Methods for generating enhanced multiplex images

Abstract

Disclosed are methods for generating enhanced multiplex images. The methods may comprise a blank image for each set of markers prior to imaging the markers and subtracting out the blank image intensity from the acquired image of interest. The methods may further comprise acquiring a pre-run blank of the sample prior to collecting an image of interest.

Description

IU 2024-155-02Quarles 144578.00457METHODS FOR GENERATING ENHANCED MULTIPLEX IMAGESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Appl. No. 63 / 687,635, filed August 27, 2024, the entire content of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under AG082275, DK106846, and DK118782 awarded by National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Bone marrow is a tissue that is of great importance to various areas of research such as hematology, oncology, bone biology, and immunology to name a few. It is also a unique tissue being gelatinous in nature but housed in a hard casing of bone. Traditionally, techniques such as flow cytometry and immunofluorescence have been employed to study cellular composition and interactions. However, it has been challenging to study bone marrow in an unperturbed state using multiple fluorescent probes to visualize the diverse cell populations in the same tissue section at a time. Therefore, there is a need in the art for improved methods to generate multiplex microscopic images of intact tissues, e.g., bone marrow.SUMMARY

[0004] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods comprise: (A) acquiring a first blank image of a sample comprising a first set of target specific markers wherein the first blank image is acquired for a first duration of time; (B) contacting the sample of (A) with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of markers; (C) acquiring a first sample image of the sample of (B) for the first duration of time. In some embodiments, the methods further comprise subtracting the first blank image from the first sample image to generate a first corrected image. In some embodiments, the first duration of time is pre-determined based on an optimized duration of exposure of the first set of reporter molecules bound to the first set of target specific markers. In someIU 2024-155-02Quarles 144578.00457 embodiments, the methods further comprise: (D) removing the first set of reporter molecules and / or removing a signal from the first set of reporter molecules from the sample; (F) acquiring an Nthblank image of the sample for an Nthset of target specific markers over an bfhduration of time, wherein N is a positive integer; (E) contacting the sample of (D) with an N11' reporter solution, wherein the N* reporter solution comprises an N111set of reporter molecules that are specific for the N111set of target specific markers; and (F) acquiring an N111sample image of the sample of (E). In some embodiments, the methods further comprise subtracting the N* blank image from the N* sample image to generate an N* corrected image. In some embodiments, the methods further comprise combining one or more of the first corrected image and the N111corrected image to generate a corrected multiplex image of the sample. In some embodiments, the method further comprises acquiring at least one pre-run blank prior to step (A) of the method. In some embodiments, the pre-run blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the composition comprising the first set of target specific markers, and the first blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the first reporter solution. In some embodiments, the pre-determined number of captures is an integer from 1 to 100. In some embodiments, the pre-determined number of captures is an integer from 1 to 20. In some embodiments, the pre-determined number of captures is an integer from 5 to 10. In some embodiments, the predetermined number of captures is 7. In some embodiments, the each of the first set of target specific markers comprise a first polynucleotide and the first set of reporter molecules each comprises a first hybridizing polynucleotide that is complementary to the first polynucleotide. In some embodiments, each of the N111set of target specific markers comprise an N* polynucleotide and the N111set of reporter molecules each comprise an N* hybridizing polynucleotide that is complementary to the first polynucleotide. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a demineralized bone sample comprising bone marrow. In some embodiments, the sample is a sectioned frozen sample embedded in tissue freezing medium. In some embodiments, acquiring comprises acquiring an image using a digital microscope with automated liquid handling. In some embodiments, at least one of the target specific markers in the first set to the N111set of target specific markers comprises a marker specific for any one of TERI 19, CD41, CD117, CD150, Grl, CD48, endomucin B, alpha actinin, B220, fibromodulin, emilin2, CD45, EFEMP2, CRTAP, LUMICAN, CD31, PEDF, CD110, CD115, fibulin2, CollA, F4 / 80, CFGRP, CD71, CD61, and Sca-1.IU 2024-155-02Quarles 144578.00457

[0005] Tn some embodiments, the methods are methods of acquiring an image of a sample, and the methods comprise: (A) acquiring a pre-run blank of the sample; (B) contacting the sample with a composition comprising a first set of target specific markers and acquiring a first blank image of the sample over a first duration of time; (C) contacting the sample of (B) with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of target specific markers; and (D) acquiring a first sample image of a sample of (C) comprising the first set of target specific markers for the first duration of time; thereby acquiring an image of the sample. In some embodiments, the methods further comprise subtracting the first blank image from the first sample image to generate a first corrected image. In some embodiments, the first duration of time is pre-determined based on an optimized duration of exposure of the first set of reporter molecules. In some embodiments, the methods further comprise: (E) removing the first set of reporter molecules and / or removing a signal from the first set of reporter molecules from the sample; (F) acquiring an N* blank image of the sample for an N* set of target specific markers over an N111duration of time, wherein N is a positive integer; (G) contacting the sample of (F) with an N* reporter solution, wherein the N* reporter solution comprises an N111set of reporter molecules that are specific for the Nlhset of target specific markers; and (H) acquiring an N111sample image of the sample of (G) comprising the first set of target specific markers; thereby acquiring an N111image of the sample. In some embodiments, the methods further comprise subtracting the N* blank image from the N* sample image to generate an Nthcorrected image. In some embodiments, the methods further comprise combining the first corrected image and the N111corrected image to generate a corrected multiplex image of the sample. In some embodiments, wherein the pre-run blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the composition comprising the first set of target specific markers, and the first blank comprises the pre-determined number of acquisitions of the sample before contacting the sample with the first reporter solution. In some embodiments, the N11’ blank image comprising the pre-determined number of acquisitions of the sample before contacting the sample with the N11' reporter solution. In some embodiments, the predetermined number of captures is an integer from 1 to 100. In some embodiments, the pre-determined number of captures is an integer from 1 to 20. In some embodiments, the pre-determined number of captures is an integer from 5 to 10. In some embodiments, the pre-determined number of captures is 7. In some embodiments, the each of the first set of target specific markers comprise a first polynucleotide and the first set of reporter molecules each comprise a first hybridizing polynucleotideIU 2024-155-02Quarles 144578.00457 that is complementary to the first polynucleotide. In some embodiments, each of the N11' set of target specific markers comprise an N* polynucleotide and the N* set of reporter molecules each comprise an N* hybridizing polynucleotide that is complementary to the first polynucleotide. In some embodiments, the sample is a fixed sample. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a demineralized bone sample comprising bone marrow. In some embodiments, the sample is a sectioned frozen sample embedded in tissue freezing medium. In some embodiments, acquiring comprises acquiring an image using a digital microscope with automated liquid handling. In some embodiments, at least one of the target specific markers in the first set to the N11' set of target specific markers comprises a marker specific for any one of TERI 19, CD41, CD117, CD 150, Grl, CD48, endomucin B, alpha actinin, B220, fibromodulin, emilin2, CD45, EFEMP2, CRTAP, LUMICAN, CD31, PEDF, CD110, CD115, fibulin2, CollA, F4 / 80, CFGRP, CD71, CD61, and Sca-1.

