein (e.g., in the section "Methods for Autofocus" on page 25 and the section "Sample Topology Mapping Methods" on page 29, including each and every specific embodiment discussed in those sections).

[0054] irradiation source The autofocus system described herein includes an illumination source. The illumination source may include at least one LED or laser diode, but may include two or more LEDs, two or more laser diodes, or a combination of LEDs and laser diodes. In certain embodiments, the two or more LEDs or laser diodes provide different characteristics that impinge on the autofocus sensor. If an LED is used, an aperture located between the LED and the sample may provide the characteristics. In certain embodiments, the device may not need to switch between autofocus and inspection apertures.

[0055] In certain embodiments, two LEDs or laser diodes may provide alternating illumination. An autofocus system may be configured so that features produced by alternating illumination overlap when the system (e.g., a laser ablation system) is in focus. Alternating laser diode radiation may be faster than alternating LED radiation. Additionally, laser diodes may produce brighter and / or smaller features than LED radiation through an aperture. However, the cost of laser diodes may be higher than LEDs.

[0056] In certain embodiments, the illumination source may be a color-multiplexed illumination source. The illumination source may include a programmable LED array. For example, the transition shift of the image generated by two-color LED illumination may be used for dynamic focus correction. Color-multiplexed autofocus is discussed in the context of whole slide imaging by Jiang, Shaowei, et al., "Fast and robust whole slide imaging based on LED array illumination and color-multiplexed single-shot autofocus," arXiv:1905.03371 (2019).

[0057] In certain embodiments, one or more of the illumination sources provide illumination at a non-zero angle relative to the sample normal. The illumination provided at the non-zero angle may be from one or more laser diodes and / or LEDs directed through an aperture. Features provided by the illumination may reflect off the sample and / or sample support and impinge on the autofocus sensor.

[0058] In certain embodiments, the autofocus system may include multiple apertures, or may optionally include only one light source, such as an LED. The system may be configured such that illumination moving through the multiple apertures results in multiple features being incident on the autofocus sensor (e.g., after reflection from the sample or sample support).

[0059] The illumination source emits radiation that is directed through multiple apertures of the autofocus component before traveling to the sample.

[0060] In some cases, the illumination source is a light emitting diode (LED). Such illumination sources are commercially available, for example, from Olympus (Japan) and Euromex (The Netherlands).

[0061] In some cases, the radiation source is a thermal radiator or an incandescent lamp, such as an incandescent tungsten halogen bulb. The lamp may comprise a sealed glass bulb filled with an inert gas and containing a tungsten wire filament excited by DC current, and may be provided with a housing comprising one or more layers of a heat sink to help dissipate excess heat. Such radiation sources are commercially available, for example, from Cairn Research (UK).

[0062] In some cases, the radiation source is an arc lamp. Examples include metal halide, mercury vapor, xenon, and zirconium arc lamps. Such radiation sources are commercially available, for example, from World Precision Instruments (UK).

[0063] In some cases, a laser source is used as the radiation source, for example an argon ion laser or a krypton laser. Such radiation sources are commercially available, for example from Laser 2000 (UK).

[0064] Autofocus Sensor Here, the autofocus sensor comprises at least one of an image sensor (such as a CCD or CMOS), a line sensor, a position-sensing photodiode, an adjacent photodiode, and a split photodiode (such as an orthogonal photodiode). In certain aspects, the autofocus sensor is an image sensor and may be shared with the inspection system (such as an optical microscope).

[0065] In certain aspects, the autofocus sensor is a line sensor. When the system includes an optical microscope, the optical microscope has a sensor separate from the autofocus sensor. Autofocus systems including a line sensor may further include a cylindrical lens.

[0066] When alternating between two or more light sources providing features, the autofocus may include correction (e.g. by adjusting the z position of the sample stage) until the features overlap on the detector. A CCD, CMOS or line scan detector may then be used to check for feature coincidence. In general, the form of detection need not be constrained to X,Y coordinates at best focus, and need not include detectors such as orthogonal photodiodes.

[0067] In certain embodiments, the illumination source may be a color-multiplexed illumination source. The illumination source may include a programmable LED array. For example, the transition shift of the image generated by two-color LED illumination may be used for dynamic focus correction. Color-multiplexed autofocus is discussed in the context of whole slide imaging by Jiang, Shaowei, et al., "Fast and robust whole slide imaging based on LED array illumination and color-multiplexed single-shot autofocus," arXiv:1905.03371 (2019).

[0068] In certain embodiments, one or more of the illumination sources provide illumination at a non-zero angle relative to the sample normal. The illumination provided at the non-zero angle may be from one or more laser diodes and / or LEDs directed through an aperture. A feature provided by the illumination may reflect off the sample and / or sample support and impinge on the autofocus sensor. The system may be pre-calibrated such that a feature impinging on a particular coordinate on the autofocus sensor indicates that the system is in focus. Autofocus may then include adjusting the focus (e.g., by moving optics and / or adjusting a movable stage), checking the incidence of the feature on the autofocus sensor, and repeating until the feature is within a tolerance of a predefined coordinate. In such a case, the autofocus sensor may be a CCD, CMOS, line array, or split photodiode.

[0069] In certain embodiments, the autofocus system may include multiple apertures, or may optionally include only one light source, such as an LED. In such cases, the autofocus sensor may be an image sensor, such as a CCD or CMOS. The system may be configured such that illumination moving through the multiple apertures results in multiple features being incident on the autofocus sensor (e.g., after reflection from the sample or sample support). The presence of the features may be used to determine whether the system is in focus and / or to determine corrections to make to focus the system. The presence of a feature may refer to the ability to detect the feature, the focus of the feature (e.g., the area in which the feature is detected and / or the distribution of intensity across that area), the intensity of the feature, and / or the consistency of the intensity of the feature. Autofocus correction based on such a system may be iterative.

[0070] The data provided by the autofocus sensor may be processed by the ASIC, FPGA or software level and used to perform autofocus corrections (e.g. by positioning optical elements and / or by moving the sample stage).

[0071] The autofocus sensor detects the radiation reflected from the sample, which is used to measure the focus score. The autofocus sensor may be a camera, for example a charge-coupled device image sensor (CCD) based camera, an active pixel sensor (APS) based camera, or any other radiation detection means within the autofocus system.

[0072] In some embodiments, the sensor is a CCD (or is based on a CCD). A CCD is a means for detecting light and converting it into digital information that can be used to generate an image. A CCD comprises a silicon chip that contains an array of light-sensitive pixels. During exposure, each pixel generates a charge proportional to the intensity of the light incident on the pixel. After exposure, a control circuit causes a series of charge transfers to generate a series of voltages. These voltages can then be analyzed to generate an image. Suitable CCDs are available, for example, from Cell Biosciences. In some embodiments, the sensor is an active pixel sensor (APS). An APS is an image sensor consisting of an integrated circuit that contains an array of pixel sensors, e.g., a CMOS sensor, where each pixel includes a photodetector and an active amplifier. Suitable APSs are available, for example, from ON Semiconductor.

[0073] In some embodiments, the sensor is a photodetector. Photodetectors may be used to image samples and / or identify features / regions of interest of interest prior to imaging by elemental mass spectrometry. Photomultiplier tubes are available, for example, from ThorLabs.

[0074] In some embodiments, the sensor is a photomultiplier tube. A photomultiplier tube comprises a vacuum chamber with a photocathode, several dynodes, and an anode. Photons incident on the photocathode cause the photocathode to emit electrons as a result of the photoelectric effect. The electrons are multiplied by the dynode due to a process of secondary emission to produce a multiplied electron current, which is then detected by the anode to provide an indication of the detection of electromagnetic radiation incident on the photocathode. Photomultiplier tubes are available, for example, from ThorLabs.

[0075] The use of a camera provides the advantage that an autofocus sensor can be used to record an image of the sample, and therefore can function as a camera as described below with respect to the various sampling and ionization system components of the apparatus of the present invention.

[0076] Further components The autofocus system may also include an objective lens, which focuses the illumination radiation onto the sample (e.g., 25x reduction). In an apparatus of the invention that includes a laser-based sampling system, radiation from the laser of the sampling system can be focused onto the sample by the same objective lens.

[0077] The autofocus system may also include a tube lens. In some embodiments, the tube lens may be positioned in the path of the illumination radiation between the autofocus component and the sample, such as between the autofocus component and one or more beam splitter components. The lens positioned in this way may function to collimate the illumination radiation after passing through the aperture of the autofocus component. In some embodiments, the tube lens may be positioned in the path of the illumination radiation between the sample and the autofocus sensor, for example, between one or more beam splitter components and the autofocus sensor. The lens positioned in this way may focus the illumination radiation reflected from the sample onto the autofocus sensor.

[0078] The autofocus system may include one or more beam splitters that split the incident radiation into two separate beams in a specified ratio, and are adapted to direct radiation from the illumination source to the sample and illumination radiation reflected from the sample to the autofocus sensor. Beam splitters generally take one of two forms: plate splitters (a thin flat glass plate coated on a first surface of a substrate) and cube splitters (constructed using two generally right-angle prisms). Beam splitters are widely available commercially from rp-photonics, Thorlabs, and Edmund optics.

[0079] The autofocus system may include a sample stage that supports the sample during operation of the system (typically the sample is on a sample carrier). The sample stage may be movable. In some embodiments, the stage may be movable in the x, y, and z axes. If the apparatus in which the autofocus system is located also includes a sampling and ionization system, the sample stage of the autofocus system is also the sample stage of the sampling and ionization system, as described below (e.g., if the sample stage is within the sample chamber).

[0080] The autofocus system may also include a controller module. The controller module controls and adjusts the moments of the components of the autofocus system. In some embodiments. The controller module receives input from the autofocus sensor and controls the position of the sample stage during the autofocus process based on the received input. The controller module may include a programmable store programmed with instructions for performing the autofocus methods of the present invention described herein, including each and every specific embodiment discussed in these sections. For example, in some embodiments, the controller module includes instructions for performing the autofocus method, which includes: determining a focus score for a first position of the sample; moving the sample to a second location; determining a focus score for the second position; Comparing the focus scores to each other; Including, Determining the focus score includes illuminating a sample with radiation from an illumination source, where the illumination radiation is passed through an autofocus component that includes a plurality of apertures, and detecting the illumination radiation reflected from the sample with an autofocus sensor, where detecting the radiation reflected from the sample includes detecting radiation at a known location (also referred to as a region of interest) on the autofocus sensor; The step of moving the sample is a movement parallel to the axis along which the illumination radiation is directed to the sample (ie, the movement is in the z-axis).

[0081] In some embodiments, the controller module includes instructions for performing an autofocus method, the autofocus method comprising: determining a direction of the focal spot at position n of the sample; moving the sample in the direction of the focus to position (n+1); Including, Determining the direction of focus includes illuminating the sample with radiation from an illumination source, where the illumination radiation is passed through an autofocus component having a plurality of apertures, at least two of the apertures of the autofocus component being offset on an axis along which the illumination radiation passes through the autofocus component, and detecting the illumination radiation reflected from the sample with an autofocus sensor, where detecting the radiation reflected from the sample includes detecting radiation at a known location (also referred to as a region of interest) on the autofocus sensor; The step of moving the sample is a movement parallel to the axis along which the illumination radiation is directed to the sample (ie, the movement is in the z-axis).

[0082] In some embodiments, the controller module includes instructions for executing a method for mapping a topology of a sample surface, the method comprising: performing an autofocus method; moving the sample in the plane of the sample (i.e., in the X-axis and / or Y-axis) to a second position in the sample plane; performing an autofocus method of the present invention to record the best focus position again at a second position in the plane of the sample, for example the autofocus method described in the previous paragraph, which is programmed in the controller module; Includes.

[0083] In some embodiments, the programmable store is a hard drive, optical disk, CD-ROM, DVD-ROM, ROM, RAM, EPROM, EEPROM, magnetic or optical cards, solid-state memory devices, or other type of media / computer readable medium suitable for storing electronic instructions.

[0084] In some embodiments, the autofocus apparatus for LA-ICP-MS may include that the apparatus does not have to switch between autofocus and inspection aperture, and may further include one or more of a movable sample stage, an optical microscope for inspection of the sample, a laser ablation sampling system, a gas conduit coupling the laser ablation sampling system to the ICP ionization system, and / or a mass spectrometer (e.g., a time-of-flight or magnetic sector mass spectrometer). The mass spectrometer may be configured to simultaneously detect mass tags (such as lanthanides in the mass tags). The autofocus system may result in multiple spots impacting the autofocus sensor. The sampling system and the autofocus system may be confocal. The system may be configured to perform autofocus during a sample run by adjusting the position of the sample stage based on multiple illumination points.

[0085] Component placement Referring to FIG. 1 (prior art system with only one aperture in the autofocus component (102)), the illumination source (101) emits illumination radiation that passes through the aperture of the autofocus component (102). This illumination radiation is then collimated by the tube lens (103) and directed to the sample by the beam splitter (104). The illumination radiation is focused by the objective lens (105) onto the sample (106) and is typically demagnified by a factor of 25. The illumination radiation reflected from the focused sample (106) back along the optical path then passes back through the objective lens (105) and through the beam splitter (104) towards the autofocus sensor (107). The reflected illumination radiation passes through the tube lens (108) before impinging on the autofocus sensor. In this arrangement, the autofocus component and the autofocus detector are confocal, so that if the sample is also in the focal plane, the illumination radiation reflected from the sample will appear as a bright, well-defined spot at the detector.

[0086] If the sample is out of focus, no bright, distinct spot will be detected. Referring to FIG. 2, the illumination source (201) emits illumination radiation that is transmitted through the aperture of the autofocus component (202). This illumination radiation is then collimated by the tube lens (203) and directed to the sample by the beam splitter (204). The illumination radiation is focused by the objective lens (205) towards the sample (206). Because the sample is not in the focal plane of the illumination radiation, the illumination radiation is defocused on the sample (206). Referring now to FIG. 3, the defocused illumination radiation is reflected back along the optical path from the out-of-focus sample (306), then passes back through the objective lens (305), passes through the beam splitter (304) towards the autofocus sensor (307), where it is further defocused. Thus, if the sample is out of focus at the autofocus sensor (207 / 307), so will the autofocus component (202 / 302), and so the illumination radiation from the aperture of the autofocus component (202 / 302) will be out of focus at the sample (206 / 306) and further out of focus at the autofocus sensor (207 / 307). By moving the sample stage, the sample can be moved into focus. However, if the particular area at which the illumination radiation is directed is not reflective, as discussed above, focusing cannot be achieved by the prior art devices and methods.

[0087] With reference to FIG. 4, the device of the present invention operates on a similar principle. However, the expected number of spots detected by the autofocus sensor (407) corresponds to the number of apertures of the autofocus component (402). Three apertures are shown in FIG. 4. Because multiple apertures are used, the beam of illumination radiation is focused (406) on distinct points on the sample and is thus reflected (407) on distinct points on the sample that are detected, these points being known (also referred to here as regions of interest). Thus, the reflected illumination radiation can be detected in the expected region (see, for example, FIG. 8, which uses a nine-aperture autofocus component with apertures arranged in a grid). With the autofocus component of the present invention, even if some points on the sample where the illumination radiation is directed do not reflect the illumination radiation back to the autofocus sensor, the other apertures do (see FIG. 9, which shows the usefulness of a nine-aperture autofocus component, since seven of the signals from the peripheral apertures are focused on a point, but still the central aperture did not return the reflected illumination radiation). In this case, the one stop autofocus component cannot focus, but the autofocus component of the present invention allows the autofocus system to function well.

[0088] In a further development, the inventors have determined that the autofocus component can comprise multiple aperture planes (502), as shown in FIG. 5. In line with the above description, the illumination source (501) emits illumination radiation that is transmitted through an aperture of the autofocus component (502) m that comprises two aperture planes equidistantly spaced on either side of a plane that is confocal with the autofocus sensor (507). Because the apertures are on different planes, the illumination radiation passing through the apertures has different focal planes at the sample, where the radiation is reflected from the sample at the autofocus sensor. The illumination radiation is then collimated by a tube lens (503) and directed to the sample by a beam splitter (504). The illumination radiation is focused towards the sample by an objective lens (505). Because the sample is not in the focal plane of the illumination radiation, the illumination radiation is defocused on the sample (506), and in this case the illumination radiation from the apertures at different focal planes is equally defocused (509). The defocused illumination is equally defocused from the aperture of each plane of the autofocus component (502) of the sample (506), which means that the sample is in focus. The defocused illumination radiation (509) is reflected from the sample (506) back along the optical path, then passes back through the objective lens (505), through the beam splitter (504) to the autofocus sensor (507), where it is also defocused, but the reflected illumination radiation detected from each plane of the aperture is equally defocused, which means that the sample (506) is in focus. When the sample (506) is out of focus, the autofocus sensor (507) detects the reflected illumination radiation from each plane of the aperture of the autofocus component (502) with different degrees of focus. The manner in which the focus differs between the illumination radiation reflected from different aperture planes of the autofocus component (502) can be used to calculate the direction in which the sample should be moved to bring the illumination radiation reflected from the different aperture planes of the autofocus component (502) to the same degree of defocus.

[0089] Thus, the key to the function of the autofocus system is to make the autofocus components and the autofocus sensor confocal. This process is generally performed when the system is manufactured, for example, by translation of the autofocus components in the axis of the illumination radiation, as shown in FIG. 6 (the components in FIG. 6 correspond to those in the previous figure, in which case simply the first digit of the component number is changed to match the figure number, where the translatable autofocus component is 602). The system also includes a laser collimating lens (609) and a further beam splitter (610) for directing the laser radiation to the sample. If the device with the autofocus system also includes a sampling and ionization system including a laser that impinges on the sample, as shown in FIG. 7, the ablation laser is also focused in the same focal plane as the autofocus components and the autofocus sensor by axial translation of the collimating lens (709). Based on observation of the size and shape of the observed ablation spot, the laser is understood to be a sharp focus in the focal plane of the autofocus system. The objective lens used to focus the illumination radiation onto the sample is also used to focus the laser radiation for laser ablation onto the sample.

[0090] Embodiments include an autofocus system and method can combine the advantages of several autofocus methods (often used for whole slide imaging) to achieve fast, reliable and accurate autofocus over a wide range of initial sample positions for imaging mass cytometry. For example, an autofocus method and / or system can combine two separate techniques, such as the projection of an aperture onto the sample plane, where the aperture is pre-aligned to be perfectly in focus when the sample is also perfectly in focus, and the illumination of this aperture involves two separately controllable light sources with a relatively small solid angle. An example giving a schematic of an autofocus setup is shown in FIG. 12. Two light sources are used, whose light is guided through a small aperture focused on the focal plane of the objective lens. When the sample is out of focus, the camera observes the image from each LED to be not only blurred but also offset according to the illumination angle of each LED at the sample.

[0091] As shown in Fig. 12, a sample 1206 may be placed near the objective focal plane. Two light sources (e.g., LEDs 1201) may be focused by a condenser lens (e.g., by one or more mirrors 1204 and / or objective 1205) through an aperture and directed toward the sample. Light reflected from the sample may impinge on an image sensor 1208, for example, after passing through a tube lens 1207. Element 1209 marks the objective back focal plane with an image of the light sources.

[0092] Each of the two light sources is switched on in turn and an image is acquired with the camera for each LED. If the sample is not perfectly focused, the two images will appear similarly blurred but offset from each other in the direction of the illumination angle (see Figure 13). The correlation between these two frames can be quickly calculated using an FFT, and this function will be maximum at some pixel offset. This pixel offset is directly proportional to the distance the sample is away from the focus, and the proportionality can be easily calibrated using an encoder on the translation stage along the focus direction that already exists. Thus, the autofocus mechanism would in principle only need to acquire two camera frames to determine the best focus position of the sample, which is orders of magnitude faster than current implementations.

[0093] There could be many shapes and sizes of aperture that could be used. One simple and robust shape is expected to be a narrow slit oriented with its small opening along the translation direction, as shown in Fig. 13. Of note, small circular or rectangular apertures could also be used. Multiple apertures (slit, circle, or other) could be used. An "inverted aperture" could be used, meaning a small blur in which the image is darkened in some areas, i.e. the sample structure is preserved in the focused image, and therefore could also be used to determine defocus. Furthermore, the aperture could be defocused relative to the perfect sample focus to create a non-zero target offset between the two LED images. Finally, the two LEDs do not have to be the same color. With two different colored LEDs, the focus data could be obtained in one frame by using one color camera, or perhaps by projecting the two colors in different areas of one monochrome camera using appropriate filters. Of note, instead of LEDs (or LEDs combined with apertures), laser diodes could be used.

[0094] The top of Figure 13 is an LED image acquired at a defocus distance of +60 μm. The two images are similarly blurred and offset along the illumination direction for each LED. Bottom: Calculated correlation function between the two images. A parabolic peak fit gives a peak position of -512.08 pixels, corresponding to a focus offset of +60 μm.

[0095] In practice, the algorithm can sacrifice some of its speed to improve robustness by performing iterative steps towards the best focus. For example, a step of 95% of the extracted defocus value can be taken and the autofocus method repeated. Subsequent iterations should converge very quickly to the correct focus position.

[0096] Critical to the high speed operation of this autofocus method is the use of an electronic trigger to trigger the camera's frame acquisition rather than using the camera in a free-running mode, which can be achieved using standard industrial vision cameras that often have asynchronous triggers as part of their feature set.

[0097] A side benefit of using a hardware trigger for the camera is that it also benefits several other IMC functions, especially the acquisition of panoramas. The current implementation of panorama acquisition (using a free-running camera) is called "stop-and-star", where the stage moves to each successive target position and waits for the next frame to arrive from the camera before proceeding. Using a hardware camera trigger based on the target stage position or together with fast alignment of the stage position, as well as strong LED illumination (allowing short integration times on the camera), the stage can be kept moving continuously while camera frames are acquired at specific positions along the motion profile. This significantly speeds up the acquisition of panoramas in IMC.

[0098] If the laser, autofocus component, and autofocus sensor are all confocal, then during use of the device, the laser can be focused by moving the sample to the focal point by moving the sample to the point where the reflected illumination radiation is focused on the autofocus sensor. Thus, the autofocus system can focus both the optical imaging component / system and the ionization and sampling system.

[0099] Methods for Autofocus Various autofocus methods are described herein, including any method that uses the autofocus systems described herein.

[0100] In certain aspects, the autofocus is based on the location of one or more spots or lines that impinge on the autofocus sensor. The autofocus system may not require pre-calibrated coordinates (e.g., best focus) at one or more spots or lines that impinge on the autofocus sensor. Alternatively, the autofocus system may be pre-calibrated such that features at best focus match known coordinates on the autofocus sensor.

[0101] Autofocus may be based on alignment of features (spots or lines) detected by an autofocus sensor. For example, autofocus (e.g., autofocus correction) can be based on offsets between spots or lines impinging on the autofocus sensor. Autofocus is based on matching features of the autofocus sensor, whereby autofocus correction is repeated until the features match. Alternating LEDs and / or laser diodes providing separate features can improve accuracy compared to constant radiation from such LEDs and / or laser diodes (e.g., slight shifts in position between features may only be apparent if they are detected separately).

[0102] In certain embodiments, multiple apertures can provide multiple characteristics from a single LED, where autofocus can be based on the number of spots detected by the autofocus sensor and / or the uniformity of the spots detected by the autofocus sensor.

[0103] Autofocus can be realized in different kinds of ways. For example, on the one hand, a retrospective determination can be made. Roughly speaking, this type of method works by scanning the sample through a range of positions in a relatively coarse way, where the distance between positions is large, and by picking the position closest to the focal plane of the autofocus system (i.e. the position with the highest focus score). Once the closest position is determined, a further refinement on that position can be performed by moving the sample again through a series of positions, where the positions are now much closer in the refinement step compared to the previous coarse step. From these positions, the position closest to the focal plane is selected (again the position with the highest focus score). This first kind of method therefore relies on scanning over a range of sample positions and retrospectively picking the optimal position.

[0104] Thus, the present invention provides determining a focus score for a first position of the sample; moving the sample to a second location; determining a focus score for the second position; Comparing the focus scores to each other; Including, determining the focus score includes illuminating a sample with radiation from an illumination source, the illumination radiation being passed through an autofocus component comprising a plurality of apertures; and detecting the illumination radiation reflected from the sample with an autofocus sensor, the detecting radiation reflected from the sample including detecting radiation at a known location (also referred to as a region of interest) on the autofocus sensor; The step of moving the sample is a movement parallel to the axis along which the illumination radiation is directed to the sample (i.e., the movement is in the z-axis); To provide an autofocus method.

