Survey mode for imaging mass cytometry

Abstract

In one embodiment, a method for imaging a biological specimen is disclosed, which includes defining a plurality of reference points across the surface of the biological specimen; directing ablation radiation to the biological specimen at a first plurality of specimen locations corresponding to a first subset of the defined reference points, thereby ablating at least a portion of the specimen at each of the locations, thereby generating a gas-phase sample from each of the specimen locations; ionizing each of the gas-phase samples to generate ions corresponding to the gas-phase sample; acquiring one or more mass signals corresponding to the ions associated with each of the ablated portions; and identifying at least one region of interest based on the analysis of the mass signals. An image of the identified region of interest can be acquired using image mass cytometry.

Description

[Technical Field]

[0001] Related applications

[0001] This application claims the interests of U.S. Provisional Patent Application No. 63 / 469,801, filed on 30 May 2023, the entire contents of which are incorporated herein by reference.

[0002]

[0002] This instruction generally relates to imaging of biological specimens using, for example, Imaging Mass Cytometry (IMC) (trademark). [Background technology]

[0003]

[0003] IMC (trademark) is used for the analysis of various samples, such as imaging of biological materials. With IMC (trademark), the sample to be analyzed is ablated with laser radiation, and the ablated material is ionized in, for example, an inductively coupled plasma to generate ions, which are then detected and analyzed by mass spectrometry. In the analysis of biological samples, IMC (trademark) can be used in combination with the use of metal-labeled antibodies that exhibit specific binding to target cells of interest to identify the cell type of interest in the sample.

[0004]

[0004] Imaging of biological specimens using IMC (trademark) has conventionally been performed using an ablation spot size of 1 micrometer or less. The use of an ablation spot size of 1 micrometer or less is considered essential for obtaining useful images from which the desired biological information can be collected.

[0005]

[0005] Despite significant progress in the field of IMC (Trademark), certain challenges remain, such as the time required to acquire low-intensity signal data per pixel for multiple low-intensity signal intensity markers. [Overview of the Initiative] [Means for solving the problem]

[0006]

[0006] In one embodiment, a method for imaging a biological specimen is disclosed, the method comprising: defining a first plurality of reference points across the surface of the biological specimen; directing ablation radiation to the biological specimen to ablate small portions of the specimen at a first plurality of specimen locations corresponding to the defined first reference points or a portion thereof, thereby ablating at least a portion of the specimen at each of the first plurality of specimen locations, thereby generating a gas-phase sample from each of the first plurality of specimen locations; ionizing each of the gas-phase samples to generate ions corresponding to the gas-phase sample; obtaining one or more mass signals corresponding to the ions associated with each of the ablated portions; and identifying at least one region of interest based on an analysis of the mass signals.

[0007]

[0007] In various embodiments, the step of identifying at least one region of interest includes comparing each of the mass signals to at least one threshold criterion defined based on at least one phenotypic signal to determine whether the sample location corresponding to that mass signal is within the region of interest.

[0008]

[0008] In various embodiments, at least one phenotypic signal comprises a plurality of phenotypic signals, and at least one threshold criterion comprises a plurality of threshold criteria, each of which corresponds to one of the phenotypic signals, and the method may further include classifying each of the ablated positions in the multidimensional signal space based on the comparison.

[0009]

[0009] In various embodiments, the method may further include performing a neighborhood analysis of the classified ablate locations to identify at least one region of interest. For example, but not limited to, in some cases, at least one region of interest can be identified without using fluorescence imaging.

[0010]

[0010] In various embodiments, the method may further include generating an image of the identified region of interest using either image mass cytometry or image mass spectrometry.

[0011]

[0011] In various embodiments, the ablation radiation is configured to ablate the upper layer of the specimen at one or more of the first plurality of locations. For example, but not limited to, the thickness of the upper layer can be in the range of about 10 nm to about 1000 nm.

[0012]

[0012] In various embodiments, an image of the identified region of interest can be obtained by directing ablation radiation to a second plurality of locations within the region of interest to generate a gas-phase sample from each of the second plurality of locations; ionizing the gas-phase sample to generate ions corresponding to each of the second plurality of sample locations; obtaining one or more mass signals corresponding to the ions associated with each of the second plurality of sample locations; and generating an image of at least a portion of the region of interest based on the mass signals associated with the second plurality of sample locations. In some such embodiments, the ablation radiation can be directed to the second plurality of locations without realigning the radiation with respect to a defined reference point.

[0013]

[0013] The biological specimen may include one or more cell types. In various embodiments, image generation may include analyzing the mass signal associated with each of the second plurality of specimen locations to identify one or more cell types of interest located at that location. For example, but not limited to, the cell types of interest may include immune cells, structural cells, tumor cells, stromal cells, germinal center cells, vascular cells, mesenchymal cells, or non-mesenchymal cells. For example, immune cells may include either CD3+ or CD45+ cells. In some cases, the second plurality of locations may be different from the first plurality of locations. In some cases, the second plurality of locations may include the first plurality of locations.

[0014]

[0014] In various embodiments, the identification of one or more cell types of interest is based on the identification of at least one phenotypic signal via analysis of mass signals associated with each of a second plurality of sample locations. For example, but not limited to, at least one phenotypic signal is associated with a target protein, target DNA, target RNA, target molecular structure, amino acid sequence, target lipid structure, target phosphorylation region, target sugar region, or a naturally occurring substance or element. For example, but not limited to, naturally occurring substances or elements include either selenium or mercury. In some cases, at least one phenotypic signal is associated with a drug-related substance. In some cases, at least one phenotypic signal is associated with platinum in a chemotherapeutic drug. In some cases, at least one phenotypic signal provides information about the protein content of different cell types or tissue compartments.

[0015]

[0015] In various embodiments, the identification of any one of one or more cell types of interest is based on the detection of a mass signal corresponding to a metal tag bound to an antibody that shows specific binding to a surface marker of the cell type of interest.

[0016]

[0016] In various embodiments, the multiple reference point locations are distributed according to a regular grid. For example, but not limited to, the regular grid can be a rectangular, square, triangular, or hexagonal grid.

[0017]

[0017] In various embodiments, both the first and second ablate positions have a maximum linear dimension in the range of more than 1 micrometer to about 20 micrometers. For example, but not limited to, the maximum linear dimension can be at least 2 micrometers, at least 3 micrometers, at least 4 micrometers, at least 5 micrometers, at least 6 micrometers, at least 7 micrometers, at least 8 micrometers, at least 9 micrometers, at least 10 micrometers, at least 11 micrometers, at least 12 micrometers, at least 13 micrometers, at least 14 micrometers, at least 15 micrometers, at least 16 micrometers, at least 17 micrometers, at least 18 micrometers, at least 19 micrometers, and at least 20 micrometers.

[0018]

[0018] In various embodiments, the first ablates are separated from each other by a distance corresponding to a multiple of the maximum linear dimension. For example, but not limited to, the multiple can be in the range of 2 to 10.

[0019]

[0019] In various embodiments, the first and second ablate positions may have substantially circular cross-sectional profiles, and the maximum linear dimension corresponds to the diameter of the substantially circular cross-sectional profile.

[0020]

[0020] In various embodiments, the second set of ablative positions can be arranged side by side.

[0021]

[0021] The method according to this disclosure can be used to obtain images of various biological specimens. For example, but not limited to, biological specimens may include tissue specimens, bone sections, blood specimens, organoid sections, and cell culture specimens.

[0022]

[0022] In various embodiments, the radiation utilized for specimen surveying and / or imaging can be laser radiation, such as pulsed laser radiation. By way of example, and not limited thereto, the wavelength of the laser radiation can be in the range of about 10 nm to about 10000 nm, for example, in the range of about 100 to about 1000 nm. In some such embodiments, the beam size of the laser radiation at the surface of the specimen can be in the range of about 1 micrometer to about 20 micrometers. Further, the fluence of the laser radiation at the specimen surface can be in the range of about 0.001 J / cm 2 ~ about 10 J / cm 2 The range can be.

[0023]

[0023] In various embodiments, the laser radiation can have a Gaussian intensity profile, and the beam size can correspond to the full width at half maximum (FWHM) of the Gaussian intensity profile.

[0024]

[0024] In various embodiments, the laser radiation can have a flat top intensity profile.

[0025]

[0025] In various embodiments, the ablation radiation is configured to ablate the plurality of positions substantially uniformly.

[0026]

[0026] In various embodiments, a mass spectrometer can be utilized to generate a mass signal corresponding to any of the first and second plurality of specimen portions. By way of example, and not limited thereto, the mass spectrometer can be a high parameter mass spectrometer such as a ToF mass spectrometer.

[0027]

[0027] In various embodiments, an optical image of a biological specimen can be generated along with a trace outlining the identified region of interest. In some such embodiments, the optical image can be presented to the user using a user interface display. Furthermore, in various embodiments, the user interface is configured to allow the user to select a position for imaging within the identified region of interest.