[0006] In some embodiments, the methods are methods of generating a corrected multiplex image of a sample, and the methods comprise: (A) acquiring a pre-run blank of the sample; (B) contacting the sample with a composition comprising a first set of target specific markers and acquiring a first blank image for a first set of target specific markers over a first duration of time; (C) contacting the sample with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of target specific markers, wherein each of the first set of target specific markers comprise a first polynucleotide and the first set of reporter molecules each comprise a first hybridizing polynucleotide that is complementary to the first polynucleotide; (D) acquiring a first sample image of a sample comprising the first set of target specific markers for the first duration of time; (E) subtracting the first blank image from the first sample image to generate a first corrected image; (F) removing the first set of reporter molecules and / or removing a signal from the first set of reporter molecules from the sample; (G) contacting the sample with an N111set of target specific markers and acquiring an N111blank image for an Nthset of target specific markers over an N* duration of time, wherein N is a positive integer; (H) contacting the sample with an Nthreporter solution, wherein the N* reporter solution comprises an N111set of reporter molecules that are specific for the N* set of target specific markers, wherein each of the N* set of target specific markers comprise an N* polynucleotide and the N* set of reporter molecules each comprise an Nthhybridizing polynucleotide that is complementary to the first polynucleotide; (I) acquiring an N111sample image of the sample comprising the first set of target specific markers; (J) subtracting the N111blank image fromIU 2024-155-02Quarles 144578.00457 the N11' sample image to generate an 14thcorrected image; and (K) combining the first corrected image and the N* corrected image to generate a corrected multiplex image of the sample, wherein acquiring comprises acquiring an image using a digital microscope with automated liquid handling. In some embodiments, the pre-run blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the composition comprising the first set of target specific markers, and the first blank comprises the pre-determined number of acquisitions of the sample before contacting the sample with the first reporter solution. In some embodiments, the pre-determined number of captures is 7. In some embodiments, the sample is a demineralized bone sample comprising bone marrow.BRIEF DESCRIPTION OF THE FIGURES

[0007] FIGs. 1A-1C show side-by-side comparison of standard image processing protocol “standard processing” and the innovative methods of processing of this disclosure “IU- image processing pipeline with extra blanks and background subtraction.”

[0008] FIGs. 2A-2F show a schematic of the Phenocycler® 2.0 multiplex imaging showing the sequential steps of oligo-barcode addition, imaging, and washing out the oligo-barcodes. The process follows these steps: (FIG. 2A) Addition of oligo-barcode conjugated antibodies on tissue section on a glass slide. (FIG. 2B) Complimentary oligo-barcode fluorophores bind to their respective barcode conjugated antibodies. (FIG. 2C) Imaging. (FIG. 2D) Wash steps to remove oligo-barcode fluorophores. (FIG. 2E) Sequential application of next set of oligo-barcode fluorophores. (FIG. 2F) Imaging.

[0009] FIGs. 3A-3E show Presence of autofluorescence due to RBCs and comparison of perfused and non-perfused femurs. (FIG. 3A) Autofluorescent RBCs in 550 nm channel, (FIG. 3B) Autofluorescent RBCs in 647 nm channel, and (FIG. 3C) Autofluorescent RBCs in 750 nm channel. Scale bar: 20 pm. Even though the RBCs fluoresce to a lesser extent in 750 nm channel, they were visible but dim. (FIG. 3D) Perfused femur showing empty spaces due to probable tissue loss. Perfusion led to the loss of vascular structures, even though, not an entire loss of the signal from CD31+ and SCA1+ cells. (FIG. 3E) Non-perfused femur had well-preserved vasculature shown here as SCA1+ and CD31+ capillary. Non-perfused femur had better tissue integrity compared to the perfused femur. Scale bar: 200 pm.IU 2024-155-02Quarles 144578.00457

[0010] FIGs. 4A and 4B shows a comparison of workflows Akoya Biosciences® image processing pipeline and customized image processing pipeline with interleaved blanks and image processing using .raw.qptiff files generated after the completion of Phenocycler® 2.0 run. (FIG. 4A) Output image from Akoya Phenocycler® 2.0 image processing pipeline (QPTiff) viewed using Phenochart ™ showing saturated signal from markers CD31, SCA1, and Endomucin (all red), CD41 (blue) and a-SMA (green). (FIG. 4B) Output image using our image processing described in our protocol of the same tissue showing specific and clear labeling of CD31, SCA1, and Endomucin (all red), CD41 (blue) and a-SMA (green).

[0011] FIG. 5 shows experimental set-up for Phenocycler® 2.0 run showing pre-run (pre-treat) blanks along with interleaved blanks for the respective markers. Pre-treat blanks (first 7 cycles without fluorescent reporter oligo-barcodes) were added for the autofluorescence to plateau before the actual cycles with antibodies start. The interleaved individual blanks corresponding to the cell or structural marker were added to get possible background subtraction using the ‘.raw.qptiff images. The blanks according to Akoya Biosciences® must still be set-up for the system to run.

[0012] FIG. 6 shows representative images of gel electrophoresis of conjugated antibodies verifying the conjugation of the oligo-barcode onto the antibody. The unconjugated antibody shows a light chain and a heavy chain band (red circles). After conjugation, there is a shift in the band size due to the addition of oligo-barcode via a maleimide bond and the band splits into multiple bands (indicated by blue squares).

[0013] FIG. 7 shows autofluorescence in murine bone marrow samples. Different fixatives and embedding techniques have different effects on the autofluorescence of murine bone marrow samples. Cryosectioned OCT-embedded bone marrow samples have reduced autofluorescence compared to paraffin-embedded samples. Methanol: Acetone (1 : 1) showed the least autofluorescence in the cryosectioned samples. Scale bar: 1000 pm.

[0014] FIG. 8 shows representative images of IF validations of in-house conjugated antibodies. Green shows the conjugated antibody and blue is DAPI for nuclear stain. Scale bar: 100 pm.

[0015] FIG. 9 shows workflow for executing steps for different stages in tissue and custom- conjugated antibodies for Phenocycler® 2.0 run.IU 2024-155-02 Quarles 144578.00457

[0016] FIG. 10 shows workflow for staining the cryosectioned tissue for Phenocycler® 2.0 run.