[0105] As mentioned above, in some embodiments of the method of the present invention, the sample is moved to several positions. Therefore, sometimes the method further comprises, before the step of comparing the focus score of the first position with the focus score of the second position, moving the sample to at least a third position and determining a focus score at at least the third position. Thus, here, the step of comparing the focus score of the first position with the focus score of the second position also includes a comparison of the third focus score with the first and second focus scores. Sometimes, the method comprises, before the step of comparing the focus score of the first position with the focus score of the second position, moving the sample to at least a fourth, fifth, sixth, seventh, eighth, ninth, tenth, fifteenth, twentieth, twenty-fifth, fiftieth, hundredth, two hundredth, five hundredth, or one thousandth position and determining a focus score thereat. Thus, herein, the step of comparing the focus score of the first location with the focus score of the second location also includes comparing the 4th, 5th, 6th, 7th, 8th, 9th, 10th, 15th, 20th, 25th, 50th, 100th, 250th, 500th or 1000th location with other determined focus scores.

[0106] Thus, the method autofocuses by searching backwards for the best focus score. The focus score is a parameter that can be determined in various ways. For example, the intensity of the reflected illumination radiation detected from multiple apertures can be summed to generate the focus score. Alternatively, the focus score can be calculated by measuring how out of focus the reflected illumination radiation is (e.g., the total number of pixels on the sensor that detect reflected illumination radiation at or around the area where the reflected illumination radiation is expected to strike the autofocus sensor when the sample is in focus, i.e., around the expected area of ​​interest).

[0107] In some embodiments, after comparing the focus scores to each other, the sample stage is moved to a first round best focus position, which is the position with the highest focus score. If saturation of the pixels of the autofocus sensor is detected, the intensity of the light source is reduced and the method is repeated.

[0108] As mentioned above, in some cases the method includes a first coarse estimate of the focus position, followed by one or more further rounds of refining the position of the sample to bring it closer to the focus plane. Thus, in some embodiments, the method further includes repeating the method with a second set of positions centered on the first round optimal position determined in the first round. In a refinement, the distance between the positions in the second set is less than the distance between the positions used to calculate the first round optimal focus position. Sometimes the distance between the positions is equal to or less than the distance between the positions used to calculate the first round optimal focus position divided by the number of positions in the second set of positions. The focus scores are then compared to generate the second round optimal focus position. In some embodiments, the second round optimal focus position is simply the second round position with the highest focus score, and the sample is moved to this position (i.e., as is done in subsequent movements after the first round). However, the step of comparing the second round focus scores to each other includes fitting a parabola to the focus scores at each position and calculating the position where the derivative of the curve is zero as the second round optimal focus position. The sample stage can then be moved to the second round best focus position determined by parabolic fitting.

[0109] In some embodiments, comparing the focus scores to each other includes fitting a parabola to the focus scores at each position and calculating the position where the derivative of the curve is zero as the first round optimal focus position. The sample stage can then be moved to the first round optimal focus position. The method can be repeated with a second set of positions centered on the position determined to be the first round optimal focus position, the distance between the positions in the second set being less than the distance between the positions used to calculate the first round optimal focus position. Sometimes the distance between the positions is equal to or less than the distance between the positions used to calculate the first round optimal focus position divided by the number of positions in the second set of positions to generate the second round optimal focus position. Thus, in some embodiments, generating the second round optimal focus position includes comparing the second round focus scores to each other, the step includes fitting a parabola to the focus scores at each position and calculating the position where the derivative of the curve is zero as the second round optimal focus position. The sample stage can then be moved to the second round optimal focus position determined by the parabolic fitting.

[0110] The present invention also provides a method of autofocus, which comprises iteratively repeating the above method.

[0111] Alternatively, a convergence algorithm can be used to focus the sample in an iterative procedure, so that reflected illumination radiation detected at any position is informative about the relative position of the sample and the focal plane. This type of method therefore works by determining a position reading, calculating the direction, and possibly estimating the distance, that the sample needs to be moved in order to move it towards the focal plane. The sample is then moved and a further reading is determined. This process is then repeated iteratively to aim at the focal plane.

[0112] The present invention relates to determining a direction of the focal spot at position n of the sample; moving the sample in the direction of the focus to position (n+1); Including, Determining the direction of focus includes illuminating the sample with radiation from an illumination source, where the illumination radiation is passed through an autofocus component having a plurality of apertures, at least two of the apertures of the autofocus component being offset on an axis along which the illumination radiation passes through the autofocus component, and detecting the illumination radiation reflected from the sample with an autofocus sensor, where detecting the radiation reflected from the sample includes detecting radiation at a known location (also referred to as a region of interest) on the autofocus sensor; The step of moving the sample is a movement parallel to the axis along which the illumination radiation is directed to the sample (i.e., the movement is in the z-axis); An autofocus method is also provided.

[0113] In some embodiments, the method includes repeating the method of the preceding paragraph at least one further time, e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, or at least 50 times.

[0114] In some embodiments, determining the direction of focus at position n includes comparing how the illumination radiation reflected from at least two offset apertures is defocused when it impinges on the autofocus sensor.

[0115] Sample Topology Mapping Method The invention also provides a method for mapping the topology of a sample surface, comprising the steps of performing an autofocus method, moving the sample in the plane of the sample (i.e. in the X-axis and / or Y-axis) to a second position in the plane of the sample, and again performing the autofocus method of the invention to record the best focus position at the second position in the plane of the sample. In some embodiments, the autofocus method is the autofocus method of the invention. In some embodiments, the method comprises moving the sample to one or more further positions in the plane of the sample, for example to at least the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 15th, 20th, 25th, 50th, 100th, 250th, 500th, or 1000th position in the plane of the sample, and recording the best focus position at each of these positions. Sometimes the method further comprises plotting the best focus position at each position in the plane of the sample. Sometimes the method further comprises the step of interpolating between the best focus positions at each location in the plane of the sample to generate a surface representation of the sample. Figure 11 provides a plot of the surface of the sample according to this method of the invention. In some embodiments, the interpolation is polynomial, spline, or Gaussian process interpolation.

[0116] In certain embodiments, the method may include creating a focus map of best focus across X, Y, or XY coordinates.

[0117] Sampling methods include the use of an autofocus system As mentioned above, an autofocus system can be used, for example, to bring the sample into optimal focus for sampling when the system is in an apparatus of the present invention.

[0118] The method of autofocus using the apparatus of any of the above embodiments may further include sampling. For example, the method of autofocus may include autofocusing a laser to ablate the sample material and may also include analysis of the sample material (e.g., by ICP-MS). The sample material may include a mass tag.

[0119] The present invention therefore provides a method for analysing a sample, such as a biological sample, comprising the steps of: performing an autofocus method of the present invention to position a sample at the focal point of a sampling and ionization system; determining the elemental composition of the sample, for example by imaging mass spectrometry or imaging mass cytometry; The present invention provides a method comprising:

[0120] The present invention also provides a method for analyzing a sample, comprising the steps of: performing an autofocusing method of the present invention to position a sample, such as a biological sample, at the focal point of a sampling and ionization system; performing sampling and ionization of a sample on a sample stage at a plurality of positions; detecting ions from locations on the sample, the detection of the ions enabling an image of the sample to be constructed, optionally the plurality of locations being a plurality of known locations; Also provided is a method comprising:

[0121] Thus, more particularly, the present invention provides a method for analyzing a sample comprising the steps of: Implementing the method of the present invention to position a sample at a laser focus for laser ablation; laser ablation of a sample on a sample stage at a plurality of locations; subjecting the plume to ionization and mass spectrometry, where detection of atoms in the plume allows an image of the sample to be constructed, and optionally the plurality of locations is a plurality of known locations; Also provided is a method comprising:

[0122] In imaging mass cytometry, the sample is labeled prior to analysis. Thus, the present invention provides a method of performing mass cytometry on a sample containing a plurality of cells, comprising: labeling a plurality of different target molecules in a sample with one or more different label atoms to provide a labeled sample; Implementing the method of the present invention to position a sample at a laser focus for laser ablation; laser ablation of a sample on a sample stage; subjecting the plume to ionization and mass spectrometry, where detection of atoms in the plume allows an image of the sample to be constructed, and optionally the plurality of locations is a plurality of known locations; Also provided is a method comprising:

[0123] As will be appreciated by those skilled in the art, laser ablation in these methods is accomplished by directing laser radiation at a sample. In some embodiments, at each location, multiple laser shots are fired at the sample, and after each shot at that location, the sample on the sample stage is moved in the Z-axis toward the laser's focal point to affect the depth of the sample ablated at that location. In some embodiments, the starting Z-axis location for ablation of the sample is the optimal focal position at that location on the sample as determined by the sample topology mapping method of the present invention, and each plume generated from each shot at the Z-axis location at that location on the sample is detected individually, for example, allowing for the construction of a 3D image of the sample.

[0124] In some embodiments, the method includes using the autofocus components of the present invention.

[0125] In some embodiments, the method includes using the autofocus system of the present invention.

[0126] In some embodiments, the method includes using an apparatus of the present invention.

[0127] Further aspects of the method Prior to the autofocus and / or autofocus-based sampling, the method of the present application may include providing a labeled biological sample. In certain embodiments, the method may include labeling the biological sample. The biological sample may be labeled with a label atom coupled to a specific binding pair (SBP) member as described herein. For example, the SBP member may include an antibody and / or the label atom may include a metal tag (such as an enriched metal isotope). Suitable samples, label atoms, SBP members, and labeling methods are further described herein.

[0128] Autofocus System Considerations A valuable feature of imaging mass cytometry systems is the ability to perform automatic focusing of the sample in the camera view. The key performance characteristics of an autofocus system are the average time required to perform autofocus, the robustness / reliability of the system across a range of samples, and the focus range over which the autofocus system produces reliable results. Most autofocus systems currently available (including those implemented in first generation IMCs) perform poorly in at least one of these three requirements.

[0129] For example, IMC autofocus systems using multiple apertures and a single LED light source are robust and generally reliable, and can find optimal focus over long translation ranges, but can be slow (e.g., milliseconds). This may not be an issue if the speeds of laser ablation and mass analysis are comparable.

[0130] Many aftermarket commercial systems suffer from the opposite limitation: they are fast and mostly robust (except for some immersion type samples), but they only work if the sample is already relatively close to optimal focus, and can suffer from focus drift over the lifetime of the device.

[0131] Autofocus systems can be categorized into two main types: hardware-based and image analysis-based. The latter category analyzes the current image of the sample in terms of sharpness, contrast, etc., and attempts to maximize those properties by moving the sample. Such systems tend to work very well for a subset of samples, but fail on samples that inherently lack contrast. Furthermore, the range over which the autofocus method works is limited, and the system must "hunt" to find the optimal focus. Overall, this type of autofocus is very fast, but is generally not reliable enough for unsupervised application in IMC.

[0132] Hardware-based autofocus systems (especially aftermarket solutions targeted at standard microscopes) often use invisible light to project an illuminated aperture onto the sample, where illumination is only emitted from one side of the back aperture of the microscope objective. The effect of this one-sided illumination is that, at the image plane of the objective, the aperture moves from side to side (in addition to becoming defocused) as the sample moves through the focus. A position-sensitive detector is used to quantify this lateral shift, and thus the optimal focus position can be maintained. Since the range over which this method works is very limited, the application of this method is usually to keep the sample in focus, rather than first finding the optimal focus. This is exacerbated by the fact that these autofocus methods have problems maintaining calibration over time, temperature, and different samples and objectives. Secondly, the structure of the sample can affect the image observed at the image plane of the objective. Thus, the output of the detector when the sample is perfectly in focus will not be the same for all samples. Again, this limits the application of these systems to "locking in" to a specific focus position and observing only one area of ​​one sample for an extended period of time.

[0133] In certain aspects, the autofocus system or method can provide fast autofocus independent of sample surface quality and can perform on-the-fly corrections to focus quality, which can mitigate sample tilt, stage flatness issues, and / or sample surface topology effects.

[0134] In certain embodiments, the light from the LED can be sent through an aperture and then to the objective lens itself (epi-illumination). The use of an aperture can improve the performance of the system by producing sharper images with higher contrast when the system is in focus.

[0135] 1. Sampling and Ionization System Sampling a sample involves removing material from the sample by a beam of particles (e.g., photons) that is focused on the sample and excites the sample, causing the material to move away from the sample. The focus of the particle beam (e.g., photons) is parfocal with the autofocus system described in the previous section, so that when the autofocus system focuses on the sample, the sample is positioned optimally on the sample.

[0136] Thus, an apparatus of the present application may include a sampling system (such as a laser ablation sampling system), an ionization system (such as an ICP system), or both. For example, an apparatus may include an autofocus system, a movable sample stage, and a laser ablation sampling system, which may be coupled to a mass spectrometer, such as an ICP-MS system. In some cases, such an apparatus may further include an ICP-MS system.

[0137] In certain embodiments, the sampling system is a laser ablation sampling system. The device may further include an ionization system, such as an ICP ionization system, coupled to the laser ablation sampling system by a gas conduit. The device may further include a mass spectrometer. The mass spectrometer may be configured to simultaneously detect multiple mass tags.

[0138] a. Laser Ablation Sampling and Ionization System Laser ablation-based analyzers generally comprise three components. The first component is a laser ablation sampling system for generating a plume of vaporous and particulate material from the sample for analysis. Before the atoms in the plume of ablated sample material (including detectable label atoms, described below) can be detected by a detector system - a mass spectrometer component (MS component; third component), the sample must be ionized (and atomized). Thus, the device comprises a second component, an ionization system that ionizes the atoms to form elemental ions and allows their detection by the MS component based on their mass / charge ratio (some ionization of the sample material may occur at the point of ablation, but space charge effects result in an almost immediate neutralization of the charge). The laser ablation sampling system is connected to the ionization system by a transfer conduit.

[0139] 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 be moved 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 is flowed 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 also be used to desorb material from the sample, as described further below.

[0140] Particularly in the case of biological samples (cells, tissue sections, etc.), samples are often heterogeneous (although heterogeneous samples are known in other application areas of the present disclosure, i.e. samples of a non-biological nature). A heterogeneous sample is one that contains sites composed of different materials, such that some sites of the sample may be ablated at a lower threshold fluence at a given wavelength than other sites. Factors that influence the ablation threshold are the extinction coefficient of the material and the mechanical strength of the material. In the case of biological tissue, the extinction coefficient has a dominant influence, as it can vary by orders of magnitude with the wavelength of the laser radiation. For example, in a biological sample, utilizing nanosecond laser pulses, sites containing proteinaceous material absorb more readily in the 200-230 nm wavelength range, while sites containing primarily DNA absorb more readily in the 260-280 nm wavelength range.

[0141] Laser ablation can be performed with a fluence close to the ablation threshold of the sample material. Ablation in this manner often improves aerosol formation, which can help improve the quality of the data after analysis. Often, a Gaussian beam is used to obtain the smallest craters and maximize the resolution of the resulting image. A cross section across the Gaussian beam records an energy density profile with a Gaussian distribution. In that case, the fluence of the beam varies with the distance from the center. As a result, the diameter of the ablation spot size is determined by two parameters: (i) the Gaussian beam waist (1 / e 2 ), and (ii) is a function of the ratio between the applied fluence and the threshold fluence.

[0142] Therefore, to ensure consistent removal of a reproducible amount of material with each ablation laser pulse, thereby maximizing the quality of the imaging data, it is useful to maintain a consistent ablation diameter, thereby adjusting the ratio of energy delivered by the laser pulse to the target to the ablation threshold energy of the material being ablated. This requirement presents a problem when ablating heterogeneous samples, where the threshold ablation energy varies across the sample, such as biological tissue, where the ratio of DNA to protein material varies, or in geological samples, where it varies with the particular composition of minerals within a region of the sample. To address this, multiple wavelengths of laser radiation can be focused at the same ablation location on the sample to more effectively ablate the sample based on the composition of the sample at that location.

[0143] Laser system for laser ablation sampling system A laser system can be set up to produce single or multiple (i.e., two or more) wavelengths of laser radiation. Generally, the wavelength of the laser radiation discussed refers to the wavelength with the highest intensity (the "peak" wavelength). If the system produces different wavelengths, those wavelengths can be used for different purposes, for example, to target different materials within a sample (where targeting means that the wavelengths selected are those that are well absorbed by the materials).

[0144] When multiple wavelengths are used, at least two of the two or more wavelengths of the laser radiation may be discrete wavelengths. Thus, when a first laser source emits radiation of a first wavelength that is discrete from radiation of a second wavelength, this means that radiation of the second wavelength is not generated by the first laser source, or that very low levels of radiation of the second wavelength are generated by the first laser source in pulses of the first wavelength, for example, at less than 10% of the intensity at the first wavelength, for example, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In general, when laser radiation of different wavelengths is generated by harmonic generation or other nonlinear frequency conversion processes, then when a specific wavelength is referred to herein, as will be understood by those skilled in the art, there will be some variation in the specific wavelength of the spectrum generated by the laser. For example, a reference to Xnm includes lasers that generate a spectrum in the range of X±10nm, for example, X±3nm, such as X±5nm.

[0145] Laser In general, the choice of wavelength and power of the laser used for ablation of the sample can 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. Generally, a power of 0.1-5 J / cm is used. 2 , e.g., 3-4 J / cm 2 or about 3.5 J / cm 2A laser fluence of 100 Hz or more is suitable, and the laser can ideally generate pulses at this fluence at a rate of 200 Hz or more. In some cases, a single laser pulse from such a laser should be sufficient to ablate cellular material for analysis, such that the laser pulse frequency matches the frequency 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 to a specific spot size, for example, as discussed below. In some embodiments of the invention, the ablation rate (i.e., the rate at which the laser ablates a spot on the surface of the sample) is 200 Hz or more, e.g., 500 Hz or more, 750 Hz or more, 1 kHz or more, 1.5 kHz or more, 2 kHz or more, 2.5 kHz or more, 3 kHz or more, 3.5 kHz or more, 4 kHz or more, 4.5 kHz or more, 5 kHz or more, 10 kHz or more, 100 kHz or more, 1 MHz or more, 10 MHz or more, or 100 MHz or more. Many lasers have repetition rates that exceed the laser ablation frequency, so that the rate of ablation can be controlled as needed using appropriate components such as a pulse picker. Thus, in some embodiments, the laser repetition rate is at least 1 kHz, e.g., at least 10 kHz, at least 100 kHz, at least 1 MHz, at least 10 MHz, about 80 MHz, or at least 100 MHz, and optionally the sampling system further comprises a pulse picker, e.g., the pulse picker is controlled by a control module that also controls the movement of the sample stage and / or positioner. In other examples, multiple closely spaced pulse bursts (e.g., a train of three closely spaced pulses) can be used to ablate one single spot.As an example, a 10 x 10 μm area may be ablated using 100 bursts of three closely spaced pulses at each spot, which may be useful in lasers that have limited ablation depth, such as femtosecond lasers, while still producing a continuous plume of ablated cellular material without loss of resolution.

[0146] 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.

[0147] At these frequencies, where it is desirable to resolve each ablated plume individually (which may not always be desirable when firing a burst of pulses at a sample, as shown below), the instrument must be able to analyze the ablated material rapidly enough to avoid substantial signal overlap between successive ablations. The overlap between signals resulting from successive plumes is preferably less than 10% in intensity, more preferably less than 5%, and ideally less than 2%. The time required to analyze the plume is determined by the washout time of the sample chamber (see Sample Chamber section below), the transit time of the plume aerosol to and through the laser ionization system, and the time it takes to analyze the ionized material. As explained in more detail below, each laser pulse can be correlated to a pixel on an image of the sample that is subsequently constructed.

[0148] In some embodiments, the laser source is a nanosecond pulse duration laser or an ultrafast laser (pulse duration of 1 ps (10 -12sec) or faster, e.g., femtosecond lasers. Ultrafast pulse durations offer many advantages as they limit heat diffusion from the ablation zone, thereby resulting in more precise and reliable ablation craters, as well as minimizing the scattering of debris from each ablation event.

[0149] In some cases, femtosecond lasers are used as laser sources. Femtosecond lasers are lasers that emit light pulses with durations of less than 1 ps. The generation of such short pulses often uses the technique of passive mode-locking. Femtosecond lasers can be generated using many types of lasers. Typical durations of 30 fs to 30 ps can be realized using passively mode-locked solid-state bulk lasers. Similarly, various diode-pumped lasers, for example based on neodymium-doped or ytterbium-doped gain media, operate in this regime. Titanium-sapphire lasers with advanced dispersion compensation are suitable for pulse widths below 10 fs, even down to about 5 fs in extreme cases. Pulse repetition rates are in most cases 10 MHz to 500 MHz, but for higher pulse energies there are low repetition rate versions with repetition rates of several megahertz (available for example from Lumentum (California, USA), Radiantis (Spain), Coherent (California, USA)). This type of laser can be accompanied by an amplifier system that increases the pulse energy.

[0150] There are also various types of ultrafast fiber lasers, which are also mostly passively mode-locked and typically provide pulse durations of 50-500 fs and repetition rates of 10-100 MHz. Such lasers are commercially available, for example, from NKT Photonics (Denmark, formerly Fianium), Amplitude Systems (France), and Laser-Femto (California, USA). The pulse energy of this type of laser can also be increased by an amplifier, often in the form of an integrated fiber amplifier.

[0151] Some mode-locked diode lasers can generate pulses with femtosecond durations. Directly at the laser output, the pulse duration is typically on the order of a few hundred femtoseconds (e.g., available from Coherent, CA, USA).

[0152] In some cases, picosecond lasers are used. Many of the types of lasers already described in the previous paragraph can also be adapted to generate pulses with durations in the picosecond range. The most common light sources are active or passive mode-locked solid-state bulk lasers, such as passive mode-locked Nd-doped YAG, glass, or vanadate lasers. Similarly, picosecond mode-locked lasers and laser diodes are commercially available (e.g., NKT Photonics (Denmark), EKSPLA (Lithuania)).

[0153] Nanosecond pulse duration lasers (gain switched and Q-switched) may also find utility with certain instrument setups (Coherent, CA, USA, Thorlabs, NJ, USA). Alternatively, continuous wave lasers may be used and externally modulated to generate sub-nanosecond duration pulses.

[0154] Typically, the laser beams used for ablation in the laser systems discussed herein have a spot size, i.e., at the sampling location, of 100 μm or less, e.g., 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less, e.g., about 3 μm or less, about 2 μm or less, about 1 μm or less, about 500 nm or less, about 250 nm or less. 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 opposing corners in the case of a square beam, the length of the longest diagonal in the case of a rectangle, etc. (As discussed 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 can be used to result in the desired ablation spot. For example, in some applications, a square ablation spot with a top-hat energy distribution can be useful (i.e., a beam with a nearly uniform fluence as opposed to a Gaussian energy distribution). This arrangement reduces the dependency of the ablation spot size on the ratio between the fluence at the peak of the Gaussian energy distribution and the threshold fluence. Ablation closer 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, positioned in the Gaussian beam to reshape the beam and generate a laser focal spot of uniform or nearly uniform fluence, such as a fluence that varies across the beam by less than ±25%, e.g., less than ±20%, ±15%, ±10%, or ±5%. Sometimes the laser beam has a square cross-sectional shape. Sometimes the beam has a top-hat energy distribution.