[0028]

[0028] In various embodiments, a digital data processor can be used to analyze the mass signal for identification of the region of interest.

[0029]

[0029] In various embodiments, the specimen can be placed in a specimen holder, and the specimen holder can be moved with respect to radiation (e.g., with respect to laser radiation pulses) during, for example, the survey period and / or the imaging period, to direct the radiation to one of a first and second plurality of specimen positions.

[0030]

[0030] In various embodiments, the radiation can be pulsed laser radiation, and multiple radiation pulses can be directed to at least one of the positions so that at least a portion of the specimen is ablated at that position. For example, but not limited to these, the laser radiation pulses can be directed to each position at a repetition rate in the range of about 10 kHz to about 10,000 kHz.

[0031]

[0031] In various embodiments, instead of moving the specimen with respect to radiation, or in addition to doing so, the radiation can be directed as a radiation beam towards the biological specimen, and the radiation beam can be moved with respect to the specimen, for example with respect to a holder on which the specimen is placed, to ablate either the first or second specimen portion.

[0032]

[0032] In a related embodiment, a method is disclosed for identifying at least one region of interest associated with a biological specimen, which includes: ablating a plurality of parts of the specimen by radiation to create a gas-phase sample from each part; ionizing at least one part of the gas-phase sample from each of the plurality of parts of the specimen to generate ions associated with two or more parts of the specimen; obtaining mass signals corresponding to the ions associated with two or more parts of the specimen; and identifying at least one region of interest based on an analysis of the mass signals associated with two or more parts of the specimen.

[0033]

[0033] In various embodiments, the step of analyzing the mass signal may include comparing each of the mass signals to a reference threshold to determine whether each part of the sample is part of the region of interest. For example, but not limited to, a part of the sample may be classified as being in the region of interest if the mass signal associated with that part of the sample is greater than or equal to a reference threshold.

[0034]

[0034] In various embodiments, the analysis of the mass signal may include comparing two or more sample portions with each other to determine whether any of the two or more sample portions is part of the region of interest.

[0035]

[0035] In various embodiments, the analysis of the mass signal may include comparing the mass signal associated with at least one of the sample portions with the mass signal associated with an adjacent sample portion.

[0036]

[0036] In various embodiments, the analysis of the mass signal may include classifying any of the sample portions as being within a region of interest when the mass signal associated with that sample portion is within a predetermined range.

[0037]

[0037] In various embodiments, the set of multiple sample parts corresponds to a small portion of the sample. In other words, the set of multiple sample parts does not collectively encompass the entire sample.

[0038]

[0038] In various embodiments, each of the sample portions has a maximum linear dimension greater than 1 μm, optionally in the range of greater than 1 μm to about 20 μm, for example, at least 2 μm, or at least 3 μm, or at least 4 μm, or at least 5 μm.

[0039]

[0039] In various embodiments, a digital data processor can be used to analyze the mass signal.

[0040]

[0040] In various embodiments, the specimen portions can be arranged relative to each other according to a regular grid, such as a grid of squares, rectangles, triangles, and hexagons.

[0041]

[0041] In various embodiments, the method may further include functionalizing the biological sample with at least one antibody tagged with at least one metal tag, the antibody exhibiting specific binding to a target surface cell marker. In such embodiments, the ionization step may include ionizing the metal tag. An image of at least a portion of the identified target region can be generated using the mass signal associated with the at least one metal tag.

[0042]

[0042] In various embodiments, the above method can be performed on various biological specimens. For example, the biological specimen may include any of the following: tissue specimens, bone sections, blood specimens, organoid sections, and cell culture specimens.

[0043]

[0043] In a related embodiment, a method is provided for identifying one or more regions of interest of a biological specimen, which includes: (a) ablating a portion of the specimen by radiation to create a gas-phase sample; (b) ionizing at least a portion of the gas-phase sample; (c) generating a mass spectrum including at least one mass signal from the ionized portion of the gas-phase sample; (d) comparing the at least one mass signal to at least one threshold defined based on at least one phenotypic signal; (e) identifying the ablated portion as part of a region of interest if the at least one mass signal is greater than the threshold to which the mass signal was compared; (f) repeating steps (a) to (e) for one or more different portions of the biological specimen; and (g) identifying one or more regions of interest of the biological specimen based on two or more of the ablated portions identified as part of a region of interest.

[0044]

[0044] In various embodiments, the above method may include at least one mass signal and a plurality of mass signals.

[0045]

[0045] In various embodiments, the above method may include at least one phenotypic signal. In some such embodiments, each of the phenotypic signals may be compared with each of the multiple thresholds.

[0046]

[0046] In various embodiments, a substantially complete image of one or more identified regions of interest can be obtained. In some such embodiments, a substantially complete image of one or more identified regions of interest can be obtained without ablating the ablated portion of the specimen again to identify one or more regions of interest.

[0047]

[0047] In various embodiments, at least one phenotypic signal is associated with a target protein, target DNA, target RNA, target molecular structure, or amino acid sequence.

[0048]

[0048] In various embodiments, ablated specimen portions to identify one or more regions of interest are separated from each other by a distance that is a multiple of the maximum linear dimension size of the ablated portion. For example, the multiple can range from about 2 to about 7.

[0049]

[0049] In a related embodiment, a method for generating a three-dimensional tomographic image of a biological specimen is disclosed, which includes defining a plurality of sections of the biological specimen. For each section, the following steps can be performed: defining a plurality of reference points across the surface of the biological specimen; ablating at least a portion of the specimen at each of the locations by directing radiation to a first plurality of samples corresponding to a first subset of the defined reference points of the biological specimen, thereby generating a gas-phase sample from each of the sample locations; ionizing each of the gas-phase samples to generate ions corresponding to the gas-phase sample; obtaining one or more mass signals corresponding to the ions associated with each of the ablated portions; identifying at least one region of interest based on the analysis of the mass signals; and generating an image of at least a portion of the identified at least one region of interest.

[0050]

[0050] In various embodiments, the step of generating an image of at least one identified region of interest may include directing ablation radiation to a second plurality of locations within the region of interest associated with a second subset of the reference point locations to generate a gas-phase sample from each of the portions; ionizing the gas-phase sample to generate ions corresponding to each of the second plurality of sample locations; obtaining one or more mass signals corresponding to the ions associated with each of the second plurality of sample locations; and generating an image of at least a portion of the region of interest based on the mass signals associated with the second plurality of sample locations.

[0051]

[0051] In a related embodiment, a method for imaging a biological specimen is disclosed, which includes acquiring an optical image of the biological specimen placed in a holder; directing ablation radiation to a plurality of parts of the biological specimen to generate a gas phase sample from each of the parts; ionizing the gas phase sample to generate ions corresponding to each of the plurality of specimen locations; acquiring one or more mass signals corresponding to the ions associated with each of the plurality of specimen locations; and generating an image of at least a portion of the biological specimen based on the mass signals associated with the plurality of specimen locations.

[0052]

[0052] The various aspects of this instruction can be better understood by referring to the detailed explanation below in conjunction with the attached drawings, which are briefly described below. [Brief explanation of the drawing]

[0053] [Figure 1A]

[0053] This is a flowchart showing the various steps of a method according to one embodiment of the teaching. [Figure 1B]

[0054] This shows a microscope slide on which a tissue sample is placed. [Figure 2A]

[0055] This is a diagram of a microscope slide with multiple reference point locations superimposed on the image of the microscope slide to define multiple locations to be ablated for fractional sampling of tissue samples. [Figure 2B]

[0056] A schematic diagram shows multiple laser shots spaced 1 micrometer apart, as used in conventional imaging mass cytometry methods. [Figure 2C]

[0057] Figure 2B schematically shows multiple laser shots with a center-to-center distance of 5 micrometers, according to one embodiment that provides 25 times the throughput compared to the conventional method shown. [Figure 2D]

[0058] Figure 2C schematically shows multiple laser shots with a center-to-center distance of 5.88 micrometers by another embodiment that provides 30 times the throughput compared to the conventional method shown. [Figure 2E]

[0059] Laser shots spaced 5 μm apart and with energies of approximately 10 dB schematically illustrate the survey modes used in the small gaps separating the laser shots. [Figure 2F]

[0060] This shows a fractional sampling survey mode that utilizes laser shots spaced approximately 25 μm apart and with an energy of approximately 10 dB. [Figure 2G]

[0061] A minimal sampling survey mode is schematically shown, in which laser shots with an energy of approximately 10 dB and an interval of approximately 25 μm were used, but the laser beam was defocused so that only the top 10% of the material was sampled within a 10 μm region. [Figure 3]

[0062] This is a diagram of a microscope slide showing the contour of a region of interest identified using a method according to one embodiment of this instruction. [Figure 4]

[0063] This is a diagram of a microscope slide showing various locations within the region of interest selected for imaging. [Figure 5]

[0064] This is a schematic diagram of an imaging system according to one embodiment of this teaching. [Figure 6]

[0065] This is an example of a workflow that can be used in several embodiments of this instruction. [Figure 7A]