[0017] FIGs. 11A-11E show a comparison of imaging results obtained by using built-in image processing vs our imaging pipeline. (FIG. 11 A) Regression analysis showing the comparison between cell percentages obtained by HALO® analysis using Phenocycler® 2.0 images vs cell percentages from flow cytometry of C57BL / 6J femurs for CD45, B220, Teri 19, GR1, and CD41 markers. Difference between the final image output using (FIG. 1 IB) built-in image processing and Phenochart™ (QPTiff) vs (FIG. 11C) image obtained by our protocol using ‘.raw.qptiff files and manual background subtraction using FIJI of the same tissue. Markers shown are CD31 SCA1 Endomucin (all red), GR1 (cyan) and CD41 (blue). (FIG. 1 ID) Higher-magnification image of the area shown in white square from built-in image processing. Scale bar 50 pm. (FIG. 1 IE) Higher- magnification image of the area shown in white square from image obtained by our protocol. Scale bar 50 pm. Signal from GR1 and CD41 markers was over- saturated in QPTiff image (FIG. 11B) and the cell types could not be segmented for HALO® image analysis.

[0018] FIGs. 12A-12B show images of different cell and structural markers in murine femur on Silane-treated slide from Phenocycler® 2.0 imaging. (FIG. 12A) Whole femur showing markers CD45, TERI 19, GR1, SCA1, Endomucin, and CD31. Scale bar: 1 mm. Inserts below show higher- magnification multiplex images of different cell types and arterioles. Scale bar: 20 pm. Individual markers for cell surface antigens and vascular structures at higher magnification are shown below the multiplex images. (FIG. 12B) Other structural and cellular markers shown at higher magnification.

[0019] FIGs. 13A-13F show comparison between Cell-Tak and Silane as tissue adhesives. (FIG. 13 A) Murine femur from C57BL / 6J on Cell-Tak™ coated slide at lower magnification and (FIG. 13B) Murine femur from C57BL / 6J on Silane-treated slide showing lymphocytes (CD45 positive), vascular structures (CD31, SCA1, and Endomucin positive) and MKs (CD41 positive) at lower magnification. Scale bar: 500 pm. (FIG. 13C) Murine femur from C57BL / 6J on Cell-Tak coated slide from region shown in red on Cell-Tak™ coated slide at higher magnification and (FIG. 13D) Murine femur from C57BL / 6J on Silane-treated slide from region shown in red on Silane-treated slide at higher magnification. Scale bar: 100 pm. Both tissue adhesives performed adequately during CODEX runs. (FIG. 13E) Murine femur from C57BL / 6J on Cell-Tak™ coated slide and (FIG. 13F) Murine femur from C57BL / 6J on Silane-treated slide showing vascular markers (CD31, SCA1, Endomucin), CD41,IU 2024-155-02Quarles 144578.00457 and Col la. Scale bar: 1 mm. Cell-Tak™ coated slide was able to retain tissues adjacent to bone marrow, such as bone matrix and muscles, better (FIG. 13E) than the Silane-treated slide (FIG. 13F) as the Silane-treated slide had few areas of the adjacent tissues and showed some Col la staining and skeletal muscle shown by the arrow.

[0020] FIGs. 14A-14D show differences between bone marrow of a wild-type mouse and inducible MK ablation Cre mouse model shown by Phenocylcer 2.0 multiplex imaging. Phenocycler® 2.0 imaging of (FIG. 14A) C57BL / 6I and (FIG. 14B) DT-injected PF4Cre; iDTR femurs at lower magnifications. (FIG. 14C) C57BL / 6J and (FIG. 14D) DT-injected PF4Cre; iDTR femurs at higher magnifications. PF4Cre; iDTR is an inducible Cre model to ablate MKs. SCA1 was selected as a marker to show vasculature and the differences between the mouse models and highlight the drastic reduction in the MKs. (FIG. 14C) CD41, a marker for MKs and platelets is seen in blue in the femur of a C57BL / 6J mouse along with SCA1, showing arterioles. (FIG. 14D) CD41 marker is not seen in the femur of a DT-injected PF4Cre; iDTR mouse, suggesting ablation of MKs.

[0021] FIGs. 15A-15C show Phenocycler® 2.0 imaging of C57BL / 6J showing CMP, CLP, and HSC cell populations. (FIG. 15 A) CMP cells were identified as Lineage- SCAl- CD117+. (FIG. 15B) CLP cells were identified as Lineage- CD117- SCA1+. (FIG. 15C) Primitive progenitors were identified as LSK (Lineage- SCAl + CD117+). Scale bar: 20 pm.DETAILED DESCRIPTION

[0022] Disclosed herein are methods for generating enhanced multiplex micrographs. The inventors discovered that interleaving blanks, i.e., a sample comprising only primary antibodies, into multiplex imaging protocols immediately preceding capturing a desired micrograph image, i.e., a micrograph with the same primary antibodies and with secondary reporter molecules, e.g., fluorescent reporter molecules, led to improved microscopy images. The inventors subtract the interleaved blank image intensities from the captured desired micrograph image to generate the improved microscopy images. The improved microscopy images could then be stacked to create improved multiplex images.

[0023] In addition, the inventors discovered that autofluorescence in captured images increased in intensity for an empirically determined number of captures, until it unexpectedly plateaued. The inventors leveraged this discovery and implemented a series of blank image captures preceding the start of the experiment, thereby preventing the accumulation of autofluorescence during the initialIU 2024-155-02Quarles 144578.00457 cycles of imaging which could confound image analysis. FIGs. 1 A, IB, and 1 C show a side-by-side comparison demonstrating a large and unexpected (1) increase in image contrast and (2) reduction in autofluorescence using the disclosed methods as compared to prior methods (3) the signal from the probes is specific to the markers and the cell percentages when quantified using Indica Labs HALO® software were found to be in agreement with the published literature determined by flow cytometry, the gold standard technique for cell population quantification.Methods

[0024] In an aspect of this disclosure, methods are provided. In some embodiments, the methods comprise (A) acquiring a first blank image of a sample comprising a first set of target specific markers wherein the first blank image is acquired for a first duration of time; (B) contacting the sample of (a) with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of markers; (C) acquiring a first sample image of the sample of (B) for the first duration of time.

[0025] The disclosed methods are designed to be used with an imaging device such as a digital microscope. Therefore, as used herein, “acquiring” refers to image acquisition using a digital microscope which can be performed with a variety of instruments and controller programs that are commercially available. Image acquisition may be described in terms of time of acquisition, e.g., milliseconds, seconds, etc. The terms “capture,” “acquire,” and “obtain” an image or a blank are used interchangeably herein.

[0026] As used herein, “marker” refers to a molecule or composition that binds to a target molecule on a sample, e.g., the marker may be an antibody, e.g., a monoclonal antibody. Markers may comprise a polynucleotide, e.g., may be chemically linked to a polynucleotide.