[0155] When used to analyze 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 tightly packed together (e.g., in a tissue section), one or more laser sources in the laser system can have a spot size no larger than these cells. This size depends on the particular cells in the sample, but generally the laser spot has a diameter of less than 4 μm, e.g., about 3 μm or less, about 2 μm or less, about 1 μm or less, about 500 nm or less, about 250 nm or less, or between 300 nm and 1 μm. To analyze a given cell with subcellular resolution, the system uses a laser spot size no larger than these cells, and more specifically, uses a laser spot size that can ablate material with subcellular resolution. In some cases, single cell analysis can be performed using a spot size larger than the size of the cells, for example, when the cells are spread on a slide with spaces between the cells. Here, a larger spot size can be used and single cell characterization can be achieved, since the additional ablated area around the cell of interest does not contain additional cells. Thus, the particular spot size used may be appropriately selected depending on the size of the cells being analyzed. In biological samples, cells are rarely all the same size, so if subcellular resolution imaging is desired, the ablation spot size should be smaller than the smallest cell if a constant spot size is maintained throughout the ablation procedure. Focusing of the laser beam can be used to achieve small spot sizes. A laser spot diameter of 1 μm corresponds to a laser focus point (i.e., the diameter of the laser beam at the focus of the beam) of 1 μm, but the laser focus point can vary by + / - 20% or more due to the spatial distribution of energy on the target (e.g., Gaussian beam shape) and variations in total laser energy relative to the ablation threshold energy. Suitable objectives for focusing the laser beam include reflective objectives, such as Schwarzschild Cassegrain (inverse Cassegrain) objectives.Refractive objectives can be used, as well as combined reflective and refractive objectives. A single aspheric lens can also be used to achieve the required focusing. A solid immersion lens or a diffractive optical element can also be used to focus the laser beam. Another means for controlling the spot size of the laser, which can be used alone or in combination with the above objectives, is to pass the beam through an aperture before focusing. Different beam diameters can be achieved by passing the beam through apertures of different diameters from an array of diameters. In some cases, there is a single aperture of variable size, for example when the aperture is an aperture. Sometimes the aperture is an iris aperture. The variation in spot size can also be achieved by dithering the optical element. One or more lenses and one or more apertures are located between the laser and the sample stage.

[0156] For completeness, as known in the art, the standard lasers for LA with intracellular resolution are excimer or exciplex lasers. Suitable results can be obtained using an argon fluoride laser (λ = 193 nm). Pulse durations of 10-15 ns with these lasers can achieve adequate ablation.

[0157] Overall, the laser pulse frequency and intensity are selected in combination with the response characteristics of the MS detector to allow unambiguous detection of individual laser ablation plumes. Rapid, high-resolution imaging is then achievable using a small laser spot combined with a sample chamber with short washout times.

[0158] Laser ablation focus For the laser to maximize the efficiency with which it ablates material from the sample, the sample should be in a suitable position relative to the focal point of the laser, e.g., at the focal point. This is because the focal point is where the laser beam has the smallest diameter and therefore the most concentrated energy. This can be achieved in a variety of ways. The first way is that the sample can be moved in 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. 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 positioned between the laser and the sample stage. A third way, which can be used alone or in combination with either or both of the two ways, is to change the position of the laser.

[0159] To assist the user of the system in positioning the sample in the most suitable position for ablation of material from the sample, a camera can be aimed at the stage holding the sample (described in more detail below). Thus, the present disclosure provides a laser ablation sampling system with a camera aimed at the sample stage. The image detected by the camera can be focused on the same point where the laser is focused. This can be achieved by using the same objective lens for both laser ablation and optical imaging. By making the two focal points coincident, the user can be confident that the laser ablation will be most effective when the optical image is in focus. Precise movement of the stage to focus on the sample can be performed using piezo activators available from Physik Instrumente, Cedrat-technologies, Thorlabs, and other sources.

[0160] In a further mode of operation, laser ablation is directed to the sample through the sample carrier. In this case, the sample support should be selected to be (at least partially) transparent to the frequency of the laser radiation used to ablate the sample. Ablation through the sample is advantageous in certain circumstances, since this ablation mode can impart additional kinetic energy to the plume of material ablated from the sample, thereby propelling the ablated material further away from the surface of the sample and thus facilitating the transport of the ablated material away from the sample for analysis at the detector. Similarly, desorption-based methods of removing slugs of sample material can also be mediated by laser radiation passing through the carrier. The additional kinetic energy imparted to the slug of material being desorbed can help to knock the slug away from the sample carrier and thus facilitate the slug being entrained in the carrier gas flowed through the sample chamber.

[0161] To achieve 3D imaging of the sample, the sample or a given region of the sample may be ablated to a first depth that does not penetrate the sample completely. Following this, the same region may be ablated again to a second depth, and so on to a third, fourth, etc. depth. In this way, a 3D image of the sample may be created. In some cases, it may be preferable to ablate the entire region to a first depth before proceeding to ablate at a second depth. Alternatively, repeated ablation may be performed at the same location to ablate through different depths before proceeding to the next location within the region for ablation. In either case, deconvolution of the MS acquired signal to the location and depth of the sample may be performed by the imaging software. Thick tissue stains may be employed and the tissue stabilized in wet conditions similar to the workflow used in confocal imaging (Clendenon et al., 2011. Microsc Microanal. 17:614-617).

[0162] Laser system optics for multiple operating modes As a matter of routine arrangement, optical components can be used to optionally direct laser radiation of different wavelengths to different relative positions. Optical components can also be arranged to optionally direct laser radiation of different wavelengths from different directions to the sample. For example, one or more wavelengths can be directed to the sample from above, and one or more wavelengths of laser radiation (optionally different wavelengths) can be directed from below (i.e. through a substrate such as a microscope slide that supports the sample, also referred to as a sample carrier). This allows multiple modes of operation with the same device. Thus, a laser system can optionally comprise an arrangement of optical components arranged to direct laser radiation of different wavelengths to the sample from different directions. Thus, optical components may be arranged such that their arrangement optionally directs laser radiation of different wavelengths to the sample from opposite directions. "Opposite" directions in this context are not limited to laser radiation directed perpendicularly to the sample from above and below (which would be 180° opposite), but include arrangements that direct laser radiation to the sample at angles other than perpendicular to the sample. Laser radiation directed to the sample from different directions does not have to be parallel. Sometimes, when the sample is on a sample carrier, the reflector arrangement may be arranged to direct laser radiation of a first wavelength directly onto the sample and laser radiation of a second wavelength through the sample carrier to the sample.

[0163] Directing laser radiation through the sample carrier to the sample can be used to ablate the sample. However, in some systems, directing laser radiation through the carrier can be used for a "LIFTing" mode of operation, as discussed in more detail below with respect to desorption-based sampling systems (although, as one skilled in the art will appreciate, ablation and LIFTing can be performed by the same device, so what is referred to herein as a laser ablation sampling system can also act as a desorption-based sampling system). The NA (numerical aperture) of a lens used to focus laser radiation from a first direction onto the sample can be different from the NA of a lens used to focus laser radiation (optionally at a different wavelength) onto the sample from a second direction. LIFTing operations (e.g., when laser radiation is directed through the sample carrier) often use a larger diameter spot size than when ablation is being performed.

[0164] Sample chamber of the laser ablation sampling system The sample is placed in a sample chamber as it undergoes laser ablation. 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 through the sample chamber from the gas inlet to the gas outlet carries away the plume of aerosolized material containing the labelled atoms that were at the ablated location. The gas carries the material to an ionization system, which ionizes the material to allow detection by a detector. The atoms in the sample, including the labelled atoms, can be distinguished by the detector, so their detection reveals the presence or absence of multiple targets in the plume and determines which targets were present in the ablated trajectory on the sample. The sample chamber thus plays a dual role in hosting the solid sample to be analysed, but is also the starting point for the transfer of the aerosolized material to the ionization and detection system. This means that the flow of gas through the chamber can affect how the plume of ablated material spreads as it passes through the system. A measure of how the ablated plume spreads is the washout time of the sample chamber. This value is a measure 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 sample chamber.

[0165] 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; the rate at which the plumes are generated versus the motion of the sample relative to the laser; and (ii) the rate at which the plumes can be analyzed relative to the rate at which they are being generated, to avoid overlapping signals from successive plumes, as discussed above. Thus, being able to analyze the plumes in as short a time as possible minimizes the chance of plumes overlapping (and therefore plumes can be generated more frequently) in the event that individual analysis of the plumes is desired.

[0166] Therefore, sample chambers with short washout times (e.g., 100 ms or less) are advantageous for use with the devices and methods disclosed herein. Sample chambers with long washout times limit the speed at which images can be generated or result in overlap between signals from consecutive sample spots (e.g., Kindness et al. (2003; Clin Chem 49:1916-23) with signal durations longer than 10 seconds). Therefore, aerosol washout times are a significant limiting factor for achieving high resolution without increasing total scan time. Sample chambers with washout times less than 100 ms are known in the art. For example, Gurevich & Hergenroder (2007; J. Anal. At. Spectrom., 22:1043-1050) disclose sample chambers with washout times 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 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 of WO 2014 / 127034 comprises a sample capture cell configured to be placed in operative proximity to a target, the sample capture cell including a capture cavity having an opening formed in a surface of the capture cell, where the capture cavity is configured to receive through the opening the target material released or generated from the laser ablation site and a guide wall exposed in the capture cavity, and configured to direct a flow of carrier gas in the capture cavity from an inlet to an 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 trapping cavity in the sample chamber of WO 2014 / 127034 is 1 cm 3 Less than 0.005 cm 3In some cases, the sample chamber has a washout time of 25 milliseconds or less, e.g., 20 milliseconds or less, 10 milliseconds or less, 5 milliseconds or less, 2 milliseconds or less, 1 millisecond or less, 500 μs or less, 200 μs or less, 100 μs or less, 50 μs or less, or 25 μs or less. For example, the washout time of the sample chamber may be 10 μs or more. Typically, the washout time of the sample chamber is 5 milliseconds or less.

[0167] For completeness, sometimes plumes can be generated from a sample more frequently than the washout time of the sample chamber, and the resulting images will be correspondingly blurred (e.g., if the highest possible resolution is not deemed necessary for the particular analysis being performed).

[0168] The sample chamber typically comprises a translation stage that holds the sample (and sample carrier) and moves the sample relative to the beam of laser radiation. For example, when a mode of operation is used that requires the direction of the laser radiation towards the sample through the sample carrier, as is the case in the LIFTing methods discussed herein, the stage holding the sample carrier should also be transparent to the laser radiation used.

[0169] Thus, the sample may be positioned on a side of the sample carrier (e.g., a glass slide) facing the laser radiation when the laser radiation is directed at the sample, such that the ablation plume is released on and captured from the same side as the laser radiation is directed at the sample, or the sample may be positioned on a side of the sample carrier opposite the laser radiation when the laser radiation is directed at the sample (i.e., the laser radiation passes through the sample carrier before reaching the sample), and the ablation plume is released on and captured from the opposite side to the laser radiation.

[0170] One feature of the sample chamber that is of particular use in which specific portions within various distinct regions of a sample are ablated is the wide range of movement that allows the sample to be moved in axes x,y (i.e., horizontally) relative to the laser (when the laser beam is directed to the sample in the z-axis), where the x and y axes are perpendicular to each other. By moving the stage within the sample chamber while the position of the laser remains fixed within the laser ablation sampling system of the device, a more reliable and accurate relative position is achieved. The wider the range of movement, the greater the distance that can be between distinct ablation regions. The sample is moved relative to the laser by moving the stage on which the sample is located. Thus, the sample stage may have a range of movement within the sample chamber of at least 10 mm in the x and y axes, e.g., 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, 75 mm in the x and y axes. In some cases, the range of movement allows it to analyze the entire surface of a standard 25 mm x 75 mm microscope slide within the chamber. Of course, to be able to achieve intracellular ablation, in addition to a wide range of motion, the motion must be precise. Thus, the stage may be configured to move the sample in the x and y axes in increments of less than 10 μm, e.g., less than 5 μm, less than 4 μm, less than 3 μm, less than 2 μm, less than 1 μm, or less than 1 μm, less than 500 nm, less than 200 nm, less than 100 nm. For example, the stage may be configured to move the sample in increments of at least 50 nm. Precise stage motion can be 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, the motorized stage can be built from components based on a positioner that provides the required range of motion and a suitably precise motion, such as the SLC-24 positioner provided by Smaract. The speed of movement of the sample stage can also affect the speed of analysis.The sample stage may therefore have a movement speed of more than 1 mm / sec, for example 10 mm / sec, 50 mm / sec or 100 mm / sec.

[0171] Of course, if the range of motion of the sample stage within the sample chamber is large, the sample must be sized appropriately to accommodate the stage motion. Thus, the size of the sample chamber depends on the size of the sample involved, which in turn determines the size of the moving sample stage. Typical sizes of sample chambers have an internal chamber of 10x10cm, 15x15cm, or 20x20cm. The depth of the chamber may be 3cm, 4cm, or 5cm. A person skilled in the art can select the appropriate dimensions following the teachings herein. The internal dimensions of a sample chamber for analyzing biological samples using a laser ablation sampler must be larger than the range of motion of the sample stage, for example at least 5mm, for example at least 10mm. This is because if the walls of the chamber are too close to the edge of the stage, the flow of carrier gas through the chamber that captures the plume of ablated material away from the sample and towards 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 carried away into the ionization system of the device, rather than remaining as a dense mass 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, and therefore ultimately to data that is poorly resolved spatially unless the rate of ablation is slowed to a rate that is no longer of experimental interest.

[0172] As noted above, the sample chamber includes a gas inlet and a gas outlet for introducing material into the ionization system. However, the sample chamber may include additional ports that act as inlets or outlets to direct the flow of gas within the chamber and / or supply 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.

[0173] camera In addition to identifying the most effective positioning of the sample for laser ablation, the inclusion of a camera (e.g., a Charged Coupled Device Image Sensor-based (CCD) camera, a Complementary Metal-Oxide-Semiconductor (CMOS) sensor, or an Active Pixel Sensor-based camera) or any other light detection means in the laser ablation sampling system enables a variety of further analyses and techniques. A CCD is a means for detecting light and converting it into digital information that can be used to generate an image. A CCD image sensor has a series of capacitors that detect light, each capacitor representing a pixel on the determined image. These capacitors allow the incoming photons to be converted into an electric charge. The CCD can then be used to read these charges and convert the recorded charges into an image. An Active Pixel Sensor (APS) is an image sensor, e.g., a CMOS sensor, that consists of an integrated circuit that contains an array of pixel sensors, each pixel containing a photodetector and an active amplifier.

[0174] A camera can be incorporated into any of the laser ablation sampling systems discussed herein. The camera can be used to scan the sample to identify specific cells of interest or specific regions of interest (e.g., cells of a particular morphology), or to identify fluorophores specific to antigens, cells, or structures. In certain embodiments, the fluorophores are histochemical stains or antibodies that also contain detectable metal tags. Once such cells are identified, a laser pulse can be directed to these specific cells to ablate material for analysis in an automated process (where the system both identifies and ablates features / regions such as cells of interest) or in a semi-automated process (where a user of the system, e.g., a clinical pathologist, identifies features / regions of interest and then the system ablates in an automated manner). This can greatly increase the speed at which analysis can be performed, since cells of interest can be specifically ablated without the need to ablate the entire sample to analyze specific cells. This translates into efficiency of the method of analyzing biological samples in terms of the time it takes to perform the ablation, but also in terms of the time it takes to interpret the data from the ablation, especially with respect to constructing an image from the data. Constructing an image from data is one of the most time-consuming parts of the imaging procedure, so minimizing the data collected to only data from relevant parts of the sample increases the overall speed of analysis.

[0175] The camera may record images from a confocal microscope. Confocal microscopy is a type of optical microscopy that offers many advantages, such as the ability to reduce interference from background information (light) away from the focal plane. This occurs by removing unfocused light and glare. Confocal microscopy can be used to assess the morphology of cells in a sample, or whether the cells are individual cells or part of a clump of cells. Often, samples are specifically labeled with fluorescent markers (such as labeled antibodies or labeled nucleic acids). These fluorescent markers can be used to stain specific cell populations (such as expression of specific genes and / or proteins) or specific morphological features on cells (such as nuclei or mitochondria), and when illuminated with the appropriate wavelength of light, can specifically identify the sample in these regions. Thus, some systems described herein can include a laser to excite fluorophores in the labels used to label the sample. Alternatively, LED light sources can be used to excite the fluorophores. Non-confocal (such as widefield) fluorescence microscopy can also be used to identify specific regions of a biological sample, but at a lower resolution than confocal microscopy.

[0176] Another imaging technique is two-photon excitation microscopy (also called nonlinear or multiphoton microscopy). This technique generally uses near-infrared light to excite a fluorophore. Two photons of IR light are absorbed for each excitation event. Scattering in tissue is minimized by IR. Furthermore, multiphoton absorption strongly suppresses background signal. Most commonly used fluorophores have excitation spectra in the 400-500 nm range, but the lasers used to excite two-photon fluorescence are in the near-infrared range. When a fluorophore simultaneously absorbs two infrared photons, the fluorophore absorbs enough energy to be raised to an excited state. The fluorophore then emits a single photon at a wavelength that depends on the type of fluorophore used, which can then be detected.

[0177] When a laser is used to excite a fluorophore for fluorescence microscopy, this laser may be the same laser that generates the laser light used to ablate material from a biological sample, but is used with a power that is not sufficient to ablate material from the sample. Sometimes, the fluorophore is excited by the wavelength of light at which the laser ablates the sample. In other cases, different wavelengths may be used, for example, by generating different harmonics of the laser to obtain different wavelengths of light, or by utilizing different harmonics generated by the aforementioned harmonic generation system apart from the harmonics used to ablate the sample. For example, when using the fourth and / or fifth harmonics of a Nd:YAG laser, the fundamental harmonic or the second to third harmonics can be used for fluorescence microscopy.

[0178] As an example of a technique combining fluorescence and laser ablation, the nuclei of cells in a biological sample can be labeled with an antibody or nucleic acid bound to a fluorescent moiety. Thus, by exciting the fluorescent label and then using a camera to observe and record the location of the fluorescence, the ablation laser can be specifically directed to the nucleus or to areas that do not contain nuclear material. Dividing the sample into nuclear and cytoplasmic regions allows for particular applications in the field of cytochemistry. By using an image sensor (such as a CCD detector or an active pixel sensor, e.g. a CMOS sensor), the process of identifying features / regions of interest of interest and then ablating them can be fully automated by using a control module (a computer or programmed chip) that correlates the location of the fluorescence with the x,y coordinates of the sample and then directs the ablation laser to that location. As part of this process, the first image taken by the image sensor may have a low objective magnification (low numerical aperture), allowing a large area of ​​the sample to be surveyed. Following this, a switch to a higher magnification objective can be used to target specific features of the object that have been determined to fluoresce by the higher magnification optical imaging. These features, recorded as fluorescent, may then be ablated by a laser.Using a low numerical aperture lens initially has the added advantage of providing a large depth of field, and therefore greater sensitivity in detecting features embedded within the sample than would be possible by first screening with a high numerical aperture lens.

[0179] In methods and systems in which fluorescence imaging is used, the emission path of the fluorescence from the sample to the camera may include one or more lenses and / or one or more optical filters. By including optical filters adapted to pass selected spectral bandwidths from one or more fluorescent labels, the system is adapted to handle chromatic aberration associated with the emission from the fluorescent labels. Chromatic aberration is a result of the inability of lenses to focus different wavelengths of light to the same focal point. Thus, by including optical filters, the background of the optical elements is reduced and the resulting optical image has higher resolution. A further method to minimize the amount of emission light of undesired wavelengths reaching the camera is to deliberately exploit the chromatic aberration of the lenses by using a series of lenses designed for the transmission and focusing of light of wavelengths transmitted by the optical filters akin to the system described in WO 2005 / 121864.

[0180] Higher resolution optical images are advantageous in this coupling of optical techniques and laser ablation sampling, since the accuracy of the optical image determines the precision with which the ablation laser can be aimed to ablate the sample.

[0181] Thus, in some embodiments disclosed herein, the device of the invention comprises a camera. This camera can be used online to identify a sample, e.g., a particular cellular feature / region, that can then be ablated (or detached by LIFTing - see below), such as by firing a burst of pulses at the feature / region of interest to ablate or detach a slug of sample material from the feature / region of interest. When the burst of pulses is directed at the sample, the resulting plume material detected can be like a continuous event (the plumes from each individual ablation effectively form a single plume, which is then executed for detection). Although each cloud of sample material formed from the collective plumes from locations within the feature / region of interest can be analyzed together, the sample material in the plumes from each different feature / region of interest is still kept discrete. That is, enough time is left between the ablations of different feature / regions of interest to allow sample material from the nth feature / region of interest to be ablated before the ablation of the (n+1)th feature / region of interest is started.

[0182] In a further mode of operation combining both fluorescence analysis and laser ablation sampling, instead of analyzing the fluorescence of the entire slide before directing the laser ablation at those locations, a pulse can be fired from the laser at a spot on the sample (at a low energy that will only excite fluorescent moieties in the sample rather than ablate it) and if fluorescent emission of the expected wavelength is detected, the sample at that spot can be ablated by firing the laser at full energy at that spot and the resulting plume analyzed by a detector as described below. This has the advantage that the raster mode of analysis is maintained, but the fluorescence is pulsed and examined and results can be obtained immediately from the fluorescence (rather than the time it takes to analyze and interpret ion data from the detector to determine if an area is of interest), thereby Again, speed is improved since only the locations of interest can be targeted for analysis. Thus, applying this strategy when imaging a biological sample containing multiple cells, the following steps can be performed: (i) labeling multiple different target molecules in the sample with one or more different label atoms and one or more fluorescent labels to provide a labeled sample, (ii) illuminating a known location on the sample with light to excite the one or more fluorescent labels, (iii) observing and recording whether there is fluorescence at that location, (iv) if there is fluorescence, directing laser ablation at that location to form a plume, (v) exposing the plume to inductively coupled plasma mass spectrometry, and (vi) repeating steps (ii)-(v) for one or more further known locations on the sample, where detection of the label atoms in the plume allows construction of an image of the sample in the ablated area.

[0183] In some cases, the sample or sample carrier may be modified to include optically detectable (e.g., by optical or fluorescent microscopy) moieties at specific locations. The fluorescence locations can then be used to positionally orient the sample within the device. The use of such marker locations finds utility, for example, when the sample may have been visually inspected "off-line" - i.e., on a piece of equipment other than the device of the present invention. Such optical images may be marked with features / areas of interest corresponding to specific cells, for example by a physician, before the optical image with the features / areas of interest is highlighted and the sample is transferred to the device of the present invention. Now, by referencing the marker locations in the annotated optical image, the device of the present invention can identify the corresponding fluorescence locations using a camera and can calculate an ablation and / or absorption (LIFTing) plan accordingly with respect to the location of the laser pulse. Thus, in some embodiments, the present invention comprises an orientation controller module capable of performing the above steps.

[0184] In some cases, selection of features / regions of interest of interest may be performed using the device of the invention based on images of the sample taken by a camera of the device of the invention.

[0185] The methods disclosed herein may also be provided as a computer program product including a non-transitory machine-readable medium having instructions stored thereon that can be used to program a computer (or other electronic device) to perform the processes described herein. Machine-readable media may include, but are not limited to, hard drives, floppy disks, optical disks, CD-ROMs, DVD-ROMs, ROMs, RAMs, EPROMs, EEPROMs, magnetic or optical cards, solid-state memory devices, or other types of media / computer-readable media suitable for storing electronic instructions. Thus, the present invention also provides a machine-readable medium including instructions for performing the methods as disclosed herein.

[0186] Optical microscope As mentioned above, the autofocus sensor may include a camera (e.g., an image sensor). This camera may be shared with an inspection system (e.g., an optical microscope) of the apparatus, such as a camera for identifying the location of the laser ablation. Alternatively, the optical microscope may include a camera (e.g., an image sensor) that is separate from the autofocus sensor, such as when the autofocus sensor has a non-camera sensor.

[0187] Optical microscopes for this application may include confocal, fluorescent, or brightfield / widefield microscopes.

[0188] In certain aspects, the focus of the laser source of the laser ablation sampling system may be confocal with the autofocus component and autofocus sensor of the autofocus system. For example, the optical microscope may be confocal with the sampling system and the autofocus system. The sampling system, the autofocus system, and the optical microscope may share at least some optical components.

[0189] transfer conduit The transport conduit forms a link between the laser ablation sampling system and the ionization system and can transport a plume of sample material generated by laser ablation of the sample from the laser ablation sampling system to the ionization system. Part (or all) of the transport conduit may be formed, for example, by drilling through a suitable material to create a lumen (e.g., a lumen having a circular, rectangular, or other cross section) for passing the plume. The transport conduit sometimes has an inner diameter in the range of 0.2 mm to 3 mm. Sometimes the inner diameter of the transport conduit may vary along its length. For example, the transport conduit may have a tapered end. The length of the transport conduit may range from 1 centimeter to 100 centimeters. In some cases, the length is 10 centimeters or less (e.g., 1 to 10 centimeters), 5 centimeters or less (e.g., 1 to 5 centimeters), or 3 centimeters or less (e.g., 0.1 to 3 centimeters). Sometimes the lumen of the transport conduit is straight along the entire distance or nearly the entire distance from the ablation system to the ionization system. Also, sometimes the lumen of the transport conduit changes direction rather than being straight over the entire distance. For example, the transport conduit may make a gradual 90 degree turn. This configuration allows the plume generated by ablation of the sample in the laser ablation sampling system to initially move in a vertical plane while the axis of the transport conduit inlet points straight up, and then move horizontally as it approaches the ionization system (e.g., an ICP torch that is typically oriented horizontally to take advantage of convection cooling). The transport conduit may be straight over a distance of at least 0.1 centimeters, at least 0.5 centimeters, or at least 1 centimeter from the inlet opening where the plume enters or is formed. Broadly speaking, the transport conduit is generally adapted to minimize the time it takes to transport material from the laser ablation sampling system to the ionization system.