[0066] This shows multiple pseudotype cells classified based on selected phenotypic signals associated with a single exemplary marker (CD3). [Figure 7B]

[0066] Multiple pseudotype cells classified based on a selected phenotypic signal associated with one exemplary marker (CD3) are shown. [Figure 7C]

[0066] Multiple pseudotype cells classified based on a selected phenotypic signal associated with one exemplary marker (CD3) are shown. [Figure 7D]

[0066] Multiple pseudotype cells classified based on a selected phenotypic signal associated with one exemplary marker (CD3) are shown. [Figure 8A]

[0067] This shows another example of pseudotype cells identified based on phenotypic signals associated with multiple markers. [Figure 8B]

[0067] Another example of pseudotype cells identified based on phenotypic signals associated with multiple markers is shown. [Modes for carrying out the invention]

[0054]

[0068] This instruction relates to a method and system for generating images of specimens, particularly biological specimens, using IMC®. Conventionally, such images are generated by ablation of micrometer and submicrometer-scale portions of the specimen to produce a gas-phase sample that generates ions that can be detected and analyzed using mass spectrometry. In the art, it is generally recognized that generating images that provide useful information about a sample, such as the distribution of various cell types in the sample, requires the use of ablation spots (hereinafter also referred to as pixels) of 1 micrometer or less in size to ensure that the sample can be examined with spatial resolution smaller than cells. However, such conventional methods result in long data acquisition times. Furthermore, conventional systems have low throughput, so users typically select only a portion of the specimen for imaging. The selection of the portion of the specimen to be imaged is done manually by the user, which can lead to selection bias and increases the overall time required to acquire images.

[0055]

[0069] This disclosure provides a method and system for selecting a region of interest (ROI) of a specimen and imaging that region, which, in contrast to conventional teachings, uses a large ablation spot size, e.g., greater than 1 micrometer, to scan the specimen. Furthermore, once the region of interest is identified, the region or a portion thereof can be imaged using ablation spots, where each ablation spot generates a gas-phase sample, which is then ionized and analyzed using mass spectrometry to obtain an image of the area under investigation. Surprisingly, it has been found that such large ablation spot sizes can be used to scan a specimen to identify a region of interest and to image that region to extract useful biological information. In particular, contrary to conventional understanding in the art, it has been surprisingly found that the mass data obtained via such large ablation spots can be processed to generate images that provide various desired information about the specimen, such as the distribution of cell types of interest within the specimen. For example, in various embodiments, "pseudo-cells" (e.g., spots with a diameter of 5 μm) can be classified, for example, based on phenotypic signals, followed by neighborhood analysis of the distribution of pseudo-cells to extract biologically useful information about the sample, as will be described in more detail below.

[0056]

[0070] Various embodiments of this instruction provide methods for imaging biological specimens using the following data acquisition and analysis modes: (a) fractional survey mode or fractional sampling mode (also referred herein as preview mode), (b) survey mode (also referred herein as tissue mode), and (c) IMC imaging mode (also referred herein as cell mode). In various embodiments, ROIs are identified in preview mode, which can provide a rapid scan of the entire tissue specimen under analysis, for example, by ablation of a collection of sparsely distributed tissue spots (e.g., tissue spots having a spot size of about 1 micrometer and an intercenter distance of about 25 micrometers), and can also identify one or more regions of interest, for example, by detection of one or more markers of interest in the ablation data (e.g., one or more antibodies showing specific binding to a particular cell surface marker). In tissue mode, following the identification of one or more regions of interest, the identified regions of interest are scanned by ablation of tissue spots having a larger size and smaller intercenter distance than those used in preview mode, and the ablation data is analyzed, for example, using pixel clustering analysis. For example, in tissue mode, the ablation spot size can be approximately 5 micrometers, and the intercenter distance between adjacent spots can also be approximately 5 micrometers. Following tissue mode, cell mode is initiated, in which single-cell resolution imaging data is acquired through the ablation of multiple tissue spots with smaller spot sizes than those used in tissue mode. For example, in cell mode, the ablation spot size can be approximately 1 micrometer, and the intercenter distance between adjacent tissue spots can be approximately 1 micrometer.

[0057]

[0071] In various embodiments, in fractional survey mode, ROIs are identified by ablation of a small portion of the specimen of interest. In various embodiments, in fractional survey mode, the spacing between ablation spot centers is on the same scale as the spot size, e.g., 150% or less, while in other embodiments, the spacing between ablation spots can be much larger. In subsequent imaging modes, imaging mass cytometry or imaging mass spectrometry is used to image the ROI or a portion thereof. As noted above and described in more detail below, it has been found that fractional survey mode can be performed with ablation spot sizes considerably larger than those conventionally employed. For example, ablation sizes larger than 1 micrometer have been found to be usable without problems in imaging mode to image ROIs and obtain desired biological information. The use of larger ablation spot sizes significantly improves system throughput and also enables certain imaging modes (e.g., 3D imaging of specimens) that would otherwise be impractical due to, for example, long data acquisition times.

[0058]

[0072] In the following explanation, various aspects of this instruction will be described in relation to tissue specimens, but it should be understood that this instruction can be applied to obtaining images of various biological specimens, such as those containing cells, including tissues, as well as body fluids and excretions (e.g., amniotic fluid, bile, whole blood, serum, plasma, cerebrospinal fluid, gastrointestinal fluid, interstitial fluid, mucus, pus, saliva, feces, tears, urine, etc.).

[0059]

[0073] In this specification, various terms are used according to their common meanings in the industry. The term “about” is intended, when used herein, to indicate a variation of up to 10% around a given number, and the term “substantially” is intended, when used herein, to indicate a variation of up to 10% from, if any, a perfect and complete state and / or condition.

[0060]

[0074] Figure 1A is a flowchart providing various steps of one embodiment of the method according to this teaching for generating an image of a biological specimen. The method includes defining a plurality of reference points across the surface of the biological specimen in preview mode, and directing ablation radiation to the biological specimen at positions corresponding to the defined reference points to ablate at least a portion of the specimen at each of those positions, thereby generating a gas phase sample from each of the specimen positions. The size of the ablation spots, e.g., the diameter, can be in the range of about 1 micrometer to about 10 micrometers. In various embodiments, the diameter of the ablation spots can be in the range of about 1 micrometer to about 5 micrometers. The distance between the centers of the ablation spots can be in the range of about 5 to about 50 times the size of the ablation spots (e.g., the diameter). In various embodiments, the distance between the centers of the ablation spots can be in the range of about 10 to about 50 times the size of the ablation spots. Thus, in some embodiments, the ablation spots can have a diameter of about 1 micrometer and be spaced about 25 times the diameter, i.e., about 25 micrometers apart from their centers. The number and size of ablation spots, as well as the distance between ablation spots, are selected so that the ablation spots cover a small portion of the area being scanned for ROI identification.

[0061]

[0075] Each gas-phase sample is ionized to generate ions corresponding to that gas-phase sample, and one or more mass signals corresponding to the ions associated with each ablation area are acquired. At least one region of interest is identified based on the analysis of the mass signals. The region of interest, or at least a portion thereof, is imaged using an ablation spot size, for example, greater than about 1 micrometer, and in various embodiments, in the range of greater than 1 micrometer to about 5 micrometers.

[0062]

[0076] Figure 1B shows a tissue sample 100 placed on a microscope slide 102. As will be discussed in more detail below, this instruction can be used to identify one or more regions of interest associated with the tissue sample 100 using imaging mass spectrometry (IMS) or imaging mass cytometry (IMC), and to generate images of the identified regions of interest. In some cases, the entire tissue sample may be the region of interest, and in other cases, only a portion of the tissue sample may be the region of interest. For example, in some cases, the region of the tissue sample where tumor cells are present may be the region of interest, if any. Various embodiments of this instruction can be used to identify regions of interest in a tissue sample and generate images of one or more of those regions.

[0063]

[0077] Referring to Figure 2A as an example, in this embodiment, a plurality of reference points 104 are defined across a portion of the slide 102, each of which identifies a location to which the ablation radiation beam is directed so that at least a portion of the material at that location is ablated. In this example, the reference points 104 are distributed across the slide as a regular rectangular grid. While a rectangular grid is used in this example, other forms of regular grids, such as squares, rectangles, triangles, or hexagons, may also be used in other embodiments. As will be discussed in more detail below, in various embodiments where the size of the ablation radiation spot on the specimen surface is in the range of more than 1 micrometer to about 10 micrometers, for example, when the beam diameter of the ablation radiation beam is in the range of more than 1 micrometer to about 10 micrometers, the use of a hexagonal grid may be particularly advantageous.

[0064]

[0078] More specifically, using a hexagonal ablation pattern allows for material ablation with fewer unablated spots remaining between ablated spots. To achieve the same level of ablation with square tessellation, overlapping ablation spots are generally required. For example, with a square ablation pattern, areas at the corners of the sample may remain unablated, requiring overlapping ablation spots to ablate the unablated areas. However, using overlapping ablation spots results in inefficient use of available laser energy. Furthermore, overlapping ablation spots can generate non-uniform signals during fractional survey mode. With hexagonal sampling and appropriate selection of ablation spot diameter and sampling distance, the amount of unablated material can be minimized, and the ablated spots show virtually no overlap.