[0027] As used herein, “reporter molecule” refers to a molecule that comprises a reporter linked to a moiety that binds to a marker. A “reporter” may be, e.g., a fluorescent molecule. The moiety that binds to a marker may be a polynucleotide, i.e., a marker may comprise a first polynucleotide and a reporter molecule may comprise a first hybridizing polynucleotide that is fully or partially complementary to the first polynucleotide such that the first polynucleotide and the first hybridizing polynucleotide hybridize to bind the reporter molecule to the marker. Similarly, the moiety that binds to a marker may be a polynucleotide, i.e., a marker may comprise an N* polynucleotide and a reporter molecule mayIU 2024-155-02Quarles 144578.00457 comprise an N* hybridizing polynucleotide that is fully or partially complementary to the N111polynucleotide such that the N111polynucleotide and the N* hybridizing polynucleotide hybridize to bind the reporter molecule to the marker.

[0028] The inventors acquired “blank” images in order to subtract background fluorescence, e.g., comprising autofluorescence, from desired micrographs. As used herein, a “blank image” refers to an image that is acquired without any reporter molecules, e.g., without any fluorescent reporter molecules. A blank image may be an image acquired after a marker is added to the sample, and before a reporter molecule is added to the sample (e.g., after an antibody binds to a target molecule, but prior to adding a fluorescent moiety to the sample). Blank images may be taken of a sample, in certain situations, where the sample comprises reporter molecules that are not intended to be captured by the instrument (e.g., imaging device or digital microscope). In some embodiments, a blank image of a sample may be captured by an instrument that is filtering out the emission spectrum, or a large proportion of the emission spectrum, e.g., the peak emission spectrum, of a given reporter molecule.

[0029] A pre-run blank is acquired before a target-specific marker and / or a reporter molecule is added to the sample. Acquiring a pre-run blank comprises exposing the sample to excitation, e.g., UV light, visible light, and / or infrared light, etc., with or without acquiring an image of the sample. Without wishing to be limited by any theory or mechanism, pre-run blanks may be used for the autofluorescence to plateau before the reporter molecules are added and / or blanks collected.

[0030] The inventors subtracted out each blank image from the acquired image of the sample after the sample was contacted with an appropriate reporter solution, thereby, reducing background and improving desired reporter signal contrast. “Subtracting” a blank image, and grammatical variations thereof, from an acquired image of interest may be performed by a variety of methods known in the art, e.g., using the Image Calculator Subtract function in Fiji / ImageJ. For example, Cycle XX_Markerl_550_O (original image for the marker) - Cycle XX_Markerl_550_B (blank image for the marker) = Cycle XX_Markerl_550_S (subtracted image for the marker).

[0031] In order to determine what constitutes true signal vs random noise in our sample, a blank that contains no fluorescent probe is acquired, and then a blank image with the probes that specifically bind our markers of interest is acquired.IU 2024-155-02Quarles 144578.00457

[0032] When blanks are captured, the base level fluorescence of the tissue, the “background” that is present constantly in the sample is obtained. There are many things that can cause this background from naturally occurring fluorescent molecules, to chemicals or chemical treatments of the tissues resulting in increased autofluorescence.

[0033] Next, a reporter solution is added (e.g., comprising probes, fluorescent probes, fluorescent moieties, etc.), which are designed to bind specifically to our protein (marker) of interest. Images are then acquired using the same settings as were used to acquire the blank image. The resulting image will have the background levels of autofluorescence that are present in the blank that was previously acquired, plus the true signal that the inventors aim to isolate and analyze. In order to get the best uncontaminated signal to investigate, the blank image (pre-run blank and / or first blank after adding target specific markers) is subtracted from the second image (background + specific probes), which results in an image with specific probes only.

[0034] To further define “subtraction”, it is important to note that every pixel in a fluorescent image has a value representing the intensity of the signal in that pixel. In some embodiments, image subtraction is performed on a pixel by pixel basis, for each marker. So when background is removed, the intensity value of each pixel is subtracted from the corresponding pixel in the “background + specific probes” image, e.g. the top left pixel in background + specific probes has a value of 200, the top left pixel in background has a value of 150, the resulting subtraction has a pixel in the top left with a value of 50 that is theoretically from only true signal.

[0035] The inventors discovered that utilizing interleaved blanking with optimized acquisition times provides for better, more accurate background subtraction In other words, the duration of exposure of a captured image is optimized for each set of markers and blank images are most effective at removing background when they are interleaved, i.e., acquired in between captures images of the sample (nonblank images that comprise reporter molecules) and acquired for the same or similar duration, e.g., about the same duration or about 5% greater or less time, about 10% greater or less time, about 15% greater or less time, or about 20% greater or less time than the acquired image of the sample. Put another way, the first duration of time may be pre-determined based on an optimized duration of exposure of the first set of reporter molecules and the first blank image is acquired for a similar amount of time.IU 2024-155-02Quarles 144578.00457

[0036] In some embodiments, the method may further include removing the first reporter molecules and / or removing a signal from the first set of reporter molecules in order to acquire a multiplex image. Removing the first set of reporter molecules may include physically removing the reporter molecules (e.g., washing the sample to remove the reporter molecules). In some embodiments, removing a signal from the first set of reporter molecules may include quenching the first set of reporter molecules, or using filters in the imaging device to block the signal from the first set of reporter molecules.

[0037] As used herein, “N” refers to a positive integer. For example, the disclosed methods may further comprise acquiring an N111blank image of the sample for an N* set of target specific markers over an N* duration of time, wherein N is a positive integer; contacting the sample of with an N* reporter solution, wherein the N* reporter solution comprises an N* set of reporter molecules that are specific for the Nlhset of target specific markers; and acquiring an N* sample image of the sample comprising the N* target specific markers and N111reporter molecules . Thus, the “N111” iteration of a “set of markers,” “duration of time,” “reporter solution,” or “image,” etc., may be the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentieth, etc. iteration of the particular element. The method may include removing the N111set of reporter molecules before continuing to the subsequent iteration of image acquisition (e.g., removing the second reporter molecules, before repeating the steps with the third set of target specific markers and third set of reporter molecules.

[0038] Markers may be contacted to a sample as a “set” where multiple markers are added at the same time. Similarly, “sets” of reporter molecules may be added to bind to the “set” of markers, where a single reporter molecule binds to a single marker, allowing for multiplexed imaging.

[0039] As used herein, “contacting” refers to contacting a sample directly or indirectly, e.g., during microscopic sample preparation. Contacting a sample may include addition of a compound or a set of compounds, e.g., a set of markers or a set of reporter molecules to a sample, encompassing incubating, sectioned samples placed on a slide or in a humidity chamber, etc.

[0040] The inventors discovered that autofluorescence in captured images increased in intensity for an empirically determined (pre-determined) number of captures, also referred to as “cycles,” until it unexpectedly plateaued. The inventors leveraged this discovery and implemented a series of blank image captures, in essence exposing the sample to several rounds of light, preceding the start of theIU 2024-155-02Quarles 144578.00457 experiment, thereby preventing spurious autofluorescence accumulation during the initial cycles of imaging which could confound image analysis. The predetermined number of captures may be an integer 1 to 100, 1-20, 5-10, or any number therein. The predetermined number of captures may be 7 captures.