[0190] Transfer conduit inlet containing sample cone The transfer conduit may include an inlet in the laser ablation sampling system (particularly in the sample chamber of the laser ablation sampling system, and therefore the inlet also corresponds to the main gas outlet of the sample chamber). The inlet of the transfer conduit receives the sample material ablated from the sample in the laser ablation sampling system and transfers it to the ionization system. In some cases, the inlet of the laser ablation sampling system is the source of all gas flow along the transfer conduit to the ionization system. In some cases, the inlet of the laser ablation sampling system that receives material from the laser ablation sampling system is an opening in the wall of the conduit through which a second "transfer" gas flows from a separate transfer inlet (e.g., as disclosed in WO2014146724 and WO2014147260). In this case, the transfer gas is a significant proportion, and often the majority of the gas flows to the ionization system. The sample chamber of the laser ablation sampling system includes a gas inlet. Flowing gas into the chamber through this inlet results in a flow of gas out of the chamber through the inlet of the transfer conduit. This gas flow captures the plume of ablated material and entrains it as it enters a transport conduit (typically the laser ablation sampling system inlet of the transport conduit is in the shape of a cone referred to herein as the sample cone) and exits the sample chamber into a conduit passing above the chamber, into which gas enters from a separate transport inlet. The transport inlet, the component that provides the laser ablation sampling system inlet and begins the transport conduit carrying the ablated sample material towards the ionization system, may also be referred to as a flow cell (as in WO2014146724 and WO2014147260).

[0191] The transport flow serves at least three purposes: it flushes the plume entering the transport conduit in the direction of the ionization system to prevent the plume material from contacting the sidewalls of the transport conduit; it creates a "protective zone" above the sample surface so that the ablation is performed under a controlled atmosphere; and it increases the flow rate in the transport conduit. Typically, the viscosity of the trapping gas is lower than that of the transport gas. This helps to confine the plume of sample material to the trapping gas in the center of the transport conduit and minimize the diffusion of the plume of sample material downstream of the laser ablation sampling system (since it is in the center of the flow, the transport rate is more constant and nearly flat). The gas may be, for example, but not limited to, argon, xenon, helium, nitrogen, or mixtures thereof. A common transport gas is argon. Argon is particularly suitable for stopping the diffusion of the plume before it reaches the walls of the transport conduit (argon also helps to improve the instrument sensitivity in instruments where the ionization system is an argon gas-based ICP). The trapping gas is preferably helium. However, the trapping gas may be replaced by or contain other gases, such as hydrogen, nitrogen, or water vapor. At 25° C., argon has a viscosity of 22.6 μPas, while helium has a viscosity of 19.8 μPas. In some cases, the trapping gas is helium and the transport gas is argon.

[0192] As described in WO2014169394, the use of a sample cone minimizes the distance between the target and the laser ablation sampling system entrance of the transport conduit. This improves capture of sample material with less turbulence and reduces the spread of the plume of ablated sample material, as the distance between the sample and the point of the cone where the capture gas can flow through the cone is reduced. Thus, the entrance of the transport conduit is the opening at the tip of the sample cone. The cone protrudes into the sample chamber.

[0193] An optional modification of the sample cone is to make it asymmetric. If the cone were symmetric, there would be zero overall flow of gas along the surface of the sample at the axis of the sample cone, since gas flow from all directions would be neutralized exactly at the center. Making the cone asymmetric would result in a non-zero velocity along the sample surface, which would aid in the washout of plume material from the sample chamber of the laser ablation sampling system.

[0194] In fact, any modification of the sample cone that induces a non-zero vector gas flow along the surface of the sample at the axis of the cone can be used. For example, the asymmetric cone may include a notch or a series of notches adapted to generate a non-zero vector gas flow along the surface of the sample at the axis of the cone. The asymmetric cone may include an orifice in the side of the cone adapted to generate a non-zero vector gas flow along the surface of the sample at the axis of the cone. This orifice unbalances the gas flow around the cone, thereby again generating a non-zero vector gas flow along the surface of the sample at the axis of the target cone. The side of the cone may include more than one orifice, or may include both one or more notches and one or more orifices. The edges of the notches and / or orifices are typically smoothed, rounded, or chamfered to prevent or minimize turbulence.

[0195] Different orientations of cone asymmetry may be appropriate for different situations, depending on the choice of trapping and transport gases and their flow rates, and it is within the ability of one of ordinary skill in the art to identify appropriate gas and flow rate combinations for each orientation.

[0196] All of the above adaptations may be present in a single asymmetric sample cone as used in the present invention, for example, the cone may be asymmetrically truncated and formed from two different elliptical cone halves, the cone may be asymmetrically truncated and equipped with one or more orifices, etc.

[0197] The sample cone is thus adapted to capture the plume of material ablated from the sample in the laser ablation sampling system. In use, the sample cone is positioned in operative proximity to the sample, for example by manipulating the sample in the laser ablation sampling system on a movable sample carrier tray, as already described above. As previously described, the plume of ablated sample material enters the transfer conduit through an opening at the narrow end of the sample cone. The diameter of the opening a) may be adjustable, b) may be dimensioned to prevent perturbations of the ablated plume as it enters the transfer conduit, and / or c) may be approximately equal to the cross-sectional diameter of the ablated plume.

[0198] Tapered Conduit In a tube with a smaller inner diameter, the same flow rate of gas moves faster. Thus, by using a tube with a smaller inner diameter, the plume of ablated sample material carried in the gas flow can be transported more quickly over a given distance at a given flow rate (e.g., from the laser ablation sampling system to the ionization system in the transport conduit). One of the key factors that determines how quickly an individual plume can be analyzed is how much the plume diffuses between its generation by ablation and its constituent ions being detected in the mass spectrometer component of the device (transient time at the detector). Thus, by using a narrow transport conduit, the time between ablation and detection is reduced, thereby reducing diffusion and ultimately reducing the transient time of each ablation plume at the detector, since there is less time for diffusion to occur. A shorter transient time means that more plumes can be generated and analyzed per unit time, thereby producing higher quality and / or faster images.

[0199] The taper may include a gradual change in the inner diameter of the transport conduit along said portion of its length (i.e., the inner diameter of the tube decreases along a cross section through the tube from the end of the portion toward the inlet (at the end of the laser ablation sampling system) toward the portion toward the outlet (at the end of the ionization system). Typically, the region of the conduit near where ablation occurs has a relatively wide inner diameter. The larger volume of the conduit before the taper facilitates containment of the material produced by ablation. When the ablated particles fly away from the ablated spot, they travel at high speeds. Gas friction slows these particles, but the plume can still spread on the scale of sub-millimeter to millimeter. Ensuring sufficient distance to the walls helps contain the plume near the center of the flow.

[0200] The wide bore section is only short (on the order of 1-2 mm) and therefore does not contribute significantly to the overall transient time if the plume spends more time in the longer section of the narrower bore transport conduit. Thus, the larger bore section is used to capture the ablation products, and the smaller bore conduit is used to quickly transport these particles to the ionization system.

[0201] The diameter of the narrow bore section is limited by the diameter corresponding to the onset of turbulence. The Reynolds number can be calculated for a round tube and a known flow. In general, Reynolds numbers above 4000 indicate turbulent flow and should be avoided. Reynolds numbers above 2000 indicate transitional flow (between non-turbulent and turbulent flow) and therefore may also be desirable to avoid. For a given mass flow rate of gas, the Reynolds number is inversely proportional to the diameter of the conduit. The inner diameter of the narrow bore section of the transport conduit is generally narrower than 2 mm, for example narrower than 1.5 mm, narrower than 1.25 mm, narrower than 1 mm, but larger than the diameter at which a flow of 4 liters of helium per minute in the conduit has a Reynolds number of more than 4000.

[0202] Rough or angular edges at the transition between the constant diameter section and the taper of the transport conduit can cause turbulence in the gas flow and are typically avoided.

[0203] Sacrificial Flow At higher flow rates, there is an increased risk of turbulence occurring in the conduits. This is especially true when the internal diameter of the transport conduit is small (e.g. 1 mm). However, high transport velocities (up to and beyond 300 m / s) can be achieved in transport conduits with small internal diameters if lighter gases such as helium or hydrogen are used as the gas transport stream instead of the conventionally used argon.

[0204] High-velocity transport presents a problem insofar as it may allow the plume of ablated sample material to pass through the ionization system without an acceptable level of ionization. Increasing the flow of cold gas may reduce the temperature of the plasma at the end of the torch, thereby reducing the level of ionization. If the plume of sample material is not ionized to an adequate level, information is lost from the ablated sample material - its components (including any labeling atomic / element tags) cannot be detected by the mass spectrometer. For example, the sample may pass so quickly through the plasma at the end of the torch in an ICP ionization system that the plasma ions do not have enough time to interact with the sample material and ionize it. This problem, caused by high flow rates, high velocity transport in a narrow bore transport conduit, may be solved by introducing a flow sacrificial system at the outlet of the transport conduit. The flow sacrificial system is adapted to receive the gas flow from the transport conduit and pass only a portion of the flow (the central portion of the flow including any plume of ablated sample material) forward into the injector leading to the ionization system. To facilitate dispersion of gas from the transport conduit in the flow sacrificial system, the transport conduit outlet may be flared outwardly.

[0205] Because the flow sacrificial system is located in close proximity to the ionization system, the length of the tube (e.g., injector) leading from the flow sacrificial system to the ionization system is short (e.g., about 1 cm in length, compared to the length of the transport conduit, which is typically on the order of tens of centimeters, such as about 50 cm in length), and therefore the lower gas velocity in the tube leading from the flow sacrificial system to the ionization system does not significantly affect the total transport time, since the slower portion of the overall transport system is much shorter.

[0206] In most configurations, it is not desirable or even conceivable to significantly increase the diameter of the tube (e.g., injector) passing from the flow sacrificial system to the ionization system as a way to slow the gas down in volumetric flow rate. For example, if the ionization system is an ICP, the conduit from the flow sacrificial system forms the injector tube in the center of the ICP torch. Using an injector with a wider inner diameter will result in poor signal quality due to the inability to precisely inject the plume of ablated sample material into the center of the plasma (the hottest and most efficiently ionizing part of the plasma). Injectors with inner diameters of 1 mm or less (e.g., inner diameters of 800 μm or less, such as 600 μm or less, 500 μm or less, or 400 μm or less) are strongly recommended. Other ionization techniques rely on material to be ionized within a relatively small volume of three-dimensional space (because the energy density required for ionization can only be achieved in a small volume), and therefore a conduit with a wide inner diameter ensures that the majority of the sample material passing through the conduit is outside of the zone where the energy density is sufficient to ionize the sample material. Therefore, small diameter tubing from the flow sacrifice system to the ionization system is also used in instruments with non-ICP ionization systems. As mentioned above, if the plume of sample material is not ionized to the proper level, information will be lost from the ablated sample material - its components (including any labeling atomic / element tags) cannot be detected by the mass spectrometer.

[0207] Pumping can be used to help ensure a desired split ratio between the sacrificial flow and the flow entering the ionization system inlet. Thus, in some cases, the flow sacrificial system includes a pump attached to the sacrificial flow outlet. A controlled restrictor can be added to the pump to control the sacrificial flow. In some cases, the flow sacrificial system also includes a mass flow controller adapted to control the restrictor.

[0208] If an expensive gas is used, the gas pumped out of the sacrificial flow outlet may be purged and recycled back into the same system using known gas purification methods. Helium is particularly suitable as a transport gas as described above, but is expensive, and therefore it is advantageous to reduce the loss of helium in the system (i.e., when it is passed to the ionization system to be ionized). Thus, a gas purification system may be connected to the sacrificial flow outlet of the sacrificial flow system.

[0209] Ionization System To generate elemental ions, it is necessary to use hard ionization techniques that can vaporize, nebulize, and ionize the nebulized sample.

[0210] Inductively Coupled Plasma Torch The apparatus of the present application may further include an inductively coupled plasma (ICP) torch or may be coupled to an ICP torch. For example, the apparatus of the present application may further include an ICP mass spectrometer (ICP-MS).

[0211] Inductively coupled plasma is commonly used to ionize the material to be analyzed before it is passed through a mass detector for analysis. Inductively coupled plasma is a plasma source that is energized by an electric current generated by electromagnetic induction. The inductively coupled plasma is maintained within a torch consisting of three concentric tubes, the innermost tube being known as the injector.

[0212] An induction coil, which provides the electromagnetic energy that sustains the plasma, is positioned around the output end of the torch. The alternating electromagnetic field reverses polarity millions of times per second. Argon gas is fed between the two outermost concentric tubes. Free electrons are introduced by the discharge and these free electrons are accelerated in the alternating electromagnetic field where they collide with argon atoms, ionizing them. At steady state, the plasma consists primarily of argon atoms with very small amounts of free electrons and argon ions.

[0213] A flow of gas between the two outermost tubes keeps the plasma away from the walls of the torch, thus keeping the ICP within the torch. A second flow of argon, introduced between the injector (center tube) and the middle tube, keeps the plasma clear of the injector. A third flow of gas is introduced into the injector in the center of the torch. The sample to be analyzed is introduced into the plasma via the injector.

[0214] The ICP may be equipped with an injector having an internal diameter of less than 2 mm and greater than 250 μm to introduce material from the sample into the plasma. The diameter of the injector refers to the internal diameter of the injector at the end proximal to the plasma. The injector may have a different diameter, e.g. a wider diameter, as it extends away from the plasma, the difference in diameter being achieved by a stepwise increase in diameter or because the injector is tapered along its length. For example, the internal diameter of the injector may be between 1.75 mm and 250 μm, e.g., between 1.5 mm and 300 μm diameter, between 1.25 mm and 300 μm diameter, between 1 mm and 300 μm diameter, between 900 μm and 300 μm diameter, between 900 μm and 400 μm diameter, e.g., about 850 μm diameter. The use of an injector with an internal diameter of less than 2 mm provides a significant advantage over injectors with a larger diameter. One advantage of this feature is that the narrower injector reduces the signal transients detected at the mass detector when a plume of sample material is introduced into the plasma (a plume of sample material is a group of specific vapor materials removed from the sample by the laser ablation sampling system). Thus, the time required to analyze the plume of sample material from its introduction into the ICP for ionization to the detection of the resulting ions at the mass detector is reduced. This reduction in the time required to analyze the plume of sample material allows more plumes of sample material to be detected in any given period of time. Also, a smaller inner diameter injector introduces the sample material more precisely into the center of the inductively coupled plasma, where more efficient ionization occurs (as opposed to a larger diameter injector that can introduce more sample material into the edge of the plasma where ionization is less efficient).

[0215] ICP torches (Agilent, Varian, Nu Instruments, Spectro, Leeman Labs, PerkinElmer, Thermo Fisher, etc.) and injectors (Elemental Scientific, Meinhard, etc.) are available.

[0216] Other Ionization Techniques Electron Ionization Electron ionization involves colliding a gas phase sample with an electron beam. An electron ionization chamber contains an electron source and an electron trap. A typical source of the electron beam is a rhenium or tungsten wire, usually operating at an energy of 70 electron volts. Electron beam sources for electron ionization are available from Markes International. The electron beam is directed into the electron trap, and a magnetic field applied parallel to the direction of electron movement causes the electrons to move in a spiral path. The gas phase sample is directed through the electron ionization chamber, where it interacts with the electron beam to form ions. Electron ionization is considered a difficult method of ionization, as the process generally fragments the sample molecules. An example of a commercially available electron ionization system is the Advanced Markus Electron Ionisation Chamber.

[0217] Optional Additional Components of the Laser Ablation-Based Sampling and Ionization System Ion Deflector Mass spectrometers detect ions when they strike the detector surface. The collision of the ions with the detector causes the liberation of electrons from the detector surface. These electrons are multiplied as they pass through the detector (the initially liberated electrons knock off more electrons in the detector, which then strike the secondary plate, further amplifying the number of electrons). The number of electrons striking the detector anode creates a current. The number of electrons striking the anode can be controlled by varying the voltage applied to the secondary plate. The current is an analog signal that can be converted by an analog-to-digital converter into a count of ions striking the detector. If the detector is operating in its linear range, the current can be directly correlated to the number of ions. There is a limit to the amount of ions that can be detected at one time (which can be expressed as the number of ions that can be detected per second). Beyond this point, the number of electrons liberated by ions striking the detector no longer correlates to the number of ions. This therefore imposes an upper limit on the quantitative capabilities of the detector.

[0218] When ions hit the detector, its surface becomes damaged by contamination. Over time, this irreversible contamination damage causes the detector surface to release fewer electrons when ions hit it, eventually requiring the detector to be replaced. This is referred to as "detector aging" and is a well-known phenomenon in MS.

[0219] Therefore, by avoiding the introduction of an overload of ions into the MS, the life of the detector can be extended. As mentioned above, when the total number of ions striking the MS detector exceeds the upper limit of detection, the signal is no longer quantitative and therefore less informative than when the number of ions is below the upper limit. It is therefore desirable to avoid exceeding the upper detection limit, as this will accelerate the aging of the detector without producing useful data.

[0220] Analysis of large packets of ions by mass spectrometry involves a particular set of challenges not found in regular mass spectrometry. In particular, typical MS techniques involve introducing a constant level of material to the detector at a low level that must not approach detection limits or accelerate detector degradation. Laser ablation and desorption based techniques, on the other hand, analyze a relatively large amount of material in a very short time window of the MS, i.e., ions from, for example, a cell-sized patch of a tissue sample, which is much larger than the small packets of ions typically analyzed in MS. In effect, it is an almost intentional overloading of the detector by the analyzed pack of ions resulting from ablation or lifting. Between analysis events, the signal is at baseline (a near-zero signal because ions from the labeled atoms are not intentionally entering the MS from the sampling and ionization system; some ions are inevitably detected because the MS is not a perfect vacuum).

[0221] Thus, the devices described herein are at high risk of accelerated detector aging, as ions from a packet of ionized sample material labeled with a large number of detectable atoms may exceed the upper limit of detection and damage the detector without providing useful data.

[0222] To address these issues, the instrument may include an ion deflector positioned between the sampling and ionization system and the detector system (mass spectrometer) and operable to control the entry of ions into the mass spectrometer. In one configuration, when the ion deflector is on, ions received from the sampling and ionization system are deflected (i.e., the path of the ions is changed so that they do not reach the detector), whereas when the deflector is off, ions reach the detector undeflected. How the ion deflector is deployed depends on the placement of the sampling and ionization system and the MS of the instrument. For example, if the portal through which ions enter the MS is not directly aligned with the path of ions exiting the sampling and ionization system, then by default, a suitably positioned ion deflector will be turned on to direct ions from the sampling and ionization system to the MS. When a packet of ionized sample material is detected that is believed to be an ionization-induced event that could overload the MS (see below), the ion deflector is switched off so that the remaining ionized material from the event is no longer deflected into the MS but can instead only strike internal surfaces of the system, thereby preserving the life of the MS detector. After preventing ions from the damaging event from entering the MS, the ion deflector is returned to its original state, allowing ions from a subsequent packet of ionized sample material to enter the MS and be detected.

[0223] Alternatively, in an arrangement where there is no change in the direction of ions leaving the sampling and ionization system before entering the MS (under normal operating conditions), the ion deflector is turned off and ions from the sampling and ionization system pass through it to be analyzed by the MS. To prevent damage when a potential overload of the detector is detected, in this configuration the ion deflector is turned on, thus diverting ions from entering the detector to prevent damage to the detector.

[0224] Ions entering the MS from ionization of a sample material (such as a plume of material produced by laser ablation or desorption) do not enter the MS simultaneously, but instead as a peak whose frequency follows a probability distribution curve near a maximum frequency. That is, from a baseline, a small number of ions first enter the MS and are detected, then the frequency of ions increases to a maximum before decreasing in number again and reaching the baseline. Instead of a slow increase in ion frequency at the start of the peak, the number of ions hitting the detector increases very rapidly, allowing the identification of events that may be damaging to the detector.

[0225] The stream of ions hitting the detector of a TOF MS, a particular type of detector described below, is not continuous during the analysis of the ions in a packet of ionized sample material. The TOF comprises a pulser that periodically releases ions into the flight chamber of the TOF MS in groups of pulses. Releasing all ions at the same known time allows for time-of-flight mass analysis. The time between the release of ion pulses for time-of-flight mass analysis is known as the extraction or push in the TOF MS. A push is on the order of microseconds. Thus, the signal from one or more packets of ions from the sampling and ionization system covers many pushes.

[0226] Thus, if the ion count reading jumps from the baseline to very high counts with one push (i.e., the first portion of ions from a particular packet of ionized sample material), it can be expected that the upper detection limit will be exceeded as the body of ions resulting from ionization of the packet of sample material becomes larger, at which point the ion deflector can be operated (either activated or deactivated depending on the system configuration, as described above) to redirect the majority of the damaging ions away from the detector.

[0227] Suitable quadrupole-based ion deflectors are available in the art (eg, from Colutron Research Corporation and Dreebit GmbH).

[0228] b. Desorption-based Sampling and Ionization System A desorption-based analyzer generally comprises three components. The first component is a desorption system for generating a slug of sample material from the sample for analysis. Before the atoms in the desorbed slug of sample material (including any detectable label atoms described below) can be detected, the sample must be ionized (and atomized). Thus, the device comprises a second component, an ionization system that ionizes the atoms to form elemental ions to enable their detection by the MS detector component (third component) based on mass / charge ratio. The desorption-based sampling system and the ionization system are connected by a transfer conduit. In many cases, the desorption-based sampling system is also a laser ablation-based sampling system.

[0229] Desorption Sampling System In some cases, rather than using laser ablation to generate a particulate and / or vaporized plume of sample material, a bulk mass of sample material is desorbed from the sample carrier on which it is placed without substantial disintegration of the sample and conversion of the sample to small particles and / or vapor (see, for example, FIG. 8 of WO2016109825, incorporated herein by reference, and the accompanying description). The term slug is used herein to refer to this desorbed material (one particular form of packet of sample material discussed herein). Slugs may have dimensions of 10 nm to 10 μm, 100 nm to 10 μm, and in some cases 1 μm to 100 μm. This process may be referred to as sample catapulting. Generally, the slug corresponds to a single cell (in which case the process may be referred to as cell catapulting).

[0230] The slug of sample material released from the sample may be a portion of the sample that has been cut into individual slugs for desorption prior to the desorption step, optionally in the process before the sample is inserted into the device. A sample that has been divided into separate slugs prior to analysis is referred to as a structured sample. Each of these individual slugs thus represents a separate portion of the sample that may be desorbed, ionized, and analyzed in the device. Analysis of the slugs from separate sites allows an image to be constructed with each slug representing a pixel of the image, in the same way that each location of the sample is ablated by the laser ablation sampling system described above.

[0231] Structured samples may be prepared in various ways. For example, a sample carrier may be used that includes topographical features configured to cut the biological sample. Here, the biological sample is applied to the surface of the carrier, whereby the topographical features cut and sever the sample, thus resulting in a structured biological sample, with sections of the biological material being held by a plurality of distinct sites between the features. Alternatively, the sample carrier may not include such topographical features (indeed, a flat surface such as a microscope slide, optionally functionalized as described below), in which case the sample may be applied to the sample carrier and the sample may be cut to define a slug of sample that can be desorbed for ionization and analysis. If the sample is a tissue section, sectioning of the sample may be achieved by a mechanical tool such as a blade or stamp. Alternatively, material around the section of the sample to be desorbed may be removed by laser ablation in the same or a separate sample preparation setup. In certain techniques, material may be removed by a setup using a focused electron or ion beam. The focused electron or ion beam produces particularly narrow cuts (potentially on the 10 nm scale) between subsections resulting in pixel sizes of the order of 1 μm or in certain cases 100 nm.