[0065]

[0079] For illustrative purposes, Figure 2B schematically shows multiple laser shots spaced 1 micrometer apart, as used in a conventional method (also known as the cell mode) for performing imaging mass cytometry. In contrast, Figure 2C schematically shows multiple laser shots with a center-to-center distance of 5 micrometers according to one embodiment, providing 25 times the throughput compared to the conventional method shown in Figure 2B. Figure 2D schematically shows multiple laser shots with a center-to-center distance of 5.88 micrometers according to another embodiment, providing 30 times the throughput compared to the conventional method shown in Figure 2C. The laser shot patterns shown in Figures 2B and 2C are based on a rectangular grid, while Figure 2D is based on a hexagonal grid, resulting in smaller unablated areas between ablated spots while providing higher throughput. The hexagonal pattern allows for high throughput, along with less material remaining on the unablated islands between ablated spots.

[0066]

[0080] Figure 2E schematically illustrates a survey mode (also called tissue mode) where laser shots with 5 μm spacing and approximately 10 dB of energy are used in small gaps separating the laser shots. Figure 2F illustrates a fractional sampling survey mode (also called preview mode) where laser shots with approximately 25 μm spacing and approximately 10 dB of energy are used. The green dots represent the first pass of fractional sampling, followed by imaging mode sampling after identification of the region of interest. The signals collected from the ablated locations in the first pass are replaced by the respective signals collected in imaging mode in the second pass. Figure 2G schematically illustrates another type of survey mode, namely minimal sampling survey mode. As an example of minimal sampling survey mode, laser shots are placed at approximately 25 μm spacing and approximately 10 dB of energy is used, but the laser beam is defocused so that only the top 10% of the material is sampled within a 10 μm area. This removes only a thin layer of the sample, leaving the sample almost intact. Once an ROI is identified, it can be imaged using any suitable imaging mass cytometry, for example, with a 5 μm spacing and an ablation spot diameter, using horizontally aligned laser shots spaced 1 μm apart or more sparsely distributed laser shots.

[0067]

[0081] The radiation beam can be aligned with respect to a reference point 104 and directed to each position identified by the reference point so that at least a portion of the material at that position is ablated.

[0068]

[0082] As an example, referring to the flowchart in Figure 6, in some embodiments, the specimen of interest can be optically imaged, and the optical image can be analyzed using methods known in the industry to identify the outline of the specimen with respect to the coordinates of the holder on which the specimen is placed (steps 1A, 1B).

[0069]

[0083] Next, to identify regions of interest within the specimen contour, the locations to be ablated during the fractional survey mode (i.e., preview mode) can be identified (Step 2A). The identified coordinates can be transferred to the ablation system, and the fractional survey of the specimen according to this instruction can proceed through ablation of the specimen locations corresponding to the identified coordinates to identify one or more regions of interest associated with the specimen (Step 2B). Alternatively, after identifying the specimen contour by optical imaging, one or more regions of interest can be identified using the conventional IMC™ workflow (i.e., cell mode) (Step 4A). Yet another approach is to proceed from Step 1B to Step 2A / 2B in minimal sampling survey mode (rather than fractional sampling survey mode), and then from Step 2B to Step 4A.

[0070]

[0084] In some cases, steps 1A and 1B can be performed on an IMC instrument by utilizing its optical subsystem. In an alternative method, step 1A can be performed on an optical slide scanner. In yet another embodiment, steps 1A and 1B can be omitted, and step 2A can be applied to an area where the specimen (e.g., a tissue section) is typically positioned, e.g., a rectangular area. In such a method, the possibility that performing step 2A will ablate a large number of empty areas (i.e., areas of the holder on which no part of the specimen is placed) is not a major concern, typically if step 2A is performed at a large ablation interval and high throughput. For example, if necessary, fractional survey sampling can be performed first at large intervals between consecutive ablation spots, e.g., 100 micrometer intervals, to speed up the task of finding the location of tissue on the holder (rather than finding ROIs in survey mode). The data can be used in step 2B, but the purpose is to identify ROIs (e.g., at 25 micrometer intervals between spots) for other rounds of step 2A. Once the second fractional survey sampling is performed, it identifies ROIs for a survey mode (i.e., tissue mode) that is performed with a spot size of, for example, 5 micrometers (see steps 3A and 3B).

[0071]

[0085] In various embodiments, the data collected during the fractional survey mode (i.e., preview mode) in step 2A can be analyzed in step 2B to identify ROIs. For example, the collected data can be normalized, and for each pixel, it can be determined whether the pixel belongs to a region of interest by analyzing the data corresponding to that pixel and those within a defined neighborhood of that pixel with respect to signals associated with one or more markers of interest (e.g., antibodies showing specific binding to one or more cell surface markers). Such identified pixels may correspond to one or more regions of interest, i.e., regions of the tissue sample from which data is acquired in the subsequent survey mode (i.e., tissue mode) in step 3A and the IMC mode (i.e., cell mode) in step 4A.

[0072]

[0086] This workflow is similar to the pyramidal data structure used in imaging large areas. The first layer of the pyramidal data contains upper-level images with coarser steps. The second layer contains data with finer steps. The third layer will contain data with a final interval such that the imaging process consumes the majority of the sample material.

[0073]

[0087] The data acquisition workflow described above is not limited to three layers. Rather, data acquisition can be performed with four or more layers as needed for specific applications. For example, a coefficient of 2 or 3 can be used when changing the interval between the final narrow and coarse intervals. An example of a four-layer pyramidal data structure aims to obtain final intervals of 5 micrometers in the fourth layer, 15 micrometers in the third layer, 45 micrometers in the second layer, and 135 micrometers in the first layer. Since the coordinates of the XY stages can be stored with a precision of more than 1 micrometer (much smaller than the minimum interval of 5 micrometers), data acquisition at each layer generates data that can also be used as data points for subsequent layers.

[0074]

[0088] In various embodiments, the radiation beam is a pulsed laser beam with a wavelength in the range of, for example, about 10 nm to about 10,000 nm. In this example, a sample holder, which is a glass slide, can be scanned with respect to the pulsed laser beam to expose each of the locations identified via a reference point to the laser radiation. In some embodiments, the laser beam may have a Gaussian profile of beam size characterized by a full width at half maximum (FWHM) in the range of more than 1 micrometer to about 5 micrometers, such as 2 micrometers, 3 micrometers, or 4 micrometers. In other embodiments, a flat-top laser beam can be used to provide substantially uniform ablation of the ablated spot, as will be described in more detail below.

[0075]

[0089] Irradiation of each position across the microscope slide, including the position on which the tissue sample is placed, will ablate at least a portion of the material at that position, that is, the radiation will vaporize at least a portion of the material at that position, thereby generating a gas-phase sample (also referred to herein as a gas-phase plume), which can be analyzed in the manner described below to determine whether that position is within the region of interest.

[0076]

[0090] More specifically, in some embodiments, each gas-phase sample can be transported with high time fidelity to an ion source, such as an inductively coupled plasma ion source, via a carrier gas, such as helium, in this case at least a portion of the gas sample, such as one or more analytes and / or tags, is ionized, and the ions are received and analyzed by a downstream mass spectrometer, such as a time-of-flight (ToF) mass spectrometer or any other suitable mass spectrometer, which generates one or more mass signals associated with these ions. In some embodiments, a mass cytometry or mass spectrometry system can record ion signals of elements naturally present in the sample, while in other embodiments, antibodies stained with metal tags are used in the mass cytometry system. In some other mass cytometry systems, an organic mass spectrometer is used for ionization and detection, and as an example, ions of mass tags formed from intact peptides may be used.

[0077]

[0091] In some embodiments, tissue samples can be functionalized with one or more antibodies containing metal stable isotope tags. For example, multiple metal-tagged antibodies exhibiting specific binding to multiple surface markers associated with target cells of interest can be utilized. For instance, metal-tagged antibodies exhibiting specific binding to CD3+ or CD45+ surface marker proteins can be used to identify specific immune cells in a tissue sample. The mass-to-charge ratio of such elemental tags can range from approximately 75 to approximately 209 m / z, but are not limited to these examples. Different cell types in a tissue sample can be identified by using different tags.

[0078]

[0092] In various embodiments, metal tags associated with each ablated tissue location are measured simultaneously and indexed at their respective locations. As will be described later, such detection of metal tags makes it possible to identify these tissue locations located within the region of interest.

[0079]

[0093] More specifically, in one implementation of this embodiment, a tissue sample is scanned position by position along a raster scan line, and the gas plume generated through ablation of the material at each position is ionized and mass-spectrometrically to identify, for example, a metal tag of interest and generate an intensity map of the target metal tag at the ablate locations.