[0041] Therefore, the methods may include acquiring a pre-determined number of acquisitions of the sample prior to contacting the sample with a composition of target molecules. The methods may further include acquiring a pre-determined number of acquisitions of the sample after contacting the sample with a composition of target molecules and before contacting the sample with a reporter solution. The methods may further include acquiring a pre-determined number of acquisitions while iterating through images (e.g., acquire a pre-determined number of N* blank images after contacting the sample with an N* composition of target specific markers, and before contacting the sample with the N111composition of reporter molecules). Thus, a “pre-run blank” may include a pre-determined number of captures or acquisitions of the sample prior to contacting the sample with the composition of target molecules. A “first blank” or an “N* blank” comprises a pre-determined number of acquisitions or captures of the sample after contacting the sample with a corresponding composition of target molecules, and prior to contacting the solution with a corresponding composition of reporter molecules.

[0042] The samples may be a fixed sample, a fixed tissue sample, a decalcified (demineralized) bone sample which may comprise bone marrow. The sample may be a sectioned frozen sample that is embedded in tissue freezing medium (also referred to as optimal cutting temperature (OCT) medium), which is commercially available.

[0043] As used herein, acquiring may comprise acquiring an image using a digital microscope with automated liquid handling, e.g., an Akoya Phenocycler®.

[0044] The markers may comprise a marker, e.g., an antibody, e.g., a monoclonal antibody, specific for any one of TERI 19, CD41, CD 117, CD 150, Grl, CD48, endomucin B, alpha actinin, B220, fibromodulin, emilin2, CD45, EFEMP2, CRTAP, LUMICAN, CD31, PEDF, CD110, CD115, fibulin2, Coll A, F4 / 80, CFGRP, CD71, CD61, and Sca-1. See also, Tables 1 and 2.IU 2024-155-02Quarles 144578.00457

[0045] This type of tissue processing, experimental design for Phenocycler® runs, and image processing have been validated in murine bones and spleens. The Akoya Phenocycler® system has been published on numerous times in various tissue across human and mouse species. In the microscopy core facility, the following tissues have been successfully run on a Phenocyclers®: bone / bone marrow, spleen, tonsil, brain, kidney, liver, pancreas, intestine, skin, uterus, and lung.

[0046] The present invention is described herein using several definitions, as set forth below and throughout the application.Further Definitions

[0047] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0048] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0049] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0050] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.IU 2024-155-02Quarles 144578.00457

[0051] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0052] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g., “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0053] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0054] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”EXAMPLES

[0055] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.IU 2024-155-02Quarles 144578.00457Example 1 - Multiplex imaging of murine bone marrow using Phenocycler® 2.0

[0056] Reference is made to SJ Karnik et al. “Multiplex imaging of murine bone marrow using Phenocycler® 2.0” Leukemia volume 39, pagesl476-1489 (2025), which is incorporated by reference herein in its entirety.

[0057] Bone marrow (BM) is a tissue that is of great importance to several areas of basic and translational research, including hematology, oncology, bone biology, and immunology. It is unique in that it is gelatinous in nature but housed in a hard casing of bone. Traditionally, flow cytometry and immunofluorescence (IF) techniques have been employed to study the composition of cellular interactions and elements of the BM. However, it has been challenging to study the BM in an unperturbed state using multiple fluorescent probes at a time to fully appreciate the diverse cell populations and their interactions and relative positioning with each other. This protocol addresses how Phenocycler® 2.0, which uses co-detection by indexing (CODEX) in conjunction with HALO® 4.0 image analysis software, can overcome the obstacles faced by traditional techniques used to study the BM in an unperturbed state.

[0058] Introduction

[0059] Bone marrow (BM) is the primary site of hematopoiesis in mammals and, therefore, is of great importance in the fields of non-malignant and malignant hematology [1, 2]. Different cells such as hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), macrophages, megakaryocytes (MKs), T cells, B cells, mast cells, endothelial cells (ECs), and others reside in the BM and interact with each other [3,4,5,6,7,8,9,10], These interactions are based on molecular signals as well as spatial relationships that cells have with each other [11, 12], Researchers currently employ established techniques such as flow cytometry and immunofluorescence (IF) to study the BM

[0013] , These techniques are useful and have been utilized to study the composition of BM tissue; however, they have drawbacks [13, 14], For example, flow cytometry uses dissociated BM to quantify the cells. Due to the dissociation of the tissue, the spatial and structural information is lost. With IF, there is a limit on the number of cell markers you can include before it becomes impossible to separate the individual signal spectrally. Typically, eight markers are viewed as the upper end of spectral unmixing. Therefore, visualizing unperturbed BM with its cellular and structural features intact, without the limitations of the number of cell markers,IU 2024-155-02 Quarles 144578.00457 can benefit hematology research by making it possible to interrogate a single section for more than eight cell markers at a time. This becomes especially beneficial in studying rare cell types such as hematopoietic stem and progenitor cells (HSPCs), which are identified by staining cells with antibodies against 5-7 different markers [15,16,17,18], An additional benefit of using a multiplex imaging platform is that different cellular and structural niches can be studied for spatial context. BM niches and their vicinity to vasculature can give researchers an idea of how cellular and structural components of the BM interact with each other.

[0060] The Phenocycler® 2.0 is available commercially from Akoya Biosciences® and achieves multiplexing by co-detection by indexing (CODEX) as a multiplex imaging technique. Multiplex imaging of murine non-hematopoietic niche was demonstrated by Coutu et al. in 2017, which used multicolor three-dimensional imaging of murine femurs to map the non-hematopoietic cells and other structural components of the BM

[0019] , Some multiplex imaging techniques have been developed to study different aspects of BM functions, such as myelopoiesis. One such technique developed by Zhang et al. in 2021 used a combination of inducible Cre mice expressing fluorescence for specific lineage markers (confetti mice), confocal imaging, and sequential building of the map of the fluorescent cells of the myeloid lineage in the murine BM

[0020] , Recently, Bandyopadhyay et al. demonstrated that CODEX can be used to image BM from human samples obtained from orthopedic hip replacement surgery

[0021] , This study showed that CODEX can be a useful tool to study and build an atlas encompassing the major cell types in healthy human and acute myeloid leukemia samples.