[0232] A slug of sample material can be released from the carrier, and each discrete portion of the sample material is sequentially introduced to a detector for analysis as a discrete event (producing a pixel of an image by techniques described below). Advantages of introducing separate materials sequentially, as opposed to randomly introducing a biological sample as in conventional mass cytometry or mass spectrometry, include higher sample throughput rates, since the slug is transported from the sample chamber to the ionization system, preferably as a single entity, so that the slug cannot spread out when the plume of ablated material is in a gas flow, especially one in which some turbulence is present.

[0233] Desorption for sampling Sample material can be desorbed from the sample by thermal energy, mechanical energy, kinetic energy, and any combination of the above. This type of sampling is particularly useful for the analysis of biological samples.

[0234] In certain instances, the sample material may be released from the sample by a thermal mechanism. For example, the surface of the sample carrier becomes hot enough to desorb a slug of sample material. The sample carrier may be coated with, for example, a polyethylene naphthalate (PEN) polymer or PMMA polymer film to facilitate the bulk desorption process. The heat may be provided by a radiation source such as a laser (such as the laser of the laser ablation sampling system described above). The energy applied to the surface must be sufficient to desorb the biological material, and preferably, if the sample material is from a biological sample, it must be sufficient to desorb the sample material without altering the sample material. Any suitable radiation wavelength may be used, which may depend in part on the absorption characteristics of the sample carrier. A surface or layer of the sample carrier may be coated with or include an absorber that absorbs the laser radiation for conversion to heat. The radiation may be delivered to a surface of the carrier other than the surface on which the sample is located or may be delivered to a surface supporting the sample, such as by penetrating the thickness of the carrier. The heated surface may be a surface layer or may be an inner layer of a multi-layer structure of the sample carrier. One example of the use of laser radiation energy is in a technique called LIFTing (Laser-Induced Forward Transfer; see, e.g., Doraiswamy et al., 2006, Applied Surface Science, 52:4743-4747; Fernandez-Pradas, 2004, Thin Solid Films 453-454:27-30; Kyrkis et al., in Recent Advances in Laser Processing of Materials, Eds. Perriere et al., 2006, Elsivier), in which the sample carrier may contain a detached film layer.The desorption membrane can absorb radiation, causing release of the desorption membrane and / or the biological sample (e.g., in some cases the sample membrane desorbs from the sample carrier along with the biological sample, in other cases the membrane remains attached to the sample carrier and the biological sample desorbs from the desorption membrane).

[0235] Thermal desorption can occur on the nanosecond, picosecond, or femtosecond time scale, depending on the laser used for desorption.

[0236] The sample may be attached to the sample carrier by a cleavable photoreactive moiety. Upon irradiation of the cleavable photoreactive moiety (e.g., from a laser in a laser system of a laser ablation sampling system), the photoreactive moiety may cleave to release the sample material. The sample carrier may comprise (i) a cleavable photoreactive moiety that bonds the sample to the sample carrier, and (ii) a desorption membrane as described above. In this situation, a first pulse of laser radiation may be used to cause cleavage of the photoreactive moiety, and a second pulse of laser radiation may be used to target the desorption membrane and separate the sample from the sample carrier by lifting (alternatively, a thermal energy pulse introduced by other means may be used to heat the desorption membrane and thus cause separation of the sample material from the sample carrier). The first and second pulses may have different wavelengths. Thus, in some methods (e.g., including both ablation and desorption), separation of the sample from the sample carrier may involve multiple laser pulses of different wavelengths. In some cases, cleavage of the photoreactive moiety and lifting may be achieved by the same laser pulse.

[0237] The sample carrier may include a coating or layer of a chemically reactive species that imparts kinetic energy to the sample to release it from the surface. For example, the chemically reactive species may liberate a gas, such as, for example, H2, CO2, N2, or a hydrochlorofluorocarbon. Examples of such compounds include foaming agents and effervescent agents that liberate a gas upon heating. The generation of gas can be used to impart kinetic energy to the desorbed sample material that can improve the reproducibility and direction of release of the material.

[0238] The sample carrier may contain a photoinitiated chemical reactant that undergoes an exothermic reaction to generate heat to desorb the sample material. The coating of the carrier described in the previous paragraph, or indeed certain chemical bonds within that carrier, which are irradiated by a laser to release a slug of sample material from the carrier, are examples of materials that can be targeted by the wavelength of laser radiation.

[0239] Generally, when performing ablation, the ablated locations are resolved as separate, non-overlapping spots. However, if desorption is used as a means to introduce sample material into the device, overlapping spots may be used, for example to ensure that all desorption films that secure the sample to the sample carrier at a particular location are removed. The inventors have confirmed that desorption of cells with a single laser pulse with a spot size large enough to completely desorb the cells from the sample carrier often causes the destruction of the slug of material. As soon as the slug of sample material is broken down into smaller parts, the transit time of the material in the ablated slug increases. This is because the material necessarily spreads as it passes from the chamber where the sample is desorbed through the transport conduit to the ionization system and then to the detector. Maintaining the integrity of the desorbed slug therefore allows the highest speed of analysis of the ablated slug, i.e., the highest speed of analysis of cells, for example when the sample is a cell smear. Desorption of single cells as separate slugs that generally maintain their integrity until ionization offers the opportunity to analyze single cells from slides at speeds similar to those made possible by the analysis of cells in solution by CyTOF (Fluidigm, CA, USA). However, desorption of individual cells from slides offers the added advantage that the cells can first be analyzed visually, thus allowing the selection of cells of interest as well as removing cells of, for example, the wrong cell type, thereby improving the efficiency of the analysis. Furthermore, it allows the slug of material to be selected so that it is in fact a single cell. In the analysis of liquid samples, there are cases where cells may aggregate into doublets, triplets of higher order multimers, or, by chance, two separate cells may be analyzed in the same event as a result of the sample introduction process. Thus, atoms from two or more cells may travel together to the ionization and detection systems, which may result in inaccurate results as well as possible damage to the instrument due to overloading the MS detector.

[0240] In many cases, features / regions of interest on a sample do not represent separate entities, such as isolated cells, at individual sites that can be easily desorbed by themselves. Instead, cells of interest may be surrounded by other cells or materials that are not needed or desired to be analyzed. Thus, when attempting to perform desorption (e.g., lifting) of a feature / region of interest of interest, both the cell of interest and the surrounding material may be desorbed together. Thus, atoms such as label atoms used in elemental tags (see below) from the surrounding regions of the sample (e.g., from other cells that are labeled) that are carried in the desorbed slug of material in addition to the particular feature / region of interest of interest (e.g., cell) may contaminate the readings regarding the location of the target.

[0241] The techniques of ablation and desorption (such as by lifting) may be combined in a single method. For example, to perform precise desorption of features / regions of interest (e.g., cells) of interest on a sample carrier, on a biological sample, for example, a tissue section sample or a cell suspension dispersion, laser ablation can be used to ablate the area around the cells of interest and remove other material from it. After removing the surrounding area by ablation, the features / regions of interest of interest can be desorbed from the sample carrier and then ionized and analyzed by mass spectrometry according to standard mass cytometry or mass spectrometry procedures. In line with the above discussion, optionally, after ablation has been used to remove the area surrounding the location to be desorbed, thermal, photolytic, chemical or physical techniques can be used to desorb material from the features / regions of interest. Lifting is often used to separate a slug of material from the sample carrier (e.g., a sample carrier coated with a desorption film to aid the lifting procedure, as described above for desorption of a separate slug of sample material).

[0242] The present invention therefore provides a method for analyzing a sample, comprising the steps of: (i) performing the method of the present invention to position a sample at the focus of a laser for laser ablation; (ii) desorbing a slug of sample material using laser radiation directed at the sample on the sample stage; (iii) ionizing a slug of sample material and detecting atoms in the slug by mass spectrometry; The present invention provides a method comprising:

[0243] The sample can be placed on a sample carrier and, optionally, laser radiation is directed through the sample carrier to desorb a slug of sample material from the sample carrier.

[0244] In some embodiments, the method further comprises performing laser ablation of the sample prior to step (i). Optionally, ablation of the sample produces one or more plumes of sample material, and the plumes are individually ionized and atoms within the plumes are detected by mass spectrometry. Optionally, the method further comprises the further step of labeling a plurality of different target molecules in the sample with one or more different labeling atomic / elemental tags prior to step (i) to provide a labeled sample. Laser ablation is used in some variations of the method to ablate material around the feature / region of interest to remove the surrounding area before the sample material of the feature / region of interest of interest is desorbed from the sample carrier as a slug of material.

[0245] The feature / region of interest of interest may be identified by another technique before laser ablation and desorption (e.g., by lifting) is performed. The inclusion of a camera (such as a charge-coupled device image sensor-based (CCD) camera or a CMOS camera or an active pixel sensor-based camera) or any other light detection means described in the previous section is one way to enable these techniques for both online and offline analysis. The camera can be used to scan the sample to identify a particular cell of interest or feature / region of interest of interest (e.g., cells of a particular morphology). Once such a location is identified, a laser pulse can be directed to the area around the feature / region of interest to remove other material by ablation before the location (e.g., cells) is lifted, followed by lifting. This process may be an automated process (where the system identifies, ablates, and lifts the feature / region of interest of interest) or a semi-automated process (where a user of the system, e.g., a clinical pathologist, identifies the feature / region of interest of interest and then the system performs the ablation and lifting in an automated manner). This allows for specific ablation of cells of interest without the need to ablate the entire sample in order to analyze specific cells, greatly increasing the speed at which analysis can be performed.

[0246] The camera can record images from a microscope (such as a confocal microscope). Identification can be performed by optical microscopy, for example by examining cell morphology or cell size, or based on whether the cells are separate single cells (as opposed to elements of a clump of cells). In some cases, the sample can be specifically labeled to identify features of interest (e.g., cells). Often, fluorescent markers are used to specifically stain the cells of interest (such as by using labeled antibodies or labeled nucleic acids), as described above in connection with the methods for ablating visually identified features of interest / regions of interest; that section will not be repeated in full here for brevity, but those skilled in the art will readily appreciate that the features of these methods are applicable to desorption-based methods, and this is within the scope of the technical teachings of this document. High-resolution optical images are advantageous in this coupling of optical techniques and lifting. This is because the precision of the optical images determines the precision with which the ablation laser source can be directed to ablate the area around the cells of interest that can then be desorbed.

[0247] The present invention also provides a method for analyzing a sample containing a plurality of cells, comprising: (i) labeling a plurality of different target molecules in a sample with one or more different label atoms to provide a labeled sample; (ii) illuminating the sample to identify one or more features of interest; (iii) recording position information of one or more features of interest on the sample; (iv) using the position information of the feature of interest to desorb a slug of sample material from the feature of interest, the step including first performing laser ablation to remove sample material surrounding the feature of interest using laser radiation before the slug of sample material is desorbed from the position using laser radiation, the laser radiation being directed towards the sample; (v) ionizing the desorbed slug of sample material; (vi) subjecting the ionized sample material to mass spectrometry for detection of label atoms in the sample material; Including, The laser radiation used to desorb the slug of sample material is focused onto the sample using the autofocus method of the present invention; A method is also provided.

[0248] The present invention also provides a variation of the above method, for example a method of performing mass cytometry involving a plurality of cells, comprising: (i) labeling a plurality of different target molecules in a sample with one or more different label atoms and one or more fluorescent labels to provide a labeled sample; (ii) irradiating the sample with laser radiation to excite one or more fluorescent labels; (iii) recording location information of one or more locations of the sample based on the pattern of fluorescence; (iv) desorbing a slug of sample material from the feature of interest using location information based on the pattern of fluorescence, the step including first performing laser ablation to remove sample material surrounding the feature of interest using laser radiation before the slug of sample material is desorbed from the location using laser radiation, the laser radiation being directed towards the sample; (v) ionizing the desorbed slug of sample material; (vi) subjecting the ionized sample material to mass spectrometry for detection of label atoms in the sample material; Including, The laser radiation used to desorb the slug of sample material is focused onto the sample using the autofocus methods described herein; A method is also provided.

[0249] In some cases, data is not recorded from the ablation performed to remove the region surrounding the location to be desorbed (e.g., cells of interest). Sometimes data is recorded from ablation of the surrounding region. Useful information that can be obtained from the surrounding region includes what target molecules, such as proteins or RNA transcripts, are present in the surrounding cells and in the intercellular environment. This can be particularly interesting when imaging solid tissue samples where direct cell-cell interactions are common and what proteins, etc. are expressed in the surrounding cells can be informative about the state of the cells of interest.

[0250] camera The camera used in the desorption-based sampling system can be as described above for the laser ablation-based sampling system, and the discussion regarding the camera for the laser ablation-based sampling system should be read here.

[0251] Sample Chamber The sample chamber used in the desorption-based sampling system can be as previously described for the laser ablation-based sampling system. If a large slug of sample material is being sampled, the skilled practitioner will appreciate that it may be necessary to increase the gas flow rate so that the slug of material is entrained in the gas flow and carried to the transfer conduit for transport to the ionization system.

[0252] transfer conduit The sample chamber used in the desorption-based sampling system can be as described above for the laser ablation-based sampling system. If large slugs of sample material are being sampled, the skilled practitioner will appreciate that the diameter of the lumen of the conduit must be appropriately sized to accommodate any slugs without the slug touching the sides of the lumen (which would cause the slug to fragment and result in overlapping signals - in this case atoms from the slug associated with the nth desorption event would diffuse into the detection window for the n+1th and subsequent slugs).

[0253] Desorption-based system ionization system In many cases, the lifting techniques described above involve the removal of relatively large slugs (10 nm to 10 μm, 100 nm to 10 μm, and sometimes 1 μm to 100 μm) of sample material that was not converted to particulate and vaporized material. Therefore, ionization techniques capable of vaporizing and nebulizing this relatively large amount of material are required.

[0254] Inductively Coupled Plasma Torch One such suitable ionization system is an inductively coupled plasma, as previously described in the section beginning on page 57 with respect to laser ablation-based sampling and ionization systems.

[0255] Optional Additional Components of a Desorption-Based Sampling and Ionization System Ion Deflector The ion deflector used in the desorption-based sampling system can be as described above for the laser ablation-based sampling system. Given the potential of desorption-based sampling to remove intact large slugs of sample material, the ion deflector can be particularly useful in this type of system to protect the detector.

[0256] c. Laser desorption / ionization system In certain embodiments, the sampling system may be a laser desorption system. Laser desorption / ionization-based analyzers generally comprise two components. The first component is a system that generates ions from a sample for analysis. In this device, this is accomplished by directing a laser beam to the sample to generate ions, which is referred to herein as a laser desorption ion generation system. These ejected sample ions (including detectable ions from labeling atoms, as described below) can be detected by a detector system (second component), such as a mass spectrometer (detectors are described in more detail below). This technique is known as laser desorption / ionization mass spectrometry (LDI-MS). LDI differs from desorption-based sampling systems, which are described in more detail below, because in desorption-based sampling systems, sample material is desorbed as a charge-neutral slug of material that is subsequently ionized to form elemental ions. Instead, here, ions are generated directly as a result of irradiation of the sample by a laser, and no separate ionization system is required.

[0257] The laser desorption ion generation system comprises a laser, a sample chamber for containing a sample onto which radiation from the laser is directed, and an ion optics element for capturing ions generated from the sample and directing them to a detector for analysis. Thus, the present invention provides an apparatus for analyzing a sample, comprising: a. a sample chamber for containing a sample; b. a laser adapted to desorb and ionize material from the sample to form ions; c. an ion optics element adapted to sample ions formed by desorption ionization and direct them away from the sample towards a detector; d. a detector for receiving ions from the ion optics element and analyzing the ions. In some embodiments, the apparatus comprises a laser adapted to desorb and ionize material from the sample to form elemental ions, and in this case a detector receives elemental ions from the sampling and ionization system and detects the elemental ions. In some cases, the LDI is matrix-assisted (i.e., MALDI).

[0258] In this process, some molecules reach an energy level where they desorb from the sample and become ionized.

[0259] Ions can be generated as primary ions directly as a result of laser irradiation or as secondary ions formed by collision of primary ions with charge-neutral species (e.g., proton transfer, cationization, electron capture). In some cases, ionization is assisted by compounds (such as matrices) added to the sample during sample preparation, as described below.

[0260] Laser A variety of different lasers can be used for LDI, including commercially available lasers such as those described above in connection with the laser of the laser ablation sampling system, which are adapted to allow for desorption of ions as needed. Thus, in some embodiments, the device includes a laser adapted to desorb and ionize material from a sample to form elemental ions, and in this case a detector receives elemental ions from the sampling and ionization system to detect the elemental ions. Sometimes, the device includes a laser adapted to desorb and ionize material from a sample to form elemental ions, and in this case a detector receives molecular ions from the sampling and ionization system to detect the molecular ions. In other cases, the device includes a laser adapted to desorb and ionize material from a sample to form both elemental and molecular ions, and in this case a detector receives ions from the sampling and ionization system to detect both the elemental and molecular ions.

[0261] Typical lasers include lasers emitting at 193 nm, 213 nm or 266 nm (deep UV lasers that can cause the release of ions from the sample without the need for a matrix to promote ionization, as in MALDI). Desorption of ions representing lichen metabolites after laser irradiation of a sample at 355 nm is demonstrated in Le Pogam et al. 2016 (Scientific Reports 6, Article number: 37807).

[0262] The femtosecond lasers mentioned above are also advantageous in certain LDI applications.

[0263] Rapid analysis of samples requires high frequency ablation, e.g., greater than 200 Hz (i.e., greater than 200 laser shots per second, giving greater than 200 ion clouds per second). Typically, the frequency of ion cloud generation by the laser system is at least 400 Hz, e.g., at least 500 Hz, at least 1 kHz, at least 10 kHz, at least 100 kHz, or at least 1 MHz. For example, the frequency of ablation by the laser system is in the range of 200 Hz to 1 MHz, in the range of 500 Hz to 100 kHz, in the range of 1 to 10 kHz.

[0264] As described above with respect to the laser ablation sampling system, the laser radiation can be directed to the sample through various optics and focused to a spot size (i.e., the size of the beam of laser radiation as it strikes the sample) of 100 μm or less, e.g., 50 μm or less, 25 μm or less, 20 μm or less, 15 μm or less, or 10 μm or less, or 1 um or less. When used to analyze biological samples including tissue sections, to analyze individual cells, the spot size of the laser beam used depends on the size and spacing of the cells. For example, if the cells are closely packed together (such as in tissue sections), the laser spot can have a spot size no larger than the cells if single cell analysis is to be performed. Although this size depends on the particular cells in the sample, generally the diameter of the laser spot for LDI is less than 4 μm, e.g., in the range of 0.1-4 μm, 0.25-3 μm, or 0.4-2 μm. To analyze certain cells with subcellular resolution, the LDI system uses a laser spot size that is no larger than these cells, and more specifically, uses a laser spot size that can ablate material with subcellular resolution. In some cases, single cell analysis can be performed using a spot size that is larger than the size of the cells, for example, when the cells are spread on a slide with spaces between the cells. Thus, the particular spot size used can be appropriately selected depending on the size of the cells being analyzed. In biological samples, cells are rarely all the same size, so if subcellular resolution imaging is desired, the laser spot size should be smaller than the smallest cell if a constant spot size is maintained throughout the ion generation procedure.

[0265] Sample Chamber The sample chamber of an LDI system shares many features in common with the sample chambers of the laser ablation-based and desorption-based sampling systems described above. The sample chamber comprises a stage for supporting the sample. The stage may be a translation stage movable in the xy or xyz axes. The sample chamber also comprises an outlet through which material removed from the sample by the laser radiation can be directed. The outlet is connected to a detector, allowing analysis of the sample ions.

[0266] The sample chamber may be at atmospheric pressure. LDI (especially MALDI) at atmospheric pressure is known, where ions generated by LDI are assisted from ionization to a high vacuum region for analysis (e.g., MS detector) by a pneumatic gas flow such as nitrogen (Laiko et al., 2000. Anal. Chem., 72:652-657).

[0267] In some cases, the sample chamber is held under vacuum or partial vacuum. Thus, in some cases, the sample chamber pressure is less than 50000 Pa, less than 10000 Pa, less than 5000 Pa, less than 1000 Pa, less than 500 Pa, less than 100 Pa, less than 10 Pa, less than 1 Pa, about 0.1 Pa, or less than 0.1 Pa, such as 0.01 Pa or less. For example, the partial vacuum pressure may be about 200-700 Pa, and the vacuum pressure may be 0.2 Pa or less.

[0268] As the skilled person will appreciate, the choice of whether the sample pressure is at atmospheric pressure, under (partial) vacuum, depends on the particular analysis to be performed. For example, at atmospheric pressure, the sample is easier to handle and softer ionization can be applied. Furthermore, the presence of gas molecules may be desired to allow the phenomenon of collisional cooling to occur, which may be interesting when the label is a large molecule and its fragmentation, e.g. molecular fragments containing label atoms or combinations thereof, is undesirable.

[0269] Keeping the sample chamber under vacuum prevents collisions between the sample ions generated by LDI and other particles in the chamber. In some cases, this may be preferable because collisions with gas molecules in the chamber can cause the generated sample ions to lose charge, which would prevent them from being detected by the instrument.

[0270] In some embodiments, the sample chamber comprises one or more gas ports arranged to allow delivery of one or more streams of gas to the location of laser desorption / ionization on the sample during laser desorption / ionization, e.g., the one or more gas ports are in the form of a nozzle. The gas port (e.g., nozzle) is operable to deliver gas at the moment of desorption and ionization to provide collisional cooling to the desorbed ions, but only at that particular time. The rest of the time, the gas port does not introduce gas into the chamber, thereby reducing the strain on the vacuum pump.

[0271] Ion Optical Elements The sample ion beam is captured from the sample via an electrostatic plate, known in the art as the extraction electrode, positioned close to the sample. The extraction electrode removes the sample ions desorbed by laser ablation from the locality of the sample. This is generally achieved by a sample and an electrode (sample electrode) positioned on the plate acting on the extraction electrode with a large potential difference. Depending on the polarity of the sample with respect to the extraction electrode, positively or negatively charged secondary ions are captured by the extraction electrode.

[0272] In some embodiments, the charge across the electrodes is constant during laser desorption / ionization. In some cases, the charge is changed following desorption / ionization, for example, in delayed extraction, the acceleration voltage is applied after some short time delay following the desorption / ionization induced by the laser pulse. This technique provides time-of-flight compensation for the spread in ion energy, where ions with higher kinetic energy move faster from the sample towards the detector than ions with lower kinetic energy. This difference in speed can therefore reduce the resolution of the detector, as not all ions are moving at the same speed. Thus, by delaying the application of the voltage across the sample and extraction electrodes, ions with lower kinetic energy remain closer to the sample electrode when the acceleration voltage is applied, and therefore begin to accelerate at a greater potential compared to ions that are further from the target electrode. With the appropriate delay time, the slower ions are accelerated sufficiently to capture ions that had higher kinetic energy after laser desorption / ionization after flying some distance from the pulse acceleration system. Ions of the same mass-to-charge ratio drift through the flight tube to the detector at the same time. Thus, in some embodiments, the sample and extraction electrodes are controllable to apply a charge across the electrodes at a set time after the laser short circuit that causes desorption / ionization of the sample.

[0273] The sample ions are then transferred to the detector through one or more further electrostatic lenses (known in the art as transfer lenses). The transfer lenses focus the beam of sample ions onto the detector. Generally, in systems with multiple transfer lenses, only one transfer lens is involved in a particular analysis. Each lens may provide a different magnification of the sample surface. Generally, between the electrode and the detector there is a set of further ion manipulation components, such as one or more apertures, mass filters or deflector plates. The electrodes, transfer lenses and any further components together form an ion optical element. Components for producing a suitable ion optical device are available from commercial suppliers, such as Agilent, Waters, Bruker, and can be appropriately positioned by one skilled in the art to deliver ions to the detector as discussed herein below.

[0274] In addition to the detectors described below, LDI can be performed to provide soft ionization (e.g., ionization without breaking bonds in the analyte molecules) and in some cases the detector may be a tandem MS, in which a first m / z separation is performed to select ions from the sample before undergoing a second m / z separation in which the selected ions are broken into their fragments and the fragments are detected.