[0080]

[0094] Subsequently, the mass signal can be used to identify the location on the microscope slide corresponding to a tissue sample in a location where no tissue is present. For example, if the mass signal corresponding to an ablate location shows no signal associated with any of the metal tags, or if the intensity of the mass signals associated with one or more metal tags is below a predetermined threshold, that location can be identified as corresponding to a microscope slide location where no tissue sample is present and therefore not part of the target region of interest. Alternatively, each ablate location can be identified as part of the region of interest by detecting the mass signal corresponding to one or more metal tags of interest.

[0081]

[0095] For example, in some embodiments where it is preferable to distinguish locations associated with a tissue sample from locations on the glass slide where no tissue is present (i.e., when the region of interest is the entire tissue sample), detection of a mass signal corresponding to at least one of the metal tags can be used to identify each location as part of the tissue sample.

[0082]

[0096] In some embodiments, the region of interest may be a portion of a tissue sample containing one or more cell types of interest. In such cases, the mass signal can be used as a phenotypic signal to identify target cells of interest. For example, if the region of interest corresponds to one or more portions of a tissue sample containing certain immune cells, e.g., immune cells exhibiting CD3+ and CD45+ surface markers, the presence of those cell types at each location can be identified, thereby identifying these locations as part of the region of interest, by utilizing the mass signals corresponding to metal tags associated with antibodies exhibiting specific binding to these surface markers. In some such cases, each location is identified as part of the region of interest only if the mass signal associated with the metal tag shows an intensity above a predetermined threshold. In some cases, each location is identified as part of the region of interest when the mass signal associated with one or more tags exceeds a first threshold and the mass signal associated with a particular other tag falls below a second threshold, and the first and second thresholds may be the same or different. As an example, consider cell types that can be characterized as CDn+, CDm-, CDo+, and CDp-. In this example, such a cell type can be identified if the signals associated with the CDn and CDo mass channel tags exceed their respective thresholds, while the signals associated with the CDm and CDp mass channels fall below their corresponding thresholds.

[0083]

[0097] In some cases, regions of interest can be identified through the detection of collocations or counterlocations of two or more cell types. For example, the presence of two or more cell types of interest within a region can be used as a criterion for identifying that region as a region of interest. Another example is the presence of one cell type and the absence of another within a region. For instance, a high abundance of immune cells, along with a high abundance of cancer cells, may indicate a region of interest. In another example, a high abundance of cancer cells and a low abundance of immune cells may indicate a region of interest.

[0084]

[0098] In this embodiment, rather than sampling the entire surface area of ​​the object of interest, only a portion of its surface is sampled via ablation at predefined locations, with the aim of identifying one or more regions of interest. For example, ablate locations can be separated from each other by a distance that is a multiple of the ablation spot size, for example, a coefficient in the range of about 2 to about 7 greater than the ablation spot size. For example, in some such embodiments, the distance between adjacent ablate locations can be about 25 micrometers, and the ablation spot size is set to 5 micrometers.

[0085]

[0099] Furthermore, surprisingly, it was found that the identification of the region of interest can be achieved using a radiation beam with a large ablation beam size, i.e., a spot size characterized by a diameter of more than approximately 1 micrometer (e.g., in the range of more than 1 micrometer to approximately 10 micrometers). In other words, the region of interest can be identified by using a beam that can simultaneously ablate adjacent portions of multiple cells at the ablation site, rather than using a beam with a subcell size. Identifying the region of interest, and especially using a large radiation beam spot size, in conjunction with sampling a small portion of the region of interest, can significantly reduce the time required for data acquisition and region identification.

[0086]

[0100] As an example, Figure 3 schematically shows an example of a region of interest 300 (shaded region) associated with a tissue sample 100 identified using one embodiment of the teaching described above. More specifically, the locations associated with reference points 104a, 104b, 104c, 104d, 104e, 104f, 104g, 104h, 104i, 104j, 104k, 104l, and 104m are identified as part of the region of interest. In this case, the region of interest is enclosed within a boundary 105, and the boundary surrounds reference points 104a to 104m.

[0087]

[0101] In this example, the region of interest is in the form of a continuous region, but in some cases, one or more discontinuous regions of interest can be identified using this instruction. For example, the region of interest may be a region containing at least one of two cell types of interest. In some such cases, one region of interest may contain one of these cell types, and the other region of interest (e.g., an overlapping or discontinuous region) may contain the other cell type.

[0088]

[0102] In some embodiments, ablation at various locations to determine whether these locations are part of a region of interest involves partial, rather than complete, ablation of the material at those locations. For example, an upper layer of the material, such as a layer with a thickness ranging from about 10 nm to about 1000 nm, may be ablated.

[0089]

[0103] In some embodiments, once one or more regions of interest are identified, an optical image of the tissue sample can be displayed to the user, for example, through a trace that draws the boundary of the region, indicating its contour. Furthermore, a user interface can be presented to the user, allowing the user to manually select, for example, a portion of the image of a specimen, in this example a tissue specimen, within the region of interest.

[0090]

[0104] Once a region of interest is identified, an image of the region (or a portion thereof) is acquired using imaging mass spectrometry. For example, referring to Figure 4, in this embodiment, a radiation beam, for example, the same radiation beam used to identify the region of interest, can be directed to multiple locations within the region of interest so that at least a portion of the tissue sample at each of those locations is ablated. Ablation of the tissue at each location generates a gas-phase sample, which can be ionized to produce ions, which can then be detected and analyzed using a mass spectrometer. For example, as with the analysis of the ablated portion used to identify the region of interest, the gas-phase sample corresponding to each location can be transported via a carrier gas, for example, helium, into an ion source of a mass spectrometer, such as an inductively coupled plasma ion source, to which at least a portion of the gas-phase sample is ionized to produce ions. For example, a metal tag associated with an antibody that shows specific binding to one or more surface markers associated with a cell type of interest can be ionized. The ions are detected via a downstream mass spectrometer, such as a ToF mass spectrometer, which generates a mass signal corresponding to the ions. The mass signals can be indexed with respect to each ablate position that produces these signals.

[0091]

[0105] In various embodiments, the mass signal can be analyzed to generate information about the presence of a cell type of interest at the ablate location. For example, as described above, the detection of ions corresponding to one or more tags can indicate the presence of one or more cell types of interest associated with those tags at a particular ablate location. Such information associated with the ablate location can be used to provide an image of the region of interest. For example, the image can provide the spatial distribution of one or more cell types of interest in the region of interest.

[0092]

[0106] For example, Figure 4 shows multiple locations (also referred to herein as imaging locations) of a tissue sample, indicated by red crosses, which are ablated, and the ablated material is analyzed to determine whether one or more target cell types of interest are present at each of these locations. More specifically, the sample holder can be moved relative to the radiation beam to sequentially expose the imaging locations to pulsed laser radiation so that they are ablated. In this embodiment, the imaging locations are arranged side by side, interleaving a portion of the locations used for identifying the region of interest.

[0093]

[0107] In some embodiments, during the imaging process (e.g., during imaging mode), locations previously ablated (e.g., during survey mode) for the identification of the region of interest are not ablated again. Rather, the mass signals previously acquired for these locations are used to generate an image of the region of interest. Alternatively, in other embodiments, during the imaging process, all locations within the region of interest, including those previously ablated for the identification of the region of interest, are ablated to generate mass signals that can be analyzed for image formation. As an example, in some embodiments, ablation of locations used for identifying the region of interest may result in the ablation of the top layer of the sample at those locations. Subsequently, during imaging mode, the remainder (or portion of the remainder) of the material at these locations may be ablated. In some embodiments, if an ablation spot overlaps with a spot previously ablated in a fractional sampling step during imaging mode, the data acquired in the second ablation is discarded, and the data acquired during the first ablation is used for imaging. In some embodiments, such an arrangement can offer certain advantages. For example, such an arrangement allows for the laser to be emitted at equal intervals without skipping any particular spots. Furthermore, in some embodiments, the ablated spots generated during the survey mode do not overlap, and therefore it is reasonable to use the data acquired during the first ablation in the survey mode to complement the data generated during the imaging mode. In other words, if the ablated spots do not overlap, the signals acquired for a particular location during the survey mode or imaging mode pass can be equally useful for image generation.

[0094]

[0108] In various embodiments, during imaging mode, whether re-ablation of previously ablated locations is not performed, or whether ablation is performed at all locations across the region of interest, including previously ablated locations, the locations identified for ablation to acquire an image of the region of interest can be selected to cover the entire region of interest.

[0095]

[0109] One advantage of this instruction is that, in imaging mode, ablation of locations within the region of interest does not require realigning the radiation, such as a laser beam, with respect to a reference point that identifies the location requiring ablation. In particular, the specimen remains on the specimen holder, and its position on the holder does not change between fractional survey mode and imaging mode for ROIs on the same sample.