[0061] Murine models are extensively used in pre-clinical research and are an important tool for understanding the pathways and mechanisms of different disease states. By manipulating specific genes in mice, one can assess how genes and / or proteins impact the development and localization of specific cells within the BM cavity with respect to other cells. Here, we show the use of the Phenocycler® 2.0 multiplex imaging platform adapted for cryosectioned murine BM tissue and cell type identification by using HALO® 4.0 (henceforth referred to as HALO®) image analysis software. Our objective was to develop a protocol for processing murine BM and adapting the Phenocycler® 2.0 for imaging cell surface proteins on HSPCs and more committed cells, as well as structural markers that constitute the murine BM microenvironment.

[0062] Phenocylcer® 2.0 multiplex imagingIU 2024-155-02Quarles 144578.00457

[0063] The Phenocycler® 2.0 is a multiplex imaging platform developed by Akoya Biosciences® that utilizes a technology called CODEX [22, 23], CODEX requires the construction of an antibody panel where each antibody has been conjugated with a unique DNA oligo tag referred to as oligobarcodes. This panel is then applied simultaneously during staining to a tissue of interest. During an imaging experiment, three complementary barcodes are added, which then bind and ‘reveal’ the antibody. After each addition, the oligo-barcodes are removed, and a new set is added and imaged [22, 23], This process is automated, with images taken in three different channels (488 or 750, 550, and 647 nm). The resulting images are stitched together by the system’s software, creating a final image (.qptiff) that displays the different markers and their location in the tissue.

[0064] FIGs. 2A-2F illustrate the steps involved in the process of Phenocycler® 2.0 multiplex imaging. One notable advantage of the Phenocycler® 2.0 is its capability to re-run the sample tissue post-run, eliminating the need to stain another tissue section, and potentially reducing the usage of antibodies, as well as saving time and resources. Additionally, the system offers the flexibility to use traditional non-fluorescent stains such as Hematoxylin and Eosin (H&E) to stain the same tissue section post-run.

[0065] Challenges involved in imaging BM and potential resolution

[0066] Imaging BM can be difficult due to the inherent nature of the tissue. The BM is gel-like and surrounded by hard, mineralized bone. Additionally, BM is highly vascular, with capillaries and blood vessels running through it. Processing the BM for imaging can damage its structure and blood vessels. The tissue’s inherent autofluorescence can also make it challenging to image certain cell markers that are found in rare cell populations. Choosing the right method for fixing, embedding, and sectioning the bone can improve the imaging process. In our protocol, we compared various commonly used fixatives for murine femurs for cryosectioning. Unlike the wide range of pre-conjugated antibodies available for human tissues, Akoya Biosciences® does not offer pre-conjugated antibodies for paraffin-embedded murine tissue. To include more cell or structural markers for imaging murine BM, custom-conjugated antibodies were created as described in this protocol. Paraffin embedding may require antigen retrieval, which can destroy delicate epitopes and distort tissue morphology [24, 25, 26, 27], Given these limitations, cryosectioning was a more suitable option for identifying the markers examined in our panel for the BM. We found that perfusion with lx PBS, a common practice forIU 2024-155-02Quarles 144578.00457 removing red blood cells from tissues, disrupts BM vasculature (FIGs. 3A-3E). Optimizing the conditions for fixing and embedding bone is crucial for obtaining good quality cryosections and reducing interference due to autofluorescence. By reducing the number of processing steps, we were able to maintain the tissue’s structural integrity, which is important for understanding cell locations and vasculature.

[0067] Rationale for improved imaging and image processing pipeline for Phenocycler® 2.0 BM imaging

[0068] The Phenocycler® 2.0 comes with built-in image processing, and the resulting image can be viewed in Phenochart® software (Akoya Biosciences®). Some of the cell and structural markers in our panel produce a dim signal. Despite selecting the best possible tissue processing conditions, the specific signal for some stem cell markers, such as CD117, remained dim, which is further complicated by the relative rarity of CD117 marker in wild-type (WT) tissues, causing it to be difficult to visualize using the manufacturer provided image processing and visualization software (FIGs. 4A-4E). Along with CD 117, there were other markers such as CD41, a-SMA, CD48, Endomucin, and many of the extracellular matrix (ECM) markers that could not be visualized accurately using the manufacturer’s recommended image processing pipeline and Phenochart™ (FIG. 4A). Manufacturer built-in image processing utilizes two blank cycles (first and last cycle of the run) for subtracting background set to a singular exposure time (150 ms as default). The exposure times for these blanks can be changed; however, they cannot be set at different exposure times for each individual marker. Due to the variety of biological markers, the abundance of antigens, and the quality of antibodies among the antibody panel, optimization of exposure times is critical. For example, our antibody panel contains markers that need higher exposure times for optimal visualization, such as Endomucin (600 ms), CD31 (800 ms), CD115 (800 ms), and other structural markers (FIG. 5). The panel also includes markers that need a shorter exposure time, such as CD41 (75 ms) (FIG. 4A). This range of exposure times poses a challenge for picking a singular exposure time suitable for background subtraction. With a low exposure blank, there is insufficient removal of background in high-exposure marker channels. With a high-exposure blank, there is an over-compensation, and we risk losing a genuine signal to this post-processing step. To overcome these obstacles for obtaining good quality multiplex images for murine BM that are also accurate, we optimized and profoundly improved the existing manufacturer’s protocol. Interleaved blanks with exposure times similar to the corresponding marker, as well as anIU 2024-155-02 Quarles 144578.00457 initial group of blanks (called “pre-treat”) were added. The images were processed from ‘.raw.qptiff fdes generated from the Akoya Biosciences® built-in image processing as explained in detail in the section on multiplex imaging and background subtraction below. The rationale for this “pre-treat” blanking scheme was to mitigate autofluorescence build-up and to increase the accuracy of background subtraction for each marker individually to obtain a better signal-to-noise ratio. This was needed to identify rare cell markers (such as CD 117), which also were low in signal intensity. Accurate visualization is important for accurate interpretation of results, especially when Phenocycler® 2.0 multiplex imaging is used in studies of diseased states of BM and the effectiveness of treatments.

[0069] Identification of cell markers for imaging the BM

[0070] A crucial step in imaging the BM is building a robust panel of antibodies and reporters with their corresponding oligo-barcodes. Three channels are available for each antibody: Atto550, AlexaFluor™ 647, and AlexaFluor™ 750. Choosing which antibody to place in which channel is an important step based on the abundance of the cell marker and the sensitivity of the channel to the camera. We avoided using 488 nm due to high autofluorescence from Red Blood Cells (RBCs) and other tissue components that exhibit strong autofluorescence (due to the porphyrin ring structures in the heme group) in the 488 nm channel

[0028] , Abundant cell markers were assigned to the AlexaFluor™ 750 channel as it is the lowest camera sensitivity channel. The least abundant cell markers were assigned to the Atto550 channel, which has the highest camera sensitivity of our three used channels. The cell markers expressed that were not rare but not too abundant were assigned to AlexaFluor™ 647 nm. The complete panel of cell markers and the cell types that we identified using these markers are listed in Table 1. Table 2 shows the channels assigned to antibodies, the cell markers, and the corresponding oligo-barcodes.