[0275] How to adopt LDI The present invention also provides a method for analyzing biological samples using LDI, in which cells are labeled with labels, and these labels are then detected in the ions generated following LDI of the sample. Thus, the present invention provides a method for performing mass cytometry on a sample containing a plurality of cells, comprising the steps of: a. labeling a plurality of different target molecules in the sample with one or more different labels to provide a labeled sample; b. performing laser desorption / ionization of the sample, where the laser desorption / ionization is performed at a plurality of locations to form a plurality of individual ion clouds; and c. exposing the ion clouds to mass spectrometry individually, where detection of the labels in the plume allows construction of an image of the sample, and optionally the plurality of locations are a plurality of known locations.

[0276] In some embodiments, the one or more labels include a label atom. In this case, the label functions as described herein below, whereby a member of a specific binding pair (e.g., an antibody that binds to a protein antigen, or a nucleic acid that binds to an RNA in a sample) is attached to an elemental tag that includes one or more label atoms (e.g., a lanthanide and an actinide). An elemental tag can include a single type of label atom (e.g., one or more atoms of a single isotope of a particular element), or can include different kinds of label atoms (e.g., different elements / isotopes), thereby generating a large number of different tags where a particular combination element / isotope acts as a label. In some cases, the label atom is detected as an elemental ion. In some embodiments, the label atom is released from the sample in a molecular ion. Thus, instead of detection in the mass channel of the label atom, the presence of the label substance in the sample is detected in the mass channel of the molecular ion (i.e., the mass channel is simply shifted by the mass of the molecule minus the label atom, for the label atom only). However, in some embodiments, the molecules that include the label atom can differ between different label atoms. In that case, ions containing the molecular residues and the label atoms are subjected to a fragmentation method that results in a more consistent mass peak for each reagent, such as by application of tandem MS. The goal of all these variations and modifications to tumor LDI imaging mass cytometry schemes is to maximize the number of available mass channels while simultaneously reducing the overlap between the mass channels.

[0277] In some embodiments, the staining reagent may be designed to facilitate the release and ionization of molecular ions that contain a single copy of the mass tag material and the individual elemental ions or tag atoms. The staining reagent may also be designed to facilitate the release and ionization of molecular ions that contain several copies of the mass tag material and the individual elemental ions or tag atoms (or combinations thereof as described above). As a further alternative, the mass of the staining reagent itself may be utilized to create detection channels for mass cytometry. In this case, rare earth isotopes are not used for staining, but the mass of the staining reagent is changed by modifying the chemistry of the staining reagent to create multiple mass channels. This can be done using carbon, oxygen, nitrogen, sulfur, phosphorus, hydrogen, and similar isotopes without the need for rare earth isotopes.

[0278] In some embodiments, the sample is also treated with a laser radiation absorber composition, which acts to enhance the absorption of laser light by the sample when irradiated, thus increasing the transfer of energy to excite the label atoms (and thus promoting the production of elemental or molecular ions containing the label atoms or combinations thereof).

[0279] Numbered embodiments related to LDI 1. An apparatus for analyzing a sample, comprising: a. a sample chamber for containing a sample; b. a laser adapted to desorb and ionize material from the sample to form ions; c. an ion optics element adapted to sample ions formed by desorption ionization and direct them away from the sample towards a detector; and d. a detector for receiving ions from the ion optics element and analyzing the ions.

[0280] 2. The apparatus of embodiment 1, wherein the apparatus comprises a laser adapted to desorb and ionize material from a sample to form elemental ions, and a detector adapted to receive elemental ions from the sampling and ionization system and analyze the elemental ions.

[0281] 3. The apparatus of embodiment 1 or 2, wherein the apparatus comprises a laser adapted to desorb and ionize material from the sample to form elemental ions, and a detector adapted to receive molecular ions from the sampling and ionization system and detect said molecular ions.

[0282] 4. The apparatus of any of embodiments 1 to 3, wherein the apparatus comprises a laser adapted to desorb and ionize material from a sample to form both elemental and molecular ions, and a detector adapted to receive ions from the sampling and ionization system to detect both the elemental and molecular ions.

[0283] 5. The apparatus of any of embodiments 1 to 4, wherein the laser is a deep ultraviolet laser, such as a laser that emits radiation at 193 nm, 213 nm, or 266 nm.

[0284] 6. The apparatus of any of embodiments 1 to 5, wherein the laser is a femtosecond laser.

[0285] 7. The apparatus of any of embodiments 1 to 6, wherein desorption / ionization occurs in the sample chamber under vacuum, partial vacuum, or atmospheric pressure.

[0286] 8. The apparatus of any of embodiments 1 to 7, wherein the sample chamber comprises one or more gas ports arranged to enable delivery of one or more streams of gas to a location of laser desorption ionization on the sample during laser desorption ionization, e.g., the one or more gas ports are in the form of a nozzle.

[0287] 9. The apparatus of embodiment 8, wherein the one or more gas ports are adapted to allow one or more pulses of gas to collisionally cool ions generated from the sample by laser radiation from the laser.

[0288] 10. A method for performing mass cytometry on a sample comprising a plurality of cells, the method comprising: a. labeling one or more distinct target molecules in the sample with one or more mass tags to result in a labeled sample; b. performing laser desorption ionization of the sample, the laser desorption ionization being performed at a plurality of known locations to form a plurality of ion clouds; and c. exposing the ion cloud to mass spectrometry, wherein detection of ions from the one or more mass tags in the cloud enables construction of an image of the sample.

[0289] 11. The method of embodiment 10, wherein the plurality of ion clouds are a plurality of individual ion clouds, each individual ion cloud formed from laser desorption ionization at a known location, and wherein exposing the ion clouds to mass analysis comprises exposing the individual ion clouds to mass analysis.

[0290] 12. The method of embodiment 10 or 11, wherein each different target is bound by a different specific binding pair member (SBP), and each different SBP is linked to a mass tag, thereby labeling each target with a specific mass tag.

[0291] 13. The method of any one of embodiments 10 to 12, further comprising treating the sample with an ionization enhancing agent composition prior to step a or between steps a and b.

[0292] 14. The method of embodiment 13, wherein the ionization promoter composition promotes ionization of the labeling atom and / or molecular ions comprising the labeling atom.

[0293] 15. The method of any one of embodiments 10 to 14, further comprising the step of treating the sample with a laser radiation absorber composition before step a or between steps a and b.

[0294] 2. Mass Detector System Typical types of mass detector systems include quadrupole, time-of-flight (TOF), magnetic sector, high resolution, single or multi-collector based mass spectrometers.

[0295] The time it takes to analyze the ionized material depends on the type of mass analyzer used to detect the ions. For example, instruments that use Faraday cups are generally too slow to analyze high speed signals. Overall, the desired imaging speed, resolution, and degree of multiplexing will determine the type of mass analyzer that should be used (or conversely, the choice of mass analyzer will determine the speed, resolution, and multiplexing that can be achieved).

[0296] Mass spectrometry instruments that detect ions at only one mass-to-charge ratio (m / Q, commonly referred to as m / z in MS) at a time, for example using a point ion detector, provide poor results in imaging detection. Firstly, the time it takes to switch between mass-to-charge ratios limits the rate at which multiple signals can be determined, and secondly, if the ions are of low abundance, signals may be lost when the instrument focuses on other mass-to-charge ratios. It is therefore preferable to use techniques that provide substantially simultaneous detection of ions with different m / Q values.

[0297] Detector Type Quadrupole Detector A quadrupole mass spectrometer comprises four parallel rods with a detector at one end. Alternating RF potentials and a predefined DC offset potential are applied between one pair of rods and the other pair, so that the rods of one pair (each of the opposing rods) have an opposite alternating potential with respect to the rods of the other pair. The ionized sample is passed 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 collide with the rods.

[0298] Magnetic Sector Detector In magnetic sector mass spectrometry, the ionized sample passes through a curved flight tube towards an ion detector. Applying a magnetic field 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 double-focusing magnetic sector instrument that analyzes ions by kinetic energy in addition to mass-to-charge ratio. In particular, multi-detectors with Mattauch-Herzog geometry can be used (e.g., the SPECTRO MS, which can simultaneously record all elements from lithium to uranium in a single measurement using a semiconductor direct charge detector). These instruments can measure multiple m / Q signals virtually simultaneously. Their sensitivity can be increased by including electron multipliers in the detectors. Array sector instruments are always applicable but are less suitable in situations where labels are present, especially in highly variable concentrations, since although they are useful for detecting increasing signals, they are less useful when the signal level is decreasing.

[0299] 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, the ionized sample molecules each have the same kinetic energy, but as they accelerate through the acceleration chamber, they are separated by their mass, where lighter ionized sample molecules move faster than heavier ions. The detector then 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.

[0300] Thus, TOF detectors can register multiple masses in a single sample nearly simultaneously. In theory, TOF techniques are not ideally suited for ICP ion sources due to their space charge characteristics, but TOF instruments can in fact analyze ICP ion aerosols quickly and sensitively enough to allow viable single cell imaging. Although TOF mass analyzers are not typically popular for atomic analysis due to the compromises required to deal with space charge effects in the TOF accelerator and flight tube, tissue imaging according to the present disclosure can be enabled by detecting only labeled atoms, thus filtering out other atoms (e.g., atoms with atomic weights less than 100). This results in a less dense ion beam, concentrating masses in the 100-250 Dalton region (e.g.) that can be more efficiently manipulated and focused, thereby facilitating TOF detection and taking advantage of the high spectral scan rates of TOF. Rapid imaging can therefore be achieved by a combination of TOF detection and the selection of labeled atoms with masses above those found in unlabeled samples, ideally by using higher mass transition elements that are not common in the sample. Therefore, using a narrower window of label masses means that TOF detection can be used for efficient imaging.

[0301] Suitable TOF instruments are available from Tofwerk, GBC Scientific Equipment (e.g., the Optimass 9500 ICP-TOFMS), and Fluidigm Canada (e.g., the CyTOF™ and CyTOF™ 2 instruments). These CyTOF™ instruments are more sensitive than the Tofwerk and GBC instruments, and are known for use in mass cytometry due to their rapid and sensitive detection of ions in the rare earth metal mass range (particularly the m / Q range of 100-200; see Bandura et al. (2009; Anal. Chem., 81:6813-22)). Therefore, they are preferred instruments for use in the present disclosure and can be used in imaging with instrumental settings already known in the art, for example, Bendall et al. (2011; Science 332, 687-696) and Bodenmiller et al. (2012; Nat. Biotechnol. 30:858-867). Their mass spectrometers can detect multiple markers quasi-simultaneously at high mass spectrum acquisition frequencies on the time scale of high frequency laser ablation or sample desorption. Their mass spectrometers can measure the abundance of labeled atoms with a detection limit of about 100 per cell, thereby allowing for sensitive construction of images of tissue samples. Due to these features, mass cytometry can be used to meet the need for sensitivity and multiplexing in tissue imaging with subcellular resolution. Combining mass cytometry instruments with high resolution laser ablation sampling systems and fast transit low dispersion sample chambers could allow for construction of highly multiplexed images of tissue samples on practical time scales.

[0302] The TOF may be coupled with a mass assignment corrector. The majority of ionization events are M +ions, in this case a single electron is knocked out of the atom. Due to the mode of operation of the TOF MS, sometimes there is some bleeding (or crosstalk) of ions of one mass (M) into channels for adjacent masses (M±1), especially when many ions of mass M are entering the detector (i.e., the ion count is high but not so high that an ion deflector located between the sampling and ionization system and the MS will prevent the ions from entering the MS, if the instrument is to include an ion deflector). + The arrival times of ions follow a probability distribution about the mean (known for each M), so that the mass M + When the number of ions is large, it is usually M-1 + Or M+1 + Some arrive at a time associated with the ion. However, because each ion has a known distribution curve as it enters the TOF MS, the overlap of an ion of mass M into the M±1 channel can be determined based on the peak in the mass M channel (compared to the known peak shape). This calculation is particularly applicable to TOF MS because the peaks of 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 detected ions to the M channel. Such corrections have particular application in correcting imaging data due to the nature of the large packets of ions generated by sampling and ionization systems such as those disclosed herein that involve laser ablation (or desorption, as described below) as a technique for removing material from the sample. Programs and methods for improving data quality by deconvoluting data from TOF MS are discussed in WO 2011 / 098834, U.S. Pat. No. 8,723,108, and WO 2014 / 091243.

[0303] Dead Time Corrector As mentioned above, the signal in the MS is detected based on collisions between ions and a detector and the release of electrons from the detector surface that the ions strike. When a large number of ions are detected by the MS and a large number of electrons are released, the detector of the MS temporarily fatigues, which results in the analog signal output from the detector being temporarily suppressed for one or more of the subsequent packets of ions. In other words, if the number of ions in a packet of ionized sample material is particularly large, the process of detecting ions from the packet of ionized sample material will release many electrons from the detector surface and secondary multiplier, so that when ions in a subsequent packet of ionized sample material strike the detector, fewer electrons are available to be released until the electrons in the detector surface and secondary multiplier are replenished.

[0304] Based on the characteristics of the detector behavior, this dead time phenomenon can be compensated for. The first step is to analyze the ion peaks of the analog signal resulting from the detection of the nth packet of ionized sample material by the detector. The magnitude of the peak may be determined by the peak height, the peak area, or a combination of the peak height and the peak area.

[0305] The magnitude of the peak is then compared to see if it exceeds a predetermined threshold. If the magnitude is below this threshold, no correction is necessary. If the magnitude exceeds the threshold, correction of the digital signal from at least one subsequent packet of ionized sample material is performed (at least the (n+1)th packet of ionized sample material, but possibly further packets of ionized sample material such as the (n+2)th, (n+3)th, (n+4)th, etc.) to compensate for the temporary degradation of the analog signal from these packets of ionized sample material due to detector fatigue caused by the nth packet of ionized sample material. The larger the magnitude of the peak in the nth packet of ionized sample material, the more peaks from subsequent packets of ionized sample material need to be corrected, and the larger the magnitude of the correction needs to be. Methods for correcting for such phenomena are discussed in Stephan et al. (1994; Vac. Sci. Technol. 12:405), Tyler and Peterson (2013; Surf Interface Anal. 45:475-478), Tyler (2014; Surf Interface Anal. 46:581-590), WO 2006 / 090138, and U.S. Pat. No. 6,229,142, and these methods may be applied to data by a dead-time corrector as described herein.

[0306] Analyser device based on optical emission spectrum detection 1. Sampling and Ionization System a. Laser ablation-based sampling and ionization system The laser ablation sampling system previously described in connection with mass-based analyzers may be used with OES detector-based systems. For detection of atomic emission spectra, an ICP is used to ionize the sample material removed from the sample, although any hard ionization technique capable of producing elemental ions may be used.

[0307] As will be appreciated by those of skill in the art, certain optional additional components of the laser ablation-based sampling and ionization system described above with respect to avoiding overload of the mass-based detector may not be applicable to all OES detector-based systems, and may not be incorporated by those of skill in the art if inappropriate.

[0308] b. Desorption-based Sampling and Ionization System The desorption-based sampling systems described above in connection with mass-based analyzers may be used with OES detector-based systems. For detection of atomic emission spectra, ICP is used to ionize sample material removed from the sample, although any hard ionization technique capable of producing elemental ions may be used.

[0309] As will be appreciated by those of skill in the art, certain optional additional components of the desorption-based sampling and ionization system described above with respect to avoiding overload of the mass-based detector may not be applicable to all OES detector-based systems, and may not be incorporated by those of skill in the art if inappropriate.

[0310] 2. Photodetector Typical types of photodetectors include photomultiplier tubes and charge-coupled devices (CCDs). Photodetectors may be used to image samples and / or identify features of interest / regions of interest prior to imaging by elemental mass spectrometry.

[0311] A photomultiplier tube comprises a vacuum chamber containing a photocathode, several dynodes, and an anode. Photons incident on the photocathode cause the photocathode to emit electrons as a result of the photoelectric effect. The electrons are multiplied by the dynodes due to a process of secondary emission to produce a multiplied electron current, which is then detected by the anode to provide an indication of the detection of electromagnetic radiation incident on the photocathode. Photomultiplier tubes are available, for example, from ThorLabs.

[0312] A CCD comprises a silicon chip containing an array of light-sensitive pixels. During exposure, each pixel generates an electric charge proportional to the intensity of light incident on the pixel. After exposure, control circuitry causes a series of charge movements to generate a series of voltages. These voltages can then be analyzed to generate an image. Suitable CCDs are available, for example, from Cell Biosciences.

[0313] Image construction The above described device can provide a signal to multiple atoms in a packet of ionized sample material removed from the sample. Detection of the atoms in the packet of sample material reveals their presence at the location of the ablation, either because the atoms are naturally present in the sample or because the atoms have been localized to that location by a labeling reagent. By generating a series of packets of ionized sample material from known spatial locations on the surface of the sample, the detector signal reveals the location of the atoms on the sample, so that the signal can be used to construct an image of the sample. By labeling multiple targets with identifiable labels, the location of the labeled atoms can be related to the location of cognate targets, allowing the method to create complex images and reach levels of multiplexing far beyond those achievable using traditional techniques such as fluorescence microscopy.

[0314] Assembling the signals into an image can be accomplished using a computer using known techniques and software packages. For example, the GRAPHIS package provided by Kylebank Software can be used, or other packages such as TERAPLOT can be used. Imaging using MS data from techniques such as MALDI-MSI is known in the art, for example, Robichaud et al. (2013; J Am Soc Mass Spectrom 24 5:718-21) discloses the "MSiReader" interface for viewing and analyzing MS imaging files on a Matlab platform, and Klinkert et al. (2014; Int J Mass Spectrom http: / / dx.doi.org / 10.1016 / j.ijms.2013.12.012) discloses two software tools, 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.

[0315] Images obtained using the methods disclosed herein can be further analyzed, for example, in the same way that IHC results are analyzed. For example, images can be used to delineate cell subpopulations within a sample and can provide information useful for clinical diagnosis. Similarly, SPADE analysis can be used to extract cellular hierarchies from the high-dimensional cytometry data provided by the methods of the present disclosure (Qiu et al. (2011; Nat. Biotechnol. 29:886-91)).

[0316] sample Certain aspects of the present disclosure provide a method for imaging a biological sample. Such a sample can include a plurality of cells that can be subjected to imaging mass cytometry (IMC) to provide an image of these cells in the sample. In general, the present invention can be used to analyze tissue samples that are currently studied by immunohistochemistry (IHC) techniques, but involves the use of labeling atoms suitable for detection by mass spectrometry (MS) or optical emission spectrometry (OES).

[0317] Any suitable tissue sample can be used in the methods described herein. For example, the tissue can 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 can be an immortalized cell line or primary cells obtained from a living subject. The tissue can be from a tumor for diagnostic, prognostic, or experimental (e.g., drug development) purposes. In some embodiments, the sample can be from a known tissue, but it may be unknown whether the sample contains tumor cells. Imaging can reveal the presence of targets indicative of the presence of a tumor, thus facilitating diagnosis. Tissue from a tumor can include immune cells that are also characterized by the subject method, providing insight into tumor biology. The tissue sample can include formalin-fixed paraffin-embedded (FFPE) tissue. Tissues can be obtained from any living multicellular organism, such as a mammal, an animal research model (e.g., an animal research model of a particular disease, such as an immunodeficient rodent with a human tumor xenograft), or a human patient.

[0318] The tissue samples may be sections, for example, with a thickness in the range of 2-10 μm, for example 4-6 μm. Techniques for preparing such sections are well known from the field of IHC, such as using a microtome, including dehydration steps, fixation, embedding, permeabilization, sectioning, etc. Thus, the tissue can be chemically fixed, and then sections can be prepared in the desired plane. Frozen sectioning or laser capture microdissection can also be used to prepare tissue samples. Samples may be permeabilized to allow the uptake of reagents for labeling of intracellular targets (see above).

[0319] 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).

[0320] In addition to being useful for imaging tissue samples, the present disclosure can alternatively be used for imaging cell samples, such as monolayers of adherent cells or cells immobilized on a solid surface (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 enhancing current immunohistochemical analyses, but can also be used to enhance immunocytochemistry.

[0321] Sample Carrier In certain embodiments, the sample may be immobilized on a solid support (i.e., a sample carrier) to position the sample for imaging mass spectrometry. The solid support may be optically transparent and formed, for example, from glass or plastic. If the sample carrier is optically transparent, ablation of the sample material through the support is possible. Sometimes, the sample carrier includes features that act as fiducials for use with the devices and methods described herein, for example, to allow ablation or detachment and calculation of the relative position of the feature / region of interest of the object to be analyzed. The fiducials may be optically resolvable or may be resolvable by mass spectrometry.

[0322] Target element In imaging mass spectrometry, the distribution of one or more target elements (i.e., elements or elemental isotopes) can be important. In certain aspects, the target element is a label atom as described herein. The label atom may be added directly to the sample alone or may be covalently attached to or within a biologically active molecule. In certain embodiments, the label atom (e.g., a metal tag) may be conjugated to a member of a specific binding pair (SBP), such as an antibody (which binds to its cognate antigen), an aptamer, or an oligonucleotide for hybridizing to a DNA or RNA target, as described in more detail below. The label atom may be attached to the SBP by any method known in the art. In certain aspects, the label 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 may deplete elemental ions below the mass of metallic elements, so that abundant lighter elements do not create space charge effects and / or overwhelm the mass detector.

[0323] 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, preferably intracellular, region of the sample (hence the labeled atoms represent elemental tags). Reference to multiple different atoms means that multiple atomic species are used to label the sample. A mass detector can be used to distinguish these atomic species (e.g., the atomic species have different m / Q ratios), so that the presence of two different labeled atoms in the plume will generate two different MS signals. An optical spectrometer can also be used to distinguish the atomic species (e.g., different atoms have different emission spectra), so that the presence of two different labeled atoms in the plume will generate two different emission spectrum signals.

[0324] Mass tagged reagents The mass tagged reagent used herein comprises several components. The first component is an SBP. The second component 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 link between the SBP and the mass tag may also comprise a spacer. The mass tag and the SBP may be conjugated by various reaction chemistries. Exemplary conjugation reaction chemistries include thiol maleimide, NHS ester and amine, or click chemistry reactive (preferably Cu(I)-free chemistry), such as strained alkynes and azides, strained alkynes and nitrones, and strained alkenes and tetrazines.

[0325] Mass Tag Mass tags used in the present invention can take several forms. Generally, tags comprise at least one labeling atom, which is described below.

[0326] Thus, in its simplest form, a mass tag may comprise a metal chelating moiety, which is a metal chelating group with a metal label atom coordinated in a ligand. In some cases, it may be sufficient to detect only one metal atom per mass tag. However, in other instances, it may be desirable for each mass tag to contain multiple label atoms. This can be achieved in several ways, as described below.

[0327] A first means for generating mass tags that can include multiple label atoms is the use of polymers that include metal chelating ligands attached to multiple subunits of the polymer. The number of metal chelating groups capable of binding at least one metal atom in the polymer can be about 1 to 10,000, such as 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500, or 500 to 1,000. At least one metal atom can be bound to at least one of the metal chelating groups. The polymer can have a degree of polymerization of about 1 to 10,000, such as 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500, or 500 to 1,000. Thus, polymer-based mass tags may contain from about 1 to 10,000 label atoms, such as from 5 to 100, 10 to 250, 250 to 5,000, 500 to 2,500, or 500 to 1,000.

[0328] The polymer may be selected from the group consisting of linear polymers, copolymers, branched polymers, graft copolymers, block polymers, star polymers, and hyperbranched polymers. The polymer backbone may be derived from substituted polyacrylamides, polymethacrylates, or polymethacrylamides, and may be substituted derivatives of acrylamides, methacrylamides, acrylate esters, methacrylate esters, homopolymers or copolymers of acrylic or methacrylic acid. The polymer may be synthesized from the group consisting of reversible addition fragmentation polymerization (RAFT), atom transfer radical polymerization (ATRP), and anionic polymerization. The step of providing the polymer may include synthesis of the polymer from a compound selected from the group consisting of N-alkylacrylamides, N,N-dialkylacrylamides, N-arylacrylamides, N-alkylmethacrylamides, N,N-dialkylmethacrylamides, arylmethacrylamides, methacrylate esters, acrylate esters, and functional equivalents thereof.

[0329] The polymer can be water-soluble. This part is not limited by the chemical moiety. However, if the size of the skeleton is relatively reproducible (e.g., length, number of tag atoms, reproducible dendrimer properties, etc.), the analysis is simplified. The requirements of stability, solubility, and non-toxicity are also considered. Therefore, the preparation and characterization of functional water-soluble polymers by a synthetic strategy that places different reactive groups (linking groups) (e.g., SBPs) that can be used to connect the polymer to molecules via linkers and optionally spacers, in addition to many functional groups along the backbone, is considered. The size of the polymer can be controlled by controlling the polymerization reaction. In general, the size of the polymer is selected so that the radius of gyration of the polymer is as small as possible, such as 2 to 11 nanometers. The length of a typical SBP, an IgG antibody, is about 10 nanometers, and therefore a polymer tag that is too large relative to the size of the SBP may sterically hinder the binding of the SBP to its target.