[0096]

[0110] The combination of the invariance of the specimen's position on the holder and the typically 1-micrometer or higher precision of the XYZ movable stage used for ablation allows the spots to be ablated during imaging mode to fit comfortably within each spot ablated during survey mode, particularly because the spot size exceeds 1 micrometer. For example, if a 5-micrometer ablate spot size is employed during survey mode, positioning errors of less than 1 micrometer during data acquisition are negligible.

[0097]

[0111] As mentioned above, it has been conventionally recognized in the art that an ablation spot size of 1 micrometer or less is necessary to adequately describe the cell boundaries of typical cells and to estimate cell shape. However, surprisingly, it has been found that by using an ablation beam spot size on the sample surface that is larger than the spatial step required to resolve typical cell size, for example, a beam spot with a diameter greater than 1 micrometer, for example, a beam spot with a diameter in the range of greater than 1 micrometer to about 5 micrometers, the necessary information, such as the types of cell types present at each investigation location, can be obtained to form an image of the region of interest. This is in contrast to conventional teaching in the art that the beam spot size on the sample surface for ablation to occur must be smaller than the typical size of the cell type of interest in order to clearly identify the cell type of interest through cell segmentation and integration of signals within cell boundaries in a region of the sample. In other words, conventionally, those skilled in the art believed that different cell types could not be identified when using an ablation spot size greater than 1 micrometer.

[0098]

[0112] However, as discussed in this disclosure, it has been found that mass data generated via ablation of large portions of a biological specimen can nevertheless be processed to obtain desired imaging information, such as the distribution of specific cell types across a region of interest, identification of the region of interest, and / or information about cell populations generated by neighbor analysis algorithms.

[0099]

[0113] In some embodiments, the classification of each ablation spot larger than 1 micrometer (also referred to herein as pseudotype cells) can be achieved based on the detection or non-detection of a specific phenotypic signal, such as a specific marker, such as a protein surface marker. Such a method may be based on comparing the measured phenotypic signal to a predetermined threshold to determine, for example, whether a pseudotype cell is negative or positive for a given marker. As an example, in some embodiments, the threshold for each marker (each channel) can be selected from a template for a particular analytical run (screening using the same set of reagents).

[0100]

[0114] As an example, Figures 7A, 7B, 7C, and 7D illustrate the identification of various markers based on a comparison of the mass signal intensity of metal tags bound to antibodies that show specific binding to these markers with respect to a given threshold. More specifically, the markers in this example include CD3, CD31, and pankeratin. More specifically, Figure 7A provides an overview. Figure 7B shows the ion signal per pixel for the recorded CD3 channel. Figure 7C now shows the same data after zeroing the signal below the threshold, which appears here as black. Figure 7D shows that the data from Figure 7C is a binary image, where all pixels with values ​​above the threshold (CD3+) are assigned the value "1", and all pixels with values ​​below the threshold (e.g., CD3-) are assigned the value "0".

[0101]

[0115] In other embodiments, the classification of pseudo-cells can be achieved using clustering algorithms. For example, a clustering algorithm known as FlowSOM may be used to combine pseudo-cells into a population that lies in close proximity to each other in a multidimensional signal space.

[0102]

[0116] In various embodiments, following the classification of the phantom cells, a neighborhood analysis can be performed to identify, for example, which classes of cells are neighboring cells of a cell type of interest. The compartment of interest can be identified using neighborhood analysis of the classified pixels. Additionally, in some embodiments, a neural network can be trained to classify compartments with barcoded pixels. Marker expression can be calculated within each compartment.

[0103]

[0117] In some embodiments, the desired imaging information can include, for example, the density of a particular cell type, the probability of the co - location and counter - location of a particular cell type, and other metrics that can be based on neighborhood analysis. As an example, such imaging information can be used, inter alia, for medical diagnosis, prognosis associated with treatment, survival rate, and recommended medical interventions.

[0104]

[0118] As another example, FIG. 8A shows a plurality of phantom cells generated in accordance with an embodiment of the present teachings. By using two mass channels, pixels of four categories are generated, namely, (1) black pixels corresponding to CD3 - and CD31 - ; (2) green pixels corresponding to CD3 + and CD31 - ; (3) red pixels corresponding to CD3 - and CD31 + ; and (4) yellow pixels corresponding to CD3 + and CD31 + . Using the information encapsulated by the classification of the phantom cells based on the above phenotypic signals, various metrics can be derived. As an example, the frequency of different pixel populations can be identified and plotted as a frequency map. The co - location and counter - localization of various cell types can be identified based on the above phenotypic signals. Further, a neighborhood analysis of the pixels can be performed.

[0105]

[0119] Figure 8B provides another example of pseudo-cell classification generated according to one embodiment of this teaching using three mass channels, thereby yielding the following eight pixel categories: (1) Black CD3-, CD31-, Pank-; (2) Green CD3+, CD31-, Pank-; (3) Red CD3-, CD31+, Pank-; (4) Yellow CD3+, CD31+, Pank-; (5) Blue CD3-, CD31-, Pank+; (6) Turquoise CD3+, CD31-, Pank+; (7) Magenta CD3-, CD31+, Pank+; and (8) White CD3+, CD31+, Pank+. Various metrics can be derived from such pseudo-cell classifications, as described above.

[0106]

[0120] For example, one advantage of the method described in this instruction for imaging biological specimens using the aforementioned method is that it requires processing fewer pixels than conventional methods, thus improving data acquisition and analysis speed while simultaneously providing useful data analysis.

[0107]

[0121] By detecting regions of interest, using larger ablation spots and wider spacing between ablation spots for region detection, and imaging those regions, the imaging of biological specimens is significantly accelerated compared to conventional techniques. At the same time, it is possible to form images with sufficient accuracy and resolution, for example, resolutions higher than 5 micrometers, so that the images can be used for the analysis of various biological samples such as tissue, blood, and bone section samples. For example, surprisingly, in various embodiments, it has been found that acquiring images with a spatial resolution of 5 micrometers can provide substantially the same amount of information about a biological specimen as can be acquired using a 1-micrometer spacing, while simultaneously improving the speed of image acquisition by 25 times.

[0108]

[0122] In some embodiments, three-dimensional (3D) tomographic images of a biological specimen, such as a tissue specimen, can be generated by imaging a continuous series of compartments of the specimen using imaging mass cytometry or imaging mass spectrometry. For each compartment, for example, a layer with a thickness ranging from about 30 nm to about 10 μm can be examined during the fractional survey mode according to this teaching to identify a region of interest within that compartment. More specifically, as described above, multiple reference locations for ablation can be identified, so that the set of ablate locations forms a small portion of the layer. In other words, the ablation locations correspond to a small portion of the layer rather than the entire layer.

[0109]

[0123] Mass signals associated with ablate locations are analyzed by the method disclosed herein to identify those locations which are part of the region of interest, which in the case of 3D can be considered as the volume of interest (VOI). Subsequently, an image of the region of interest is generated based on ablation of multiple locations within that region and analysis of the gas-phase sample generated by ablation using, for example, imaging mass cytometry or imaging mass spectrometry, using the teachings herein. In other words, pseudo-cellular data relating to the VOI can be generated after the survey mode. Such data can be processed by cell classification and 3D neighbor analysis. Examples of VOIs in biological samples are blood vessels or fibers or nerve fibers.

[0110]

[0124] This process is repeated for each section, or layer, to form an image of that layer. Images of different layers are then indexed relative to the position of that layer (for example, the depth of each layer relative to the surface of the biological sample) to form a 3D image of the biological sample.

[0111]

[0125] Figure 5 schematically shows an example of an imaging system 1000 according to one embodiment, which can be configured to perform identification and imaging of a region of interest in various embodiments. The imaging system 1000 includes a sample holder 1002, such as a microscope slide, held on an XYZ positioning stage. In this embodiment, the X and Y axes are controlled with positioning repeatability of, for example, 50 nm using high-precision piezo motors or any other suitable linear motor, while a piezo-driven flexure actuator controls the Z axis with positioning accuracy of, for example, 300 nm. The imaging system further includes an airtight sealed ablation cell filled with high-purity helium gas or any other suitable mixed gas (e.g., a mixture of H2, 3% and He, 97%). The output of a fifth-harmonic Nd:YAG (213 nm), diode-pumped solid-state (DPSS) laser is focused onto the surface of the microscope slide through a UV-grade fused silica window using a Schwarzschild objective lens.

[0112]

[0126] A laser beam attenuator is used to adjust the energy of the laser pulses reaching the sample placed on the microscope slide. An LED illumination source that generates visible radiation can be used for visual inspection of the specimen during analysis. The radiation generated by the LED illumination source passes through an aperture and reaches a beam splitter. A portion of the irradiation radiation passes through the beam splitter and microscope tube, is reflected by a silver mirror, propagates collinearly with UV radiation, and is focused onto the sample holder via the objective lens. The other portion of the LED light is directed by the beam splitter to the silver mirror, which reflects this light towards the camera. The ablation chamber is in communication with the mass cytometer 1004. The controller 1006 controls the movement of the sample holder so that various positions on the surface of the sample holder are exposed to UV radiation.