[0071] Table 1 : Cell markers, cell types, and structures ofBM.IU 2024-155-02Quarles 144578.00457

[0072] Table 2: Antibodies against the cell markers, channels, and oligo-barcode numbers (manufacturer assigned)IU 2024-155-02Quarles 144578.00457IU 2024-155-02Quarles 144578.00457

[0073] Materials

[0074] Animals: 12-14-week-old male C57BL / 6J mice (In Vivo Therapeutics Core, Indiana University Simon Comprehensive Cancer Center). C57BL / 6J mice were housed in a pathogen-free facility at Indiana University School of Medicine, Indianapolis. All animal studies were conducted with approval from the Indiana University Laboratory Animal Resource Center.

[0075] Reagents: Methanol (ThermoFisher®, cat #176840010); Acetone (ThermoFisher®, cat # L10407); 16% w / v aqueous solution of Paraformaldehyde (Thermofisher®, cat # 043368.9 M); 2x Laemmli sample buffer (Bio-Rad, cat# 1610737); Novex™ Tris-Glycine Mini Protein Gels, 4-20%, 1.0 mm, WedgeWell™ format (ThermoFisher®, cat # XP04205BOX); Coomassie Brilliant Blue G 250 (Sigma, cat# 115444); Glacial acetic acid (ThermoFisher®, cat# 9526-33); 10% Neutral Buffered Formalin (EKI, cat# 4499-GAL); EDTA (ThermoFisher®, cat# 17892); Optimal Cutting Temperature (O. C. T.) Compound (Fisher Scientific®, cat# 23-730-571); Sucrose (Fisher Scientific®, cat# S5-3); Anti-mouse CD61 (Biolegend® 104325); Anti-mouse CD150 (Biolegend® 115949); Anti-mouse Ly- 6G / Ly-6C (Grl) (Biolegend® 108435); Anti-mouse Teri 19 (Biolegend® 116253); Anti-mouse CD41 (Biolegend® 133939); Anti-mouse CD117(c-kit) (Biolegend® 135114); Anti-mouse Seal (ThermoFisher® 14-5981-82); Pierce Antibody Clean-up Kit (ThermoFisher ®44600); Oligo-barcode and reporter information (refer to Table 2); Akoya® pre-conjugated antibody anti-mouse CD31- BX002 (Akoya®, cat# 4250001); Akoya® pre-conjugated antibody anti-mouse CD45-BX007 (Akoya®, cat# 4450002); Akoya® pre-conjugated antibody anti-mouse CD45R / B220-BX010 (Akoya®, cat# 4450006); Akoya® pre-conjugated antibody anti-mouse CD71-BX027 (Akoya®, cat# 4550111); Akoya® antibody conjugation kit (Akoya®, cat# 7000009); Akoya® staining kit (Akoya®, cat# 7000008); Akoya® 96-well plates for Phenocycler® (Akoya®, cat# 7000006); Akoya® 96-well plate seals for Phenocycler® (Akoya®, cat# 7000007); Corning Cell-Tak™ (Corning® CLS354240);IU 2024-155-02 Quarles 144578.00457ProLong Diamond antifade mountant with DAPI (Invitrogen®, cat# P36962); Rabbit F (ab’)2 Antimouse FITC IgG (H+L) secondary antibody (Southern Biotech cat# 6120-02); Rabbit F (ab’)2 Antirat FITC IgG (H+L) secondary antibody (Southern Biotech® cat# 6130-02); Rabbit F (ab’)2 Antigoat FITC IgG (H+L) secondary antibody (Southern Biotech® cat# 6020-02); Bovine Serum Albumin (Sigma-Aldrich® cat# A3733).

[0076] Equipment: BZ-X810 fluorescent microscope (Keyence, Itasca, Illinois); Gel Electrophoresis (Bio-Rad® ChemiDoc MP Imaging System, Hercules, California); Phenocycler® 2.0 (Akoya, Marlborough, Massachussetts).

[0077] Software programs: HALO® 4.0 (Indica Labs, Albuquerque, New Mexico).

[0078] Procedure

[0079] After the channels and oligo-barcodes were assigned to the cell markers, we next custom- conjugated the cell markers that were not available commercially to their respective oligo-barcodes.

[0080] The process described below is adapted from

[0023] ,

[0081] Custom conjugating antibodies to Phenocycler® Fusion oli go-barcodes. The manufacturer uses Phenocycler® Fusion to refer to the reagents used for imaging assays and associated processes for Phenocycler® Fusion microscope. The automated platform, including the microscope, fluidics, software to run the imaging assay, and image output, is collectively referred to as Phenocycler® 2.0 by the manufacturer.

[0082] The protocol to custom conjugate the antibodies to the oligo-barcodes was obtained from Akoya, and the manufacturer’s instructions were followed. The steps are as follows:

[0083] 1. Purified stock solution of antibodies in l x PBS was prepared. Antibodies were free of carrier proteins and sodium azide. If antibodies contained carrier proteins and sodium azide, they were purified using a protein purification kit.

[0084] 2. Volume of the solution corresponding to 50 pg of antibody was calculated.IU 2024-155-02Quarles 144578.00457

[0085] 3 The following reagents were retrieved at the start of the process: Reduction solutions 1&2; fdter blocking solution.

[0086] 4. The following reagents were retrieved ~1 h after starting the process: Conjugation solution; barcodes.

[0087] 5 The following reagents were retrieved in ~3 h: Purification solution, antibody storage solution, purified antibody.

[0088] a 5-0 kDa MWCO filter was labeled for each antibody.

[0089] b. 500 pl of Filter Blocking Solution was added to the top of each 50 kDa MCSO filter. The collection tubes were then centrifuged at 12,000 x g for 2 min.

[0090] c. All the liquid that was left was removed and discarded.

[0091] d. 50 pg of the purified antibody in a volume of 100 pl or greater was added to the filters and collection tubes and then centrifuged 12,000 x g for 8 min. Flow-through was discarded .

[0092] e. Antibody Reduction Master Mix was prepared based on the number of Phenocycler® Fusion antibody conjugates as shown below in Table 3.

[0093] Table 3: Preparation of reduction master mix for antibody conjugation.IU 2024-155-02Quarles 144578.00457

[0094] We next verified if the conjugation of the antibodies to their respective oligo-barcodes was successful and if the conjugated antibodies remained functional after the chemical modification during the conjugation steps. Verification steps included gel electrophoresis and immunofluorescence validation to not only confirm the success of the conjugation to the barcodes but also to verify if the binding sites for the antibodies are not blocked or rendered unusable for imaging.