[0330] The metal chelating group capable of binding at least one metal atom can include at least four acetate groups. For example, the metal chelating group can be a diethylenetriaminepentaacetate (DTPA) group or a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group. Another group includes ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).

[0331] The metal chelating groups may be attached to the polymer via an ester or amide. Examples of suitable metal chelating polymers include X8 polymer and DM3 polymer available from Fluidigm Canada, Inc.

[0332] The polymers can be water soluble. Because of their hydrolytic stability, N-alkylacrylamides, N-alkylmethacrylamides, and methacrylate esters or functional equivalents can be used. A degree of polymerization (DP) of about 1-1000 (1-2000 backbone atoms) encompasses most of the polymers of interest. Larger polymers are within the scope of the invention with the same functionality and can be envisioned as would be understood by one of ordinary skill in the art. Generally, the degree of polymerization is 1-10,000, e.g., 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000. The polymers may be amenable to synthesis by routes that result in relatively narrow polydispersity. The polymers can be synthesized by atom transfer radical polymerization (ATRP) or reversible addition fragmentation (RAFT) polymerization, which should result in values ​​of Mw (weight average molecular weight) / Mn (number average molecular weight) in the range of 1.1-1.2. An alternative strategy involves anionic polymerization, which gives polymers with Mw / Mn of about 1.02-1.05. In either method, the end groups can be controlled by the choice of initiator or terminator. This allows the synthesis of polymers to which linkers can be attached. A strategy can be employed to prepare polymers containing functional pendant groups on the repeat units to which liganded transition metal units (e.g., Ln units) can be attached in a later step. This embodiment has several advantages. It avoids the complications that can arise from carrying out the polymerization of ligand-containing monomers.

[0333] To minimize charge repulsion between the pendant groups, (M 3+ ) the targeting ligand must impart a net charge of -1 to the chelate.

[0334] Polymers that may be used in the present invention include:

[0335] -Random copolymer poly(DMA-co-NAS): The synthesis of a 75 / 25 molar ratio random copolymer of N-acryloxysuccinimide (NAS) and N,N-dimethylacrylamide (DMA) by RAFT with Mw / Mn of about 1.1 and excellent molar mass control in the range of 5000-130,000 with high conversion is reported in Relogio et al. (2004) (Polymer, 45, 8639-49). The active NHS ester reacts with a metal chelating group bearing a reactive amino group to produce a metal chelating copolymer synthesized by RAFT polymerization.

[0336] - Poly(NMAS): NMAS can be polymerized by ATRP to give polymers with average molecular weights ranging from 12 to 40 KDa and Mw / Mn of approximately 1.1 (see, for example, Godwin et al., 2001; Angew. Chem. Int. Ed, 40:594-97).

[0337] - Poly(MAA): Polymethacrylic acid (PMAA) can be prepared by anionic polymerization of its t-butyl or trimethylsilyl (TMS) esters.

[0338] - Poly(DMAEMA): Poly(dimethylaminoethyl methacrylate) (PDMAEMA) can be prepared by ATRP (see Wang et al, 2004, J. Am. Chem. Soc, 126, 7784-85). It is a well-known polymer and is conveniently prepared with average Mn values ​​ranging from 2 to 35 KDa, with Mw / Mn of about 1.2. This polymer can also be synthesized by anionic polymerization with a narrower size distribution.

[0339] Polyacrylamide or polymethacrylamide.

[0340] The metal chelating groups can be attached to the polymer by methods known to those skilled in the art, for example, the pendant groups may be attached via esters or amides. For example, for methyl acrylate-based polymers, the metal chelating groups can be attached to the polymer backbone by first reacting the polymer with ethylenediamine in methanol, followed by reaction with DTPA anhydride under alkaline conditions in carbonate buffer.

[0341] The second avenue is to generate nanoparticles that can function as mass tags. The first route to generate such mass tags is the use of nanoscale particles of metals coated with polymers. Here, the metal is sequestered and protected from the environment by the polymer and does not react if the polymer shell can be reacted, for example by functional groups incorporated in the polymer shell. This type of mass tag can be plugged in a simple modular way into the synthetic strategies described above, since the functional groups can be reacted with linker components (optionally incorporating spacers) to attach click chemistry reagents.

[0342] Grafting-to and grafting-from are the two main mechanisms for generating polymer brushes around nanoparticles. In grafting-to, the polymer is synthesized separately, so the synthesis is not constrained by the need to keep the nanoparticles colloidally stable. Here, reversible addition-fragmentation chain transfer (RAFT) synthesis excels due to the wide variety of monomers and easy functionalization. Chain transfer agents (CTAs) can be easily used as functional groups themselves, functionalized CTAs can be used, or the polymer chains can be post-functionalized. Chemical reactions or physical adsorption are used to attach the polymer to the nanoparticles. One of the disadvantages of grafting-to is that the grafting density is usually low due to the steric repulsion of the coiled polymer chains during attachment to the particle surface. All grafting-to methods have the disadvantage that rigorous workup is required to remove excess free ligand from the functionalized nanocomposite particles. This is generally achieved by selective precipitation and centrifugation. In the grafting-from approach, molecules such as initiators for atom transfer radical polymerization (ATRP) or CTAs for (RAFT) polymerization are immobilized on the particle surface. A drawback of this method is the development of new initiator coupling reactions. Moreover, in contrast to grafting-to, the particles must be colloidally stable under the polymerization conditions.

[0343] A further means of generating mass tags is through the use of doped beads. Chelated lanthanide (or other metal) ions can be used in miniemulsion polymerization to create polymer particles with chelated lanthanide ions embedded in the polymer. The chelating group is selected so that the metal chelate has negligible solubility in water but reasonable solubility in the monomer for miniemulsion polymerization, as known to those skilled in the art. Typical monomers that can be used are styrene, methylstyrene, various acrylates and methacrylates, among others, as known to those skilled in the art. For mechanical robustness, the glass transition temperature (Tg) of the metal-tagged particles is higher than room temperature. In some cases, core-shell particles are used, in which case metal-containing particles prepared by miniemulsion polymerization are used as seed particles for seeded emulsion polymerization to control the nature of the surface functionality. Surface functionality can be introduced by selecting the appropriate monomer for this second stage polymerization. Furthermore, acrylate (and possibly methacrylate) polymers are advantageous over polystyrene particles because ester groups can bind or stabilize unfilled ligand sites on lanthanide complexes. A typical method for forming such doped beads is: (a) combining at least one labeled atom-containing complex in a solvent mixture containing at least one organic monomer (such as styrene and / or methyl methacrylate in one embodiment) in which the at least one labeled atom-containing complex is soluble, with at least one different solvent in which the organic monomer and the at least one labeled atom-containing complex are poorly soluble; (b) emulsifying the mixture of step (a) for a sufficient time to result in a uniform emulsion; (c) initiating polymerization and continuing the reaction until a significant portion of the monomer is converted to polymer; (d) incubating the product of step (c) for a sufficient time to obtain a latex suspension of polymer particles in which at least one labeled atom-containing complex is incorporated in or on the particle, the at least one atom-containing complex being selected such that upon interrogation of the polymer mass tag, a distinct mass signal is obtained from the at least one labeled atom.By using two or more complexes containing different labeled atoms, doped beads containing two or more different labeled atoms can be made. Furthermore, controlling the ratio of complexes containing different labeled atoms allows the production of doped beads with different ratios of labeled atoms. By using multiple labeled atoms, and also with different radios, the number of unambiguously identifiable mass tags is increased. In core-shell beads, this may be achieved by incorporating a first labeled atom-containing complex in the core and a second labeled atom-containing complex in the shell.

[0344] Yet another approach is the generation of polymers that contain labeling atoms in the polymer backbone rather than as coordinated metal ligands. For example, Carerra and Seferos (Macromolecules 2015, 48, 297-308) disclose polymers containing tellurium in the backbone. Other polymers that incorporate atoms that can function as labeling atoms include those that incorporate tin, antimony, and bismuth. Such molecules are discussed, among others, in Priegert et al., 2016 (Chem. Soc. Rev., 45, 922-953).

[0345] Thus, a mass tag can comprise at least two components: a label atom and a polymer that chelates, contains, or is doped with the label atom. Additionally, the mass tag includes an attachment group (if not conjugated to an SBP) that forms part of a chemical bond between the mass tag and the SBP following reaction of the two components in a click chemistry reaction consistent with the discussion above.

[0346] Polydopamine coating can be used as an additional method to attach SBPs, for example, to doped beads or nanoparticles. Given the range of functions of polydopamine, SBPs can be conjugated to mass tags formed from PDA-coated beads or particles by reaction of amine or sulfhydryl groups on the SBP, such as antibodies. Alternatively, the PDA functionality can be reacted with reagents such as bifunctional linkers that introduce further functionality for reaction with SBPs. In some cases, the linkers can include spacers, as described below. These spacers increase the distance between the mass tag and the SBP, minimizing steric hindrance of the SBP. Thus, the present invention includes mass-tagged SBPs, including SBPs, and mass tags, including polydopamine, where the polydopamine comprises at least part of the link between the SBP and the mass tag. Nanoparticles and beads, particularly polydopamine-coated nanoparticles and beads, may be useful for signal enhancement for detecting low abundance targets, as they can have thousands of metal atoms and multiple copies of the same affinity reagent. The affinity reagent can be a secondary antibody that can further enhance the signal.

[0347] labeled atom Labeled atoms that can be used in the present disclosure include any species that is detectable by MS or OES and that is substantially absent in an unlabeled tissue sample. Thus, for example, 12 C atoms are not suitable as label atoms because they are abundant in nature. 11C is an artificial isotope that does not occur naturally, so it can theoretically be used in MS. In many cases, the labeled atom is a metal. However, in a preferred embodiment, the labeled atom is a transition metal, such as a rare earth metal (the 15 lanthanides plus scandium and yttrium). These 17 elements (distinguishable by OES and MS) provide many different isotopes that can be easily distinguished (by MS). These various elements are available in enriched isotopic form, for example, samarium has 6 stable isotopes and neodymium has 7 stable isotopes, all of which are available in enriched form. The 15 lanthanide elements provide at least 37 isotopes with non-redundantly unique masses. Examples of elements suitable for use as labeling atoms include lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc), and yttrium (Y). In addition to rare earth metals, other metal atoms such as gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), and bismuth (Bi) are suitable for detection. The use of radioisotopes is not preferred because they are inconvenient to handle and unstable, for example, Pm is not a preferred labeling atom among the lanthanides.

[0348] 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 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 can be used to provide rapid imaging at biologically significant levels.

[0349] 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 can be attached to a single SBP element. The more label atoms attached to the SBP element, the greater the sensitivity can be. For example, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more than 100 label atoms can be attached to the SBP element, such as up to 10,000, e.g., 5-100, 10-250, 250-5,000, 500-2,500, or 500-1,000 label atoms. As mentioned above, monodisperse polymers can be used that contain multiple monomer units, each containing a chelating agent such as diethylenetriaminepentaacetic acid (DTPA) or DOTA. For example, DTPA chelates 3+ lanthanide ions at approximately 10 -6The polymers bind with a dissociation constant of M. These polymers can be terminated with thiols that can be used to attach to the SBPs by reaction with maleimides to attach click chemistry reactivity along the lines of the previous description. Other functional groups, such as amine-reactive groups such as N-hydroxysuccinimide esters, or groups reactive to carboxyl groups or to glycosylation of antibodies, can also be used for conjugation of these polymers. Any number of polymers may 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™ drugs. The use of metal nanoparticles can also be used to increase the number of atoms in the label, as previously described.

[0350] In some embodiments, all the labeled atoms in a mass tag have the same atomic mass. Alternatively, the mass tags can contain labeled atoms of different atomic masses. Thus, in some cases, a labeled sample may be labeled with a series of mass-tagged SBPs, each containing only a single type of labeled atom (each SBP binds to its cognate target, so that each type of mass tag is localized to a specific, e.g., antigen, on the sample). Alternatively, in some cases, a labeled sample may be labeled with a series of mass-tagged SBPs, each containing a mixture of labeled atoms. In some cases, the mass-tagged SBPs used to label a sample may contain a mixture of labeled atoms and mixtures of labeled atoms with a single labeled atomic mass tag in their mass tags.

[0351] Spacer As mentioned above, in some cases, the SBP is conjugated to the mass tag via a linker that includes a spacer. There may be a spacer between the SBP and the click chemistry reagent (e.g., between the SBP and the strained cycloalkyne (or azide); the strained cycloalkene (or tetrazine), etc.). There may be a spacer between the mass tag and the click chemistry reagent (e.g., between the mass tag and the azide (or strained cycloalkyne); the tetrazine (or strained cycloalkene), etc.). In some cases, there may be a spacer both between the SNP and the click chemistry reagent and between the click chemistry reagent and the mass tag.

[0352] The spacer may 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 (such as POZ, polymethyloxazoline, polyethyloxazoline, or polypropyleneoxazoline) or a C5-C20 acyclic alkyl spacer. For example, the spacer may be a PEG spacer with 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 may have 3-12 EG units, 4-10 EG units, or may have 4, 5, 6, 7, 8, 9, or 10 EG units. The linker may include cystamine or a derivative thereof, may include one or more disulfide groups, or may be any other suitable linker known to those of skill in the art.

[0353] A spacer can be advantageous to minimize steric effects of the mass tag on the conjugated SBP. Hydrophilic spacers, such as PEG-based spacers, can also act to improve the solubility of the mass-tagged SBP as well as prevent aggregation.

[0354] SBP (specific binding pair element) Mass cytometry, including imaging mass cytometry, is based on the principle of specific binding between members of a specific binding pair. A mass tag is linked to a specific binding pair member, which allows the mass tag to localize to the target / analyte, which is the other member of the pair. However, specific binding does not require binding to only one molecular species, excluding other molecular species. Rather, it specifies 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 a common epitope among many 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 may be naturally occurring, or it may be an artificial tag, such as a FLAG tag. Similarly, in the case of nucleic acids, a nucleic acid of a given sequence may not bind exclusively to a completely complementary sequence, but as one skilled in the art will appreciate, various tolerances of mismatches can be introduced under the use of hybridization conditions of different stringency. Nevertheless, this hybridization is not non-specific since it is mediated by homology between the SBP nucleic acid and the target analyte.Similarly, a ligand can specifically bind to multiple receptors; a simple example is TNFα, which binds to both TNFR1 and TNFR2.

[0355] SBPs may include any of the following: an antibody / antigen complex; a receptor / ligand pair; or an aptamer / target pair. Thus, a labeled atom can be attached to a nucleic acid probe, which is then contacted with a tissue sample so that the probe can hybridize to a complementary nucleic acid therein to form a DNA / DNA duplex, a DNA / RNA duplex, or an RNA / RNA duplex. Similarly, a labeled atom can be attached to an antibody, which is then contacted with a tissue sample so that it can bind to its antigen. A labeled atom can be attached to a ligand, which is then contacted with a tissue sample so that it can bind to its receptor. A labeled atom can be attached to an aptamer ligand, which is then contacted with a tissue sample so that it can bind to its target. Thus, labeled SBP elements can be used to detect a variety of targets in samples, including DNA sequences, RNA sequences, proteins, sugars, lipids, and metabolites.

[0356] Thus, the mass-tagged SBP can be a protein or peptide, or a polynucleotide or oligonucleotide.

[0357] 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.

[0358] The peptide may be a linear peptide or a cyclic peptide, such as a bicyclic peptide. One example of a peptide that can be used is phalloidin.

[0359] Polynucleotide or oligonucleotide generally refers to a single- or double-stranded polymer of nucleotides comprising 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 naturally occurring polynucleotides, and, optionally, modified sugar moieties or moieties other than ribose or deoxyribose. Polynucleotide analogs contain bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, and the analog backbone has 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 heterologous nucleic acids (XNA), bridged nucleic acids (BNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), yPNAs, morpholino polynucleotides, locked nucleic acids (LNA). Polynucleotide analogs include, but are not limited to, threos nucleic acids (TNAs), 2'-0-methyl polynucleotides, 2'-0-alkylribosyl substituted polynucleotides, phosphorothioate polynucleotides, and boronophosphate polynucleotides. Polynucleotide analogs may 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.

[0360] Antibody SBP element In a typical embodiment, the labeled SBP element is an antibody. Labeling of the antibody can be achieved through the conjugation of one or more label atoms that bind the molecule to the antibody, for example, by attachment of a mass tag using NHS-amine chemistry, sulfhydryl-maleimide chemistry, or click chemistry (strained alkynes and azides, strained alkynes and nitrones, strained alkenes and tetrazines, etc.). Antibodies that recognize cellular proteins useful for imaging are already widely available for use in IHC, and these known antibodies can be easily adapted for use in the methods disclosed herein by using label atoms instead of current labeling techniques (e.g., fluorescence), but with the advantage of improving multiplexing capabilities. The antibody can recognize targets on the cell surface or targets within the cell. Antibodies can recognize a variety of targets, for example, antibodies can specifically recognize individual proteins, or they can recognize multiple related proteins that share a common epitope, or they can recognize specific post-translational modifications of proteins (e.g., to distinguish between tyrosine and phosphotyrosine, to distinguish between lysine and acetyl-lysine, to detect ubiquitination, etc.) of a protein of interest. After binding to its target, a label atom conjugated to the antibody can be detected to reveal the location of the target in a sample.

[0361] The labeled SBP element usually interacts directly with the target SBP element in the sample. However, in some embodiments, the labeled SBP element can interact indirectly with the target SBP element, for example, a primary antibody can bind to the target SBP element and a labeled secondary antibody can then bind to the primary antibody in a sandwich assay. However, usually, the method relies on direct interaction, as this is easier to achieve and allows a higher degree of multiplexing. However, in either case, the sample is contacted with an SBP element that can bind to the target SBP element in the sample, and in a subsequent step, the label attached to the target SBP element is detected.

[0362] Nucleic acid SBP and changes in labeling methods RNA is another biological molecule that the methods and devices disclosed herein can detect in a particular sensitive and, if necessary, quantitative manner. In the same manner as described above for the analysis of proteins, RNA can be detected by using SBP elements labeled with elemental tags that specifically bind to RNA (e.g., polynucleotides or oligonucleotides of complementary sequence as described above, including locked nucleic acid (LNA) molecules of complementary sequence, peptide nucleic acid (PNA) molecules of complementary sequence, plasmid DNA of complementary sequence, amplified DNA of complementary sequence, fragments of RNA of complementary sequence, and fragments of genomic DNA of complementary sequence). RNA includes not only mature mRNA, but also RNA processing intermediates and nascent pre-mRNA transcripts.

[0363] In certain embodiments, both RNA and protein are detected using the methods of the claimed invention.

[0364] 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 embodiments, cells are fixed and permeabilized prior to the hybridization step. Cells are provided to be fixed and / or permeabilized. Cells may be fixed by cross-linking fixatives such as formaldehyde, glutaraldehyde, etc. Alternatively or additionally, cells may be fixed using precipitating fixatives 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 (a group of amphiphilic glycosides), or chemicals such as methanol or acetone. In some cases, fixation and permeabilization may be performed using the same reagent or set of reagents. Fixation and permeabilization techniques are discussed by Jamur et al. in "Permeabilization of Cell Membranes" (Methods Mol.Biol., 2010).

[0365] Detection of target nucleic acids in cells or "in-situ hybridization" (ISH) has previously been performed using fluorophore-tagged oligonucleotide probes. As discussed herein, mass-tagged oligonucleotides combined with ionization and mass spectrometry can be used to detect target nucleic acids in cells. Methods for in-situ hybridization are known in the art (see Zenobi et al., "Single-Cell Metabolomics: Analytical and Biological Perspectives," Science 342, no. 6163, 2013). Hybridization protocols are also described in U.S. Pat. No. 5,225,326 and U.S. Pat. Pub. Nos. 2010 / 0092972 and 2013 / 0164750, which are incorporated herein by reference.

[0366] Prior to hybridization, cells in suspension or immobilized on a solid support may be fixed and permeabilized as described above. Permeabilization may allow cells to retain the target nucleic acid while allowing target hybridization oligonucleotides, amplification oligonucleotides, and / or mass-tagged oligonucleotides to enter the cells. Cells may be washed after any hybridization step, for example, after hybridization of the target hybridization oligonucleotide to the nucleic acid target, after hybridization of the amplification oligonucleotide, and / or after hybridization of the mass-tagged oligonucleotide.

[0367] The cells may be in suspension throughout all or most steps of the method for ease of handling. However, the method is also applicable to cells in solid tissue samples (e.g., tissue sections) and / or cells immobilized on a solid support (e.g., a slide or other surface). Thus, in some cases, the cells may be suspended in the sample and during the hybridization step. In other cases, the cells may be immobilized on a solid support during hybridization.

[0368] Target nucleic acids include any nucleic acid of interest that is sufficiently present in a cell to be detected by the method. Target nucleic acids may 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 1000 or more copies of the target RNA may be present in a cell. Target RNAs may be messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (IncRNAs), or any other type of RNA known in the art. Target RNAs may 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, 1000 nucleotides or more, 20-1000 nucleotides, 20-500 nucleotides, 40-200 nucleotides, etc.

[0369] In certain embodiments, mass-tagged oligonucleotides may be directly hybridized to the target nucleic acid sequence, however, hybridization of an additional oligonucleotide may allow for improved specificity and / or signal amplification.

[0370] In certain embodiments, two or more target hybridization oligonucleotides may hybridize to proximal regions on the target nucleic acid and may combine together to provide sites for hybridization of additional oligonucleotides in a hybridization scheme.

[0371] In certain embodiments, the 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 to provide multiple hybridization sites to which the mass-tagged oligonucleotides can bind. One or more amplification oligonucleotides with or without mass-tagged oligonucleotides can be provided as multimers that can hybridize to two or more target hybridization oligonucleotides.

[0372] The use of more than one target hybridization oligonucleotide improves specificity, while the use of an amplification oligonucleotide increases the signal. Two target hybridization oligonucleotides are hybridized to a target RNA in a cell. The two target hybridization oligonucleotides combine together to provide a hybridization site to which an amplification oligonucleotide can bind. Hybridization and / or subsequent washing of the amplification oligonucleotides may be performed at a temperature that allows hybridization to two proximal target hybridization oligonucleotides, but above the melting temperature of hybridization of the amplification oligonucleotide to only one target hybridization oligonucleotide. A first amplification oligonucleotide provides multiple hybridization sites to which a second amplification oligonucleotide can bind to form a branched pattern. A mass-tagged oligonucleotide may bind to multiple hybridization sites provided by a second amplification oligonucleotide. These amplification oligonucleotides (with or without a mass-tagged oligonucleotide) combined together are referred to herein as "multimers". Thus, the term "amplification oligonucleotide" includes oligonucleotides that provide multiple copies of the same binding site to which additional oligonucleotides can anneal. By increasing the number of binding sites for other oligonucleotides, the final number of labels that can be found on the target increases. Thus, multiple labeled oligonucleotides are indirectly hybridized to a single target RNA. This allows the detection of low copy number RNA by increasing the number of detectable atoms of the element used per RNA.

[0373] One particular method for carrying out this amplification involves using the RNAscope® method from Advanced Cytodiagnostics, as discussed in more detail below. A further alternative is the use of a method that adapts the QuantiGene® FlowRNA method (Affymetrix eBioscience). This analysis is based on the design of oligonucleotide pair probes with branched DNA (bDNA) signal amplification. There are over 4,000 probes in the catalog, or you can request a custom set at no extra cost. In line with the previous paragraph, this method works by hybridization of a target hybridization oligonucleotide to a target, followed by the formation of a branched structure that includes a first amplification oligonucleotide (referred to as a preamplification oligonucleotide in the QuantiGene® method), forming a stem to which multiple second amplification oligonucleotides can anneal (referred to simply as amplification oligonucleotides in the QuantiGene® method). Multiple mass-tagged oligonucleotides can then be attached.