[0113]

[0127] Ablation at the exposed location occurs when the intensity of the focused UV laser light exceeds the ablation threshold of the material. Helium flows from the ablation chamber through a small aperture, where it intersects with an argon gas stream that redirects the ablated material and transports it through a tube to an inductively coupled plasma (ICP).

[0114]

[0128] The analysis module 1007, for example, a standalone computer or computing platform integrated with a mass cytometer, can be configured to process the mass signal generated via the detection of ions produced by ICP, for example, in the manner discussed herein, to form an image of the region of interest of a biological specimen.

[0115]

[0129] A graphical user interface (GUI) 1008 allows the user to communicate with the imaging system. For example, the GUI can provide a display for presenting identified regions of interest to the user, thereby allowing the user to manually select the portion of the region of interest to be imaged.

[0116]

[0130] Defocusing a laser spot on a target can be achieved by various methods. In one embodiment, the Z position is shifted from the position recommended by the autofocus module for the tightest focusing. In another embodiment, an optical element inserted into a beam expander is positioned along its axis to defocus the laser beam at the Z position recommended by the autofocus module. In yet another configuration, a series of beam shaping optics are inserted into the laser's optical path to uniformly distribute energy across the slide surface at the Z position recommended by the autofocus module.

[0117]

[0131] In some embodiments, fractional sampling surveys may be omitted, and the biological sample on the holder (microscope slide) can be identified using optical imaging. This allows imaging modes to be performed on the sample using ablation spot sizes larger than 1 micrometer. Even without fractional sampling surveys, a larger spot size used for imaging allows for faster data acquisition. Such optical detection can also be used to identify various sections of the biological sample when performing 3D imaging, as described above.

[0118]

[0132] In contrast to ablation spots smaller than 1 micrometer, the use of larger ablation spots offers a greater selection of viable radiation beam profiles. For example, in some embodiments, suitable optics can be used to ensure substantially uniform ablation of the ablation spot. In other words, the amount of material ablated across the ablation spot can be substantially uniform. For example, in some embodiments, a flat-top radiation beam can be used to provide a substantially uniform ablation spot. As an example, a flat-top beam with a circular cross-sectional profile with a diameter greater than 1 micrometer, e.g., in the range of about 5 or about 7 micrometers, can be used for this purpose. In some embodiments, the laser beam can be shaped into a uniform (flat-top) distribution of a hexagonal or square profile instead of a conventional circular profile. For example, when the spot size is significantly larger than 1 μm, such as in the case of a 5 μm spot, it becomes easier to create a suitable shape for illumination.

[0119]

[0133] In some embodiments, each location in a sample can be irradiated with multiple radiation pulses, such as multiple laser pulses, to achieve a desired ablation of the material at that location. In other words, a single pixel can be ablated by multiple laser shots. In some embodiments, this can be achieved using a high repetition rate laser, for example, in the range of about 10 kHz to about 10,000 kHz. For example, in some embodiments, such a laser can be directed to each location to be ablated so that each shot ablates a specific layer of the material at that location. In other embodiments, the laser spot is smaller than the area of ​​the pixel, and the area of ​​the pixel can be filled with laser shots using a high-speed steering optical system. For example, such high-speed steering can be implemented using the teachings provided in U.S. Patent Application Publication No. 2021 / 0333173A1, entitled "High-Speed ​​Modulated Sample Imaging Apparatus and Method," which is incorporated herein by reference in its entirety.

[0120]

[0134] Those skilled in the art will see that various modifications can be made to the above embodiments without departing from the scope of this teaching.

Claims

1. A method for imaging biological specimens, Defining a first set of reference points across the surface of the biological specimen, Directing ablation radiation to ablate a small portion of the sample at a first plurality of sample locations corresponding to the defined first reference position point or a subset thereof, thereby ablating at least a portion of the sample at each of the first plurality of sample locations, and thereby generating a gas phase sample from each of the first plurality of sample locations, Each of the aforementioned gas phase samples is ionized to generate ions corresponding to that gas phase sample, Acquiring one or more mass signals corresponding to the ions associated with each of the ablated portions, Based on the analysis of the aforementioned mass signal, at least one region of interest is identified, A method that includes this.

2. The method according to claim 1, wherein identifying the at least one region of interest includes comparing each of the mass signals to at least one threshold criterion defined based on at least one phenotypic signal to determine whether the sample location corresponding to the mass signal lies within the region of interest.

3. The aforementioned at least one phenotypic signal includes a plurality of phenotypic signals, The at least one threshold criterion includes a plurality of threshold criteria, each corresponding to one of the phenotypic signals, The method according to claim 2, further comprising classifying each of the ablated positions in a multidimensional signal space based on the comparison.

4. The method according to claim 3, further comprising performing a neighborhood analysis of the classified ablate locations to identify the at least one region of interest.

5. The method according to claim 1, wherein the at least one region of interest is identified without using fluorescence imaging.

6. The method according to claim 1, further comprising generating an image of the identified region of interest using either image mass cytometry or image mass spectrometry.

7. The method according to claim 1, wherein the ablation radiation is configured to ablate the upper layer of the specimen at one or more of the first plurality of locations.

8. The method according to claim 7, wherein the thickness of the upper layer is in the range of about 10 nm to about 1000 nm.

9. Acquiring an image of the identified region of interest means Directing ablation radiation to a second plurality of locations within the region of interest, thereby generating a gas phase sample from each of the second plurality of portions, The gas phase sample is ionized to generate ions corresponding to each of the second plurality of sample positions, Acquiring one or more mass signals corresponding to ions associated with each of the second plurality of sample positions, Based on the mass signals associated with the second plurality of sample locations, an image of at least a portion of the region of interest is generated. The method according to claim 6, including the method described in claim 6.

10. The method according to claim 9, wherein directing the ablation radiation to the second plurality of locations is performed without realigning the radiation with respect to the defined reference position points.

11. The method according to claim 9, wherein the biological specimen comprises one or more cell types.

12. The method according to claim 11, wherein generating the image comprises analyzing the mass signal associated with each of the second plurality of sample locations to identify one or more cell types of interest at that location.

13. The method according to claim 12, wherein the cell type of interest includes any one of immune cells, structural cells, tumor cells, stromal cells, germinal center cells, vascular cells, mesenchymal cells, or non-mesenchymal cells.

14. The method according to claim 13, wherein the immune cells include either CD3+ or CD45+ cells.

15. The method according to claim 12, wherein the step of identifying one or more cell types of interest is based on the identification of at least a phenotypic signal via the analysis of the mass signal associated with each of the second plurality of sample locations.

16. The method according to claim 15, wherein the at least one phenotypic signal is associated with any of the following: a target protein, target DNA, target RNA, target molecular structure, amino acid sequence, target lipid structure, target phosphorylation region, target sugar region, or a naturally occurring substance or element.

17. The method according to claim 16, wherein the naturally occurring substance or element comprises either selenium or mercury.

18. The method according to claim 16, wherein the at least one phenotypic signal is associated with a drug-related substance.

19. The method according to claim 16, wherein the at least one phenotypic signal is associated with platinum in the chemotherapeutic agent.

20. The method according to claim 16, wherein the at least one phenotypic signal provides information regarding the protein content of different cell types or tissue compartments.

21. The method according to claim 12, wherein the identification of any one of the one or more cell types of interest is based on the detection of a mass signal corresponding to a metal tag bound to an antibody that exhibits specific binding to a surface marker of the cell type of interest.

22. The method according to claim 9, wherein the second plurality of positions are different from the first plurality of positions.

23. The method according to claim 9, wherein the second plurality of positions includes the first plurality of positions.

24. The method according to claim 1, wherein the plurality of reference point locations are distributed according to a regular grid.

25. The method according to claim 24, wherein the regular grid includes any of the grids of rectangles, squares, triangles, and hexagons.

26. The method according to claim 9, wherein both the first and second ablate positions have a maximum linear dimension in the range of more than 1 micrometer to about 20 micrometers.

27. The method according to claim 26, wherein the maximum linear dimension is any one of the following: at least 2 micrometers, at least 3 micrometers, at least 4 micrometers, at least 5 micrometers, at least 6 micrometers, at least 7 micrometers, at least 8 micrometers, at least 9 micrometers, at least 10 micrometers, at least 11 micrometers, at least 12 micrometers, at least 13 micrometers, at least 14 micrometers, at least 15 micrometers, at least 16 micrometers, at least 17 micrometers, at least 18 micrometers, at least 19 micrometers, and at least 20 micrometers.

28. The method according to claim 27, wherein the first ablative position is separated by a distance corresponding to a multiple of the maximum linear dimension.

29. The method according to claim 28, wherein the multiple is in the range of 2 to 10.

30. The first and second ablate positions have substantially circular cross-sectional profiles. The method according to claim 27, 28, or 29, wherein the maximum linear dimension corresponds to the diameter of the substantially circular cross-sectional profile.

31. The method according to claim 9, wherein the set of the second ablate positions is arranged in a horizontal line.

32. The method according to claim 1, wherein the biological specimen includes any of a tissue specimen, a bone section, a blood specimen, an organoid section, or a cell culture specimen.

33. The method according to claim 1, wherein the radiation includes laser radiation.

34. The method according to claim 33, wherein the wavelength of the laser radiation is in the range of about 10 nm to about 10,000 nm.

35. The method according to claim 34, wherein the wavelength of the laser radiation is in the range of about 100 nm to about 1000 nm.

36. The method according to claim 34, wherein the beam size of the laser radiation on the surface of the specimen is in the range of about 1 micrometer to about 20 micrometers.

37. The fluence of the laser radiation on the sample surface is approximately 0.001 J / cm². ２ ~About 10J / cm ２ The method according to claim 29, which is within the range of the present invention.

38. The method according to any one of claims 33 to 37, wherein the laser emission has a Gaussian intensity profile, and the beam size corresponds to the full width at half maximum (FWHM) of the Gaussian intensity profile.

39. The method according to any one of claims 33 to 37, wherein the laser emission has a flat-top intensity profile.

40. The method according to claim 1, wherein the ablation radiation is configured to perform substantially uniform ablation at the plurality of locations.

41. The method according to claim 1, further comprising using a mass spectrometer to generate the mass signals corresponding to any of the first plurality of sample portions and the second plurality of sample portions.

42. The method according to claim 41, wherein the mass spectrometer includes a high-parameter mass spectrometer, and optionally the high-parameter mass spectrometer includes a ToF mass spectrometer.

43. The method according to claim 1, further comprising generating an optical image of the biological sample along with a trace outlining the identified region of interest.

44. The method according to claim 43, further comprising presenting the optical image to the user using a user interface display.

45. The method according to claim 44, wherein the user interface is configured to allow the user to select a location within the identified region of interest for imaging.

46. The method according to claim 1, further comprising using a digital data processor to analyze the mass signal for identification of the region of interest.

47. The method according to claim 7, wherein the sample is placed on a sample holder.

48. The method according to claim 47, wherein the sample holder is moved with respect to the radiation so that the radiation is directed toward one of the first and second sample positions.

49. The method according to claim 1, wherein the radiation includes pulsed radiation, and a plurality of radiation pulses are directed toward at least one of the positions such that at least a portion of the specimen is ablated at the position.

50. The method according to claim 49, wherein the radiated pulse is directed to at least one of the positions at a repetition rate in the range of about 10 kHz to about 10,000 kHz.

51. The aforementioned radiation is directed towards the biological specimen as a radiation beam. The method according to claim 47, wherein the radiation beam is moved relative to the specimen holder to ablate either the first or second specimen portion.

52. A method for identifying at least one region of interest associated with a biological specimen, Multiple parts of the aforementioned sample are ablated by radiation, and gas-phase samples are prepared from each part. Ionizing at least a portion of the gas phase sample from each of the plurality of sample portions to generate ions associated with two or more of the sample portions, To obtain the mass signal corresponding to the ion associated with the two or more sample portions, Based on the analysis of the mass signals associated with the two or more sample portions, at least one region of interest is identified. A method that includes this.

53. The method according to claim 52, wherein analyzing the mass signals includes comparing each of the mass signals with a reference threshold to determine whether each portion of the sample is part of the region of interest.

54. The method according to claim 53, further comprising classifying any of the sample portions as being within the region of interest when the mass signal associated with the sample portion is greater than the reference threshold.

55. The method according to claim 52, wherein analyzing the mass signal includes comparing the two or more sample portions to determine whether any of the two or more sample portions is part of the region of interest.

56. The method according to claim 52, wherein analyzing the mass signal includes comparing the mass signal associated with at least one of the sample portions with the mass signal associated with an adjacent sample portion.

57. The method according to claim 52, wherein analyzing the mass signal includes classifying any of the sample portions as being within the region of interest when the mass signal associated with the sample portion is within a predetermined range.

58. The method according to claim 52, wherein the collection of the plurality of sample portions corresponds to a small portion of the sample.

59. The method according to claim 52, wherein each of the sample portions has a maximum linear dimension greater than 1 μm, and optionally in the range of greater than 1 μm to about 20 μm.

60. The method according to claim 52, wherein each of the sample portions has a maximum linear dimension of at least 2 μm.

61. The method according to claim 52, wherein each of the sample portions has a maximum linear dimension of at least 3 μm.

62. The method according to claim 52, wherein each of the sample portions has a maximum linear dimension of at least 4 μm.

63. The method according to claim 52, wherein each of the sample portions has a maximum linear dimension of at least 5 μm.

64. The method of claim 52, further comprising using a digital data processor to carry out the step of analyzing the mass signal.

65. The method according to claim 52, wherein the sample portions are arranged according to a regular grid with respect to each other.

66. The method according to claim 65, wherein the grid includes any of the grids of squares, rectangles, triangles, and hexagons.

67. The biological sample is further functionalized with at least one antibody tagged with at least one metal tag, The method according to claim 52, wherein the antibody exhibits specific binding to a target surface cell marker.

68. The method according to claim 67, wherein the ionization step includes ionizing the metal tag.

69. The method according to claim 67, further comprising generating an image of at least a portion of the identified target region using a mass signal associated with the at least one metal tag.

70. The method according to claim 52, wherein the biological specimen includes any of a tissue specimen, a bone section, a blood specimen, an organoid section, and a cell culture specimen.

71. A method for identifying one or more regions of interest in a biological specimen, (a) Ablate a portion of the sample with radiation to create a gas phase sample, (b) Ionizing at least a portion of the gas phase sample, (c) To generate a mass spectrum including at least one mass signal from the ionized portion of the gas phase sample, (d) Comparing the at least one mass signal with at least one threshold defined based on at least one phenotypic signal, (e) Identifying the ablated portion as part of the region of interest when at least one mass signal is greater than or equal to a threshold for comparison, (f) Repeating steps (a) to (e) for one or more different parts of the biological specimen, (g) Identifying one or more regions of interest of the biological specimen based on two or more of the ablated portions identified as part of the region of interest, A method that includes this.

72. The method according to claim 71, wherein the at least one mass signal includes a plurality of mass signals.

73. The method according to claim 71, wherein the at least one phenotypic signal includes a plurality of phenotypic signals.

74. The method according to claim 73, wherein the comparison step includes comparing each of the phenotypic signals with each of a plurality of thresholds.

75. The method according to claim 71, further comprising obtaining a substantially complete image of one or more identified regions of interest.

76. The method of claim 75, wherein the substantially complete image of the identified one or more regions of interest is obtained without re-ablating the portion of the specimen ablated to identify the one or more regions of interest.

77. The method of claim 75, wherein the substantially complete image of the identified one or more regions of interest is obtained by re-ablating the portion of the specimen ablated to identify the one or more regions of interest.

78. The method according to claim 71, wherein the at least one phenotypic signal is associated with a target protein, target DNA, target RNA, target molecular structure, or amino acid sequence.

79. The method according to claim 59, wherein the sample portions ablated to identify one or more regions of interest are separated from each other by a distance that is a multiple of the maximum linear dimension size of the ablated portion.

80. The method according to claim 67, wherein the multiplier is in the range of approximately 2 to approximately 7.

81. A method for generating three-dimensional tomographic images of a biological specimen, To define multiple compartments of the aforementioned biological specimen, For each of the aforementioned sections, The steps include defining a plurality of reference points across the surface of the biological specimen, The steps include directing ablation radiation to the biological sample at a first plurality of sample locations corresponding to a first subset of the defined reference position points, thereby ablating at least a portion of the sample at each of the locations, and thereby generating a gas-phase sample from each of the sample locations, The steps include ionizing each of the aforementioned gas phase samples to generate ions corresponding to the gas phase sample, The steps include acquiring one or more mass signals corresponding to the ions associated with each of the ablated portions, The steps include identifying at least one region of interest based on the analysis of the mass signal, The steps include generating an image of at least a portion of the identified at least one region of interest, To perform, A method that includes this.

82. The step of generating the image of the identified at least one region of interest is: Directing ablation radiation to a second set of locations within the region of interest associated with a second subset of the reference point, thereby generating gas-phase samples from each of the portions, The gas phase sample is ionized to generate ions corresponding to each of the second plurality of sample positions, To acquire one or more mass signals corresponding to ions associated with each of the second plurality of sample positions, Based on the mass signals associated with the second plurality of sample positions, an image of at least a portion of the region of interest is generated. The method according to claim 81, including the method described in claim 81.

83. A method for imaging biological specimens, To acquire optical images of biological specimens placed on a holder, Directing ablation radiation to multiple parts of the biological specimen and generating a gas-phase sample from each of the parts, The aforementioned gas-phase sample is ionized to generate ions corresponding to each of the multiple sample positions, To acquire one or more mass signals corresponding to ions associated with each of the aforementioned plurality of sample locations, Based on the mass signals associated with the plurality of sample locations, an image of at least a portion of the biological sample is generated. A method that includes this.

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