[0374] Another means of amplifying the RNA signal relies on rolling circle amplification means (RCA). There are various means by which this amplification system can be introduced into the amplification process. In the first case, a first nucleic acid is used as a hybridization nucleic acid, in which case the first nucleic acid is circular. The first nucleic acid may be single-stranded or double-stranded. The first nucleic acid comprises a sequence complementary to the target RNA. Following hybridization of the first nucleic acid to the target RNA, a primer complementary to the first nucleic acid is hybridized to the first nucleic acid and used for primer extension, typically using a polymerase and a nucleic acid that are exogenously added to the sample. However, in some cases, when the first nucleic acid is added to the sample, a primer for extension may already be hybridized to it. As a result of the first nucleic acid being circular, when primer extension completes a complete round of replication, polymerase can displace the primer and continue extension (i.e., without 5'→3' exonuclease activity), which further ligates and produces further linear copies of the complement of the first nucleic acid, thereby amplifying the nucleic acid sequence.Thus, the oligonucleotides containing element tags (such as RNA or DNA, or LNA or PNA) as described above can be hybridized to the linear copies of the complement of the first nucleic acid.Thus, the degree of amplification of RNA signal can be controlled by the length of time allocated to the step of amplifying circular nucleic acid.

[0375] In another application of RCA, the RCA is not a first, e.g., circular, oligonucleotide hybridizing to the target RNA, but a first portion with a sequence complementary to the target and a second portion selected by the user, which may be linear. A circular RCA template with a sequence homologous to the second portion may then be hybridized to this first oligonucleotide, and RCA amplification may be performed as described above. The application of a first, e.g., oligonucleotide with a target-specific portion and a user-selected portion is that the user-selected portion can be selected to be common among a variety of different probes. This allows for high reagent efficiency, since the same subsequent amplification reagents can be used in a series of reactions to detect different targets. However, as the skilled artisan will appreciate, when adopting this strategy, for individual detection of a specific RNA in a multiplex reaction, each first nucleic acid hybridizing to the target RNA must have a unique second sequence, and thus 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.

[0376] Other configurations for effecting RCA analysis are known to those skilled in the art. In some cases, 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 the subsequent amplification and hybridization steps.

[0377] Additionally, hybridization chain reaction (HCR) can be used to amplify RNA signals (see, e.g., Choi et al., 2010, Nat. Biotech, 28:1208-1210). Choi describes an HCR amplifier consisting of two nucleic acid hairpin species that will not polymerize without an initiator. Each HCR hairpin consists of an input domain with an exposed single-stranded toe and an output domain with a single-stranded toe hidden in the folded hairpin. Hybridization of an initiator to the input domain of one of the two hairpins opens the hairpin to expose its output domain. Hybridization of this (previously hidden) output domain to the input domain of a second hairpin opens that hairpin, exposing its output domains in the same order as the initiator. Regeneration of the initiator sequence provides the basis for a chain reaction of alternating first and second hairpin polymerization steps that leads to the formation of a nicked double-stranded "polymer." Either or both of the first and second hairpins may be labeled with element tags in the application of the methods and devices disclosed herein. Because the amplification procedure relies on the output domain of a specific sequence, various separate amplification reactions using separate sets of hairpins can be performed independently in the same process. This amplification therefore also allows for the amplification of multiple RNA species in multiplex analysis. As Choi mentions, HCR is an isothermally triggered self-assembly process. Thus, the hairpins need to penetrate into the sample before undergoing in situ triggered self-assembly, suggesting the possibility of deep sample penetration and high signal-to-background ratio.

[0378] 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 under which hybridization can occur. Hybridization may be performed in saline-sodium citrate (SCC) buffer, phosphate buffered saline (PBS), saline-sodium phosphate-EDTA (SSPE) buffer, TNT buffer (Tris-HCl, sodium chloride, and Tween 20), or any other suitable buffer. Hybridization may be performed at about or below the melting temperature of hybridization of one or more oligonucleotides.

[0379] Specificity may be improved by performing one or more washes following hybridization to remove unbound oligonucleotides. Increasing the stringency of the washes improves specificity but reduces the overall signal. The stringency of the washes may be increased by increasing or decreasing the concentration of the wash buffer, increasing the temperature, and / or increasing the duration of the washes. RNAse inhibitors may be used in any or all of the hybridization incubations and subsequent washes.

[0380] A first set of hybridization probes, including one or more target hybridization 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, hybridized and washed to reduce or prevent hybridization between oligonucleotides of different sets. Furthermore, each set of mass-tagged oligonucleotides can provide a unique signal. Thus, multiple sets of oligonucleotides can be used to detect 2, 3, 5, 10, 15, 20 or more distinct nucleic acid targets.

[0381] Sometimes the different nucleic acids detected are splice variants of a single gene. Mass tagged oligonucleotides can be designed to hybridize within the sequence of an exon to detect all transcripts containing that exon (either directly or indirectly via other oligonucleotides as described below) or they can be designed to bridge splice junctions to detect specific variants (e.g., if a gene has three exons and there are 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 through the exon 1-3 junction).

[0382] histochemical stain The histochemical staining reagent with one or more unique metal atoms can be combined with other reagents and methods of use as described herein. For example, the histochemical staining solution may be co-localized with metal-containing drugs, metal-labeled antibodies, and / or accumulated heavy metals (e.g., at cellular or subcellular resolution). In certain embodiments, the one or more histochemical staining solutions may be used at lower concentrations (e.g., less than half, 1 / 4, 1 / 10, etc.) than those used for other imaging methods (e.g., fluorescence microscopy, light microscopy, or electron microscopy).

[0383] A wide range of histological stains and indicators are available and well characterized to visualize and identify structures. Metal-containing stains may affect the acceptance of imaging mass cytometry by pathologists. Certain metal-containing stains are well known to reveal cellular components and are suitable for use in the present invention. Furthermore, distinct stains can be used in digital image analysis to provide contrast for feature recognition algorithms. These features are of strategic importance to the development of imaging mass cytometry.

[0384] In many cases, the morphological structure of tissue sections can be contrasted using affinity products such as antibodies. Affinity products are expensive and require an additional labeling step using metal-containing tags compared to using histochemical stains. This technique was used in pioneering studies of imaging mass cytometry using antibodies labeled with available lanthanide isotopes, thus depleting the mass (e.g. metal) tags for functional antibodies to answer biological questions.

[0385] The present invention expands the catalog of available isotopes to include elements such as Ag, Au, Ru, W, Mo, Hf, and Zr (including compounds such as ruthenium red used to identify myxoid stroma, trichrome stains to identify collagen fibers, and osmium tetroxide as a cellular counterstain). Silver stains are used for karyotyping. Silver nitrate stains nucleolus organizer region (NOR)-associated proteins to produce dark areas where silver is deposited, indicating activity of rRNA genes within the NOR. To adapt to IMC, protocols (e.g., oxidation with potassium permanganate, 1% silver concentration) must be modified to use lower concentrations of silver solutions, such as 0.5%, 0.01%, and less than 0.05% silver solutions.

[0386] Autometallographic amplification techniques have evolved into important tools in histochemistry. Many endogenous and toxic heavy metals form sulfide or selenide nanocrystals that can be autocatalytically amplified by reaction with Ag ions. Larger Ag nanoclusters can be easily visualized by IMC. Now, robust protocols have been established for silver-amplified detection of Zn-S / Se nanocrystals and detection of selenium by formation of silver-selenium nanocrystals. Moreover, commercially available quantum dots (detection of Cd) are also autocatalytically active and can be used as histochemical labels.

[0387] Aspects of the invention may include histochemical stains and the use of histochemical stains in imaging by elemental mass spectrometry. Any histochemical stain that is resolvable by elemental mass spectrometry may be used in the present invention. In certain aspects, the histochemical stain comprises one or more atoms with a mass greater than the cutoff of the elemental mass spectrometer used to image the sample, e.g., greater than 60 amu, 80 amu, 100 amu, or 120 amu. For example, the histochemical stain may comprise a metal tag (e.g., a metal atom) as described herein. The metal atom may be chelated to the histochemical stain or covalently bound within the chemical structure of the histochemical stain. In certain aspects, the histochemical stain may be an organic molecule. The histochemical stain may be polar, hydrophobic (e.g., lipophilic), ionic, or may comprise groups with different properties. In certain aspects, the histochemical stain may comprise multiple chemicals.

[0388] Histochemical stains include small molecules less than 2000, 1500, 1000, 800, 600, 400, or 200 amu. Histochemical stains may bind to samples via covalent or non-covalent (e.g., ionic or hydrophobic) interactions. Histochemical stains may provide contrast to reveal the morphology of biological samples, for example, to help identify individual cells, intracellular structures, and / or extracellular structures. Subcellular structures that may be resolved by histochemical stains include cell membranes, cytoplasm, nucleus, Golgi apparatus, endoplasmic reticulum, mitochondria, and other organelles. Histochemical stains may have affinity for types of biomolecules, such as nucleic acids, proteins, lipids, phospholipids, or carbohydrates. In certain embodiments, histochemical stains may bind molecules other than DNA. Suitable histochemical stains also include stains that bind extracellular structures (e.g., structures of the extracellular matrix) including stroma (e.g., mucosal stroma), basement membranes, interstitial stroma, collages or proteins such as elastin, proteoglycans, non-proteoglycan polysaccharides, extracellular vesicles, fibronectin, laminin, and the like.

[0389] In certain embodiments, the histochemical stains and / or metabolic probes may indicate the state of the cell or tissue. For example, the histochemical stains may include vital stains such as cisplatin, eosin, propidium iodide, etc. Other histochemical stains may stain for hypoxia, e.g., may only bind or deposit under hypoxic conditions. Probes such as rhododeoxyuridine (IdU) or derivatives thereof may stain cell proliferation. In certain embodiments, the histochemical stains may not indicate the state of the cell or tissue. Probes that detect the state of the cell (e.g., viability, hypoxia, and / or cell proliferation) but are administered in vivo (e.g., to a living animal or cell culture) are used in any of the present methods but do not qualify as histochemical stains.

[0390] The histochemical stain may have an affinity for types of biological molecules such as nucleic acids (e.g., DNA and / or RNA), proteins, lipids, phospholipids, carbohydrates (e.g., sugars such as mono- or disaccharides or polyols; oligosaccharides; and / or polysaccharides such as starch or glycogen), glycoproteins, and / or glycolipids. In certain aspects, the histochemical stain may be a counterstain.

[0391] Below are examples of specific histochemical stains and their use in the present methods.

[0392] Ruthenium red staining solution as a metal-containing staining agent solution for mucinous stroma detection may be used as follows: Immunostained tissue (e.g., deparaffinized FFPE or frozen sections) may be treated with 0.0001-0.5%, 0.001-0.05%, less than 0.1%, less than 0.05%, or about 0.0025% ruthenium red (e.g., at 4-42°C for at least 5 minutes, at least 10 minutes, at least 30 minutes, or about 30 minutes, or at about room temperature). The biological sample may be rinsed, for example, with water or a buffer solution. The tissue may then be dried prior to imaging by elemental mass spectrometry.

[0393] Phosphotungstic acid (e.g., as a trichrome stain) may be used as a metal-containing stain for collagen fibers. Tissue sections (deparaffinized FFPE or frozen sections) on slides may be fixed in Bouin's solution (e.g., for at least 5 minutes, at least 10 minutes, at least 30 minutes, or about 30 minutes at 4-42°C or near room temperature). The sections may then be treated with 0.0001% to 0.01%, 0.0005% to 0.005%, or about 0.001% phosphotandic acid (e.g., for at least 5 minutes, at least 10 minutes, at least 30 minutes, or about 15 minutes at 4-42°C or near room temperature). Samples may then be rinsed with water and / or buffer and dried as necessary prior to imaging by elemental mass spectrometry. Trichrome stains may be used at dilutions (e.g., 5-fold, 10-fold, 20-fold, 50-fold, or greater dilutions) compared to the concentrations used for imaging by optical (e.g., fluorescence) microscopy.

[0394] In some embodiments, the histochemical stain is an organic molecule. In some embodiments, the second metal is covalently bound. In some embodiments, the second metal is chelated. In some embodiments, the histochemical stain specifically binds cell membranes. In some embodiments, the histochemical stain is osmium tetroxide. In some embodiments, the histochemical stain is lipophilic. In some embodiments, the histochemical stain specifically binds extracellular structures. In some embodiments, the histochemical stain specifically binds extracellular collagen. In some embodiments, the histochemical stain is a trichrome stain comprising phosphotungstic acid / phosphomolybdic acid. In some embodiments, after contacting the sample with the antibody, the trichrome stain is used, for example, at a lower concentration than that used for optical imaging, for example, in this case, the concentration is 50 times or more diluted trichrome stain.

[0395] Metal-containing drugs Metals in medicine are a new and exciting field in pharmacology. Little is known about the cellular structures involved in the temporary storage of metal ions before incorporation into metalloproteins, nucleic acid metal complexes or metal-containing drugs, or about the final fate of metal ions upon protein or drug degradation. An important first step to elucidate the regulatory mechanisms involved in the transport, storage and distribution of trace metals corresponds to the identification and quantification of metals, ideally in tissues, cells or even in the context of their native physiological environment at the level of individual organelles and subcellular compartments. Histological studies are generally performed on thin sections of tissues or cultured cells.

[0396] Many metal-containing drugs, i.e., cisplatin, ruthenium imidazole, metallocene-based anticancer drugs with Mo, tungstenose with W, B-diketonate complexes with Hf or Zr, auranofin with Au, polyoxomolybdate drugs, are used to treat various diseases, but their mechanism of action or biodistribution is not fully understood. Many metal complexes are used as MRI contrast agents (Gd(III) chelates). Characterization of the uptake and biodistribution of metal-based anticancer drugs is crucial to understand and minimize the underlying toxicity.

[0397] The atomic weight of a particular metal present in a drug falls into the mass cytometry range. In particular, Pt complexes such as cisplatin (iproplatin, lobuplatin) are widely used as chemotherapeutic drugs to treat various cancers. The nephrotoxicity and myelotoxicity of platinum-based anticancer drugs are well known. Now, using the methods and reagents described herein, their subcellular localization within tissue sections and colocalization with mass (e.g., metal) tagged antibodies and / or histochemical stains can be examined. Chemotherapeutic drugs may be toxic to certain cells, such as proliferating cells, through direct DNA damage, inhibition of DNA damage repair pathways, radioactivity, etc. In certain embodiments, chemotherapeutic drugs may target tumors via antibody intermediates.

[0398] In certain aspects, the metal-containing drug is a chemotherapeutic drug. The method may include administering the metal-containing drug to a living animal, such as an animal research model or a human patient, before obtaining the biological sample, as described above. The biological sample may be, for example, a biopsy of cancerous tissue or primary cells. Alternatively, the metal-containing drug may be added directly to the biological sample, which may be an immortalized cell line or primary cells. If the animal is a human patient, the method may include adjusting a treatment regimen that includes the metal-containing drug based on detecting the distribution of the metal-containing drug.

[0399] Method steps for detecting metal-containing drugs may include intracellular imaging of the metal-containing drug by elemental mass spectrometry and may also include detecting retention of the metal-containing drug in intracellular structures (such as membranes, cytoplasm, nucleus, Golgi apparatus, ER, mitochondria, and other cellular organelles) and / or extracellular structures (such as stroma, mucosal stroma, basement membrane, interstitial stroma, collage or proteins such as elastin, proteoglycans, non-proteoglycan polysaccharides, extracellular vesicles, fibronectin, laminin, etc.).

[0400] Histochemical stains and / or mass (e.g., metal) tagged SBPs that resolve (e.g., bind to) one or more of the above structures may be co-localized with metal-containing drugs to detect retention of the drug at specific intracellular or extracellular structures. For example, a chemotherapeutic drug such as cisplatin may be co-localized with structures such as collagen. Alternatively or additionally, drug localization may be related to the presence of markers of cell viability, cell proliferation, hypoxia, DNA damage response, or immune response.

[0401] In some embodiments, the metal-containing drug comprises a non-endogenous metal, for example, the non-endogenous metal is platinum, palladium, cerium, cadmium, silver or gold.In certain aspects, the metal-containing drug is cisplatin, ruthenium imidazole, metallocene-based anticancer drug with Mo, tungsten cen with W, B-diketonate complex with Hf or Zr, auranofin with Au, polyoxomolybdate drug, N-myristoyltransferase-1 inhibitor with Pd (tris(dibenzylideneacetone)dipalladium) or one of their derivatives.For example, the drug may comprise Pt, and may be, for example, cisplatin, carboplatin, oxaliplatin, iproplatin, lobaplatin or their derivatives. Metal-containing drugs may include non-endogenous metals such as platinum (Pt), ruthenium (Ru), molybdenum (Mo), tungsten (W), hafnium (Hf), zirconium (Zr), gold (Au), gadolinium (Gd), palladium (Pd) or their isotopes. Gold compounds (e.g., auranofin) and gold nanoparticle bioconjugates for photothermal therapy of cancer may be identified in tissue sections.

[0402] Multiple analysis One feature of the present disclosure is its ability to detect multiple (e.g., 10 or more, even up to 100 or more) different target SBP elements in a sample to detect multiple different proteins and / or multiple different nucleic acid sequences. To allow differential detection of these target SBP elements, each of them must carry a different label atom so that the signals can be distinguished. For example, if 10 different proteins are being detected, 10 different antibodies (each specific to a different target protein) can be used, each of which has a unique label so that the signals from the different antibodies can be distinguished. In some embodiments, it is desirable to use multiple different antibodies against a single target, e.g., recognizing different epitopes on the same protein. Thus, the method may use more antibodies than targets due to this type of redundancy. In general, however, the present disclosure uses multiple different label atoms to detect multiple different targets.

[0403] When multiple labeled antibodies are used in the present disclosure, it is preferred that the antibodies have similar affinities for their respective antigens. This helps ensure that the relationship between the amount of labeled atoms detected and the abundance of target antigens in tissue samples is consistent across different SBPs (especially at high scanning frequencies). Similarly, it is preferred that the labels of various antibodies have the same efficiency, so that the antibodies each carry a comparable amount of labeled atoms.

[0404] In some cases, the SBP may carry a fluorescent label as well as an elemental tag. The fluorescence of the sample may then be used to determine the region of the sample, such as a tissue section containing the material of interest, which may then be sampled for detection of the labeled atom. For example, the fluorescent label may be conjugated to an antibody that binds to an antigen that is abundant in cancer cells, and any fluorescent cells may then be targeted to determine the expression of other cellular proteins by the SBP that is conjugated to the labeled atom.

[0405] If the target SBP element is intracellular, it is generally necessary to permeabilize the cell membrane before or during contacting the sample with the label. For example, if the target is a DNA sequence but the labeled SBP element cannot penetrate the membrane of living cells, the cells of the tissue sample can be fixed and permeabilized. The labeled SBP element can enter the cell and form SBP with the target SBP element. In this regard, known protocols for use with IHC and FISH can be utilized.

[0406] The method may be used to detect at least one intracellular target and at least one cell surface target. However, in some embodiments, the present disclosure can be used to detect multiple cell surface targets while ignoring intracellular targets. Overall, the selection of targets is determined by the information desired from the method, as the disclosure provides an image of the location of selected targets within a sample.

[0407] As further described herein, specific binding partners (i.e., affinity reagents) comprising label atoms can be used to stain (contact) biological samples. Suitable specific binding partners include antibodies (including antibody fragments). Label atoms may be distinguished (i.e., have different masses) by mass spectrometry. Label atoms may be referred to herein as metal tags if they comprise one or more metal atoms. Metal tags may comprise a polymer having a carbon backbone and multiple pendant groups, each of which binds to a metal atom. Alternatively or additionally, metal tags may comprise metal nanoparticles. Antibodies may be tagged with metal tags by covalent or non-covalent interactions.

[0408] The antibody stain may be used to image proteins at cellular or subcellular resolution. Aspects of the invention include contacting the sample with one or more antibodies that specifically bind to proteins expressed by cells of the biological sample, the antibodies being tagged with a first metal tag. For example, the sample may be contacted with 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more antibodies, each having an identifiable metal tag. The sample may be further contacted with one or more histochemical stains before, during (e.g., to facilitate workflow), or after (e.g., to avoid altering the antigen target of the antibody) staining the sample with the antibody. The sample may further include one or more metal-containing drugs and / or accumulated heavy metals, as described herein.

[0409] The metal-labeled antibodies used in the present invention can specifically bind to metal-free metabolic probes (e.g., EF5). Other metal-tagged antibodies can specifically bind to ta...

Claims

1. A movable sample stage; an optical microscope for examining the sample; A sampling system; The autofocus system, Equipped with The autofocus system comprises: A radiation source; and An autofocus sensor; an autofocus optical component; Equipped with one of the autofocus optical components comprises a plurality of apertures; the plurality of apertures are arranged in a regular shape, and in the regular shape, in addition to apex apertures at apexes of the regular shape, a centroid aperture is present at a centroid of the regular shape; the centroid aperture is configured to deliver illumination radiation to a point on the sample where ablation of the sample occurs such that a single focal point at the point on the sample where ablation of the sample occurs can be measured; a plurality of said vertex apertures are configured to transmit illumination radiation such that a plurality of additional focal points around the point on said sample at which ablation of said sample occurs can be measured; the centroid aperture is configured to facilitate calculation of the focal point from readings from the point on the sample where ablation of the sample occurs; the autofocus system is configured to provide redundancy in case a measurement of focus from illumination radiation passing through the one or more apertures fails; the sampling system and the autofocus system are confocal; Device.

2. the optical microscope is confocal with the sampling system and the autofocus system; The autofocus optical component having the plurality of apertures is a hybrid mask, the hybrid mask comprising: an opaque region including the plurality of apertures, the opaque region blocking illumination radiation except for the plurality of apertures; an area that allows passage of irradiating radiation; The apparatus of claim 1 , further comprising:

3. The apparatus of claim 1 , wherein the sampling system, the autofocus system, and the optical microscope all share at least some optical components.

4. The apparatus of claim 1 , wherein the apparatus is configured to perform autofocus by moving the sample stage in response to a readout from the autofocus optical components.

5. 5. The apparatus of claim 1, wherein the apparatus performs sample-independent autofocus.

6. 6. The apparatus of claim 1, wherein the autofocus system performs autofocus during a sample run.

7. An apparatus according to any preceding claim, wherein the autofocus system projects a focus map across X, Y or XY coordinates.

8. The apparatus of claim 1 , wherein the autofocus system comprises a plurality of apertures, the plurality of apertures being arranged in two dimensions.

9. 9. Apparatus according to claim 1, wherein the autofocus system provides a number of spots that can be impinged on the autofocus sensor.

10. 10. Apparatus according to any one of the preceding claims, wherein the autofocus system comprises a number of LEDs and / or laser diodes.

11. 11. Apparatus according to any one of claims 1 to 10, wherein the apparatus does not need to switch between autofocus and a test aperture.

12. 12. The apparatus of claim 1, wherein the autofocus sensor comprises an image sensor, the image sensor being shared with an optical microscope of the apparatus.

13. 13. The apparatus of claim 1, wherein the illumination source comprises at least two LEDs, the two LEDs being configured for alternating illumination.

14. 14. Apparatus according to any one of claims 1 to 13, wherein the illumination source comprises at least two laser diodes, the two laser diodes being arranged for alternating illumination.

15. 15. The apparatus of claim 1, wherein the autofocus system does not require pre-calibrated coordinates of one or more spots or lines aligned with the autofocus sensor.

16. 16. Apparatus according to any one of claims 1 to 15, wherein autofocus is based on an offset between the spots or lines.

17. 17. An apparatus according to any one of claims 1 to 16, wherein two or more spots or lines aligned with the autofocus sensor overlap at best focus.

18. 18. The apparatus of claim 1, wherein autofocus is based on the number of spots detected by the autofocus sensor.

19. 19. Apparatus according to any one of claims 1 to 18, wherein autofocus is based on spot uniformity detected by the autofocus sensor.

20. 20. The apparatus of claim 1, wherein the sampling system is a laser ablation sampling system, and wherein a focus of a laser source of the laser ablation sampling system is confocal with an autofocus component of the autofocus system and the autofocus sensor.

21. 21. The apparatus of claim 1, wherein the sampling system is a laser ablation sampling system, and the apparatus further comprises an ICP ionization system coupled to the laser ablation sampling system by a gas conduit.

22. 22. The apparatus of claim 21 further comprising a mass spectrometer.

23. A method of autofocus using an apparatus according to any one of claims 1 to 22, comprising sampling based autofocus.

24. 24. The method of claim 23, further comprising sampling the mass tags from the biological sample.

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