[0095] Gel electrophoresis

[0096] Protein gel electrophoresis was performed to verify the success of antibody conjugation for any antibodies not obtained from Akoya Biosciences®. The detailed procedure is as follows:

[0097] 1. 5 pl of each conjugated antibody and 2 pl of unconjugated antibody (used as a control) were diluted to a final volume of 10 pl and mixed with 10 pl of 2 x Laemmli sample buffer from Bio-Rad.

[0098] 2 The samples were then denatured at 95 °C in a dry bath for 10 min. Subsequently, each sample was loaded into the wells of a 10-well Novex WedgeWell 4-20% Tris-Glycine gel, and the gel was electrophoresed at 100 V for 1 h until the process was complete.

[0099] 3. After the gel run, the gels were gently removed from the cassette and rinsed once with distilled water.

[0100] 4. The gel was then stained for one hour using a Coomassie Brilliant Blue staining solution (comprising 0.1% Brilliant Blue G from Sigma, 50% methanol, and 10% glacial acetic acid).

[0101] 5. Subsequently, the gel was destained using a destaining buffer (consisting of 50% methanol and 10% glacial acetic acid) until complete destaining was achieved.

[0102] 6. Images were captured using the ChemiDoc MP Imaging System by Bio-Rad.

[0103] As shown in FIG. 6, the unconjugated antibody showed one band for the light chain of the antibody and one for the heavy chain of the antibody, whereas the conjugated antibody showed a shift due to a higher band size due to the addition of an oligo-barcode. Multiple bands are frequently observed on the conjugated antibody, suggesting multiple oligos have been conjugated. To ensure that the antibody remains functional after conjugation, tissue is prepared and IF validation performed as described below.IU 2024-155-02Quarles 144578.00457

[0104] Selection of the best fixative for Phenocycler® 2.0 imaging of murine BM

[0105] Different fixatives can be used to fix bones, depending on the area and the desired imaging feature. 10% Neutral Buffered Formalin (NBF) is a commonly used fixative that has been used to fix bones such as femurs, tibiae, ulnae, radii, cranium, sternum, and vertebral column. However, due to the nature of formalin (37% formaldehyde with 6-12% methanol), formalin containing fixative can result in autofluorescence, which can hamper signal recognition in sensitive imaging such as with the Phenocycler® 2.0.

[0106] A pilot study was conducted to see which method of fixation provided the best results with the least autofluorescence while preserving the tissue architecture and internal features of interest utilizing Phenocycler® imaging. The conditions and details for fixation, decalcification, embedding, and sectioning are provided in Table 4. All the femurs treated with the indicated fixatives were decalcified in 10% EDTA on a shaking platform for approximately 2 weeks post-fixation. The completion of decalcification was verified by x-ray imaging of the bones. Decalcification was considered complete if the x-ray was transparent, indicating the removal of mineral content. The femurs were washed in l x PBS and then put in 30% sucrose solution overnight at 4 °C before embedding in O C T. compound.

[0107] Table 4: Conditions and details for fixing femurs for cryosectioning.IU 2024-155-02Quarles 144578.00457

[0108] The metric used to assess the quality of the fixation included identifying the best fixative for bone cryosections that not only preserves the architecture and cell niches in the marrow but also generates little to no autofluorescence, To address this aspect of tissue processing, we imaged the tissue sections on a Phenocycler® 2.0 system without any antibodies before IF validation of the conjugated antibodies to test which condition would work the best with respect to autofluorescence for BM imaging. This was achieved by testing a series of fixative conditions with mock runs on the Phenocycler® 2.0 system. The results suggest that Methanol: Acetone (1 : 1) fixative resulted in the least amount of background autofluorescence from processing the tissue and the quality of cryosections. Representative images are shown in FIG. 7.

[0109] Preparation of femurs for cryosectioning

[0110] After selecting the appropriate method of fixation and processing, the femurs were embedded in O. C. T. compound for cryosectioning. The detailed steps from isolating femurs from mice to embedding and cryosectioning are described below:

[0111] 1. Femurs were Isolated from 12-15-week-old male C57BL / 6J mice. Soft tissue was removed from the femurs. Femurs were placed in 1 :1 Methanol: Acetone fixative (kept cold at -20 °C) and fixed for 30 min at -20 °C.

[0112] 2 After fixation, femurs were washed in 1 * PBS to rid the tissue of any fixative solution.

[0113] 3 Next, femurs were placed in 10% EDTA decalcification solution on a rocking platform for ~2-2.5 weeks.

[0114] 4. Complete decalcification was verified by x-ray imaging. Complete decalcification is required to ensure proper sectioning of the tissue. Incomplete decalcification may lead to poor, ruptured sections affecting BM continuity.

[0115] 5. After decalcification, femurs were washed in 1 x PBS.

[0116] 6. Femurs were then placed in 30% sucrose solution overnight at 4 °C.

[0117] 7 Next day, femurs were embedded in O. C. T. in cryomolds, and the blocks were stored at -80 °C until sectioning.IU 2024-155-02 Quarles 144578.00457

[0118] 8 Femurs were sectioned on Cell-Tak™ coated or Si lane-treated slides at 10 urn thickness, ensuring the section lies flat on the slide without any folds or creases. Folds and creases in the section can result in tissue lifting off the slide during Phenocycler® runs.

[0119] 9. Slides containing Cryosections were stored at -80 °C.

[0120] Immunofluorescent validation

[0121] For the IF validation of custom-conjugated antibodies, based on the results from the preliminary Phenocycler® run (as discussed above), Methanol: Acetone fixed cryosections of EDTA decalcified femurs of the C57BL / 6J mice were used. The detailed procedure is described below:

[0122] 1. The slides with cryosectioned tissues were equilibrated in a humidity chamber for 10 min at room temperature. For all the following steps until mounting (steps 2-6), the tissue slides were kept in a humidity chamber to prevent the tissue from drying.

[0123] 2. Blocking buffer (1% bovine serum albumin in 1 x PBS) was added to the tissues for 30 min at room temperature.

[0124] 3. Primary antibodies diluted to the appropriate dilution factor in blocking buffer were added to the tissues and kept overnight at 4 °C (Table 2).

[0125] 4. The tissues were washed via gentle pipetting with 1 x PBS to remove any unbound primary antibody. Caution: Tissue can be easily damaged during washing steps and is susceptible to drying.

[0126] 5. Appropriate secondary antibodies, diluted in the blocking buffer (1 :500), were added to the tissues and kept for 30 min at room temperature in the dark.

[0127] 6. The tissues were washed in 1 x PBS twice for 10 min at room temperature in the dark. This was done gently to remove any unbound secondary antibodies.

[0128] 7. After the washes, an antifade mounting medium with DAPI was used to stain the nuclei and to protect the tissues from fading during fluorescence imaging.

[0129] 8. After curing for 24 h, fluorescence images were captured using a Keyence BZ-X810 fluorescent microscope.IU 2024-155-02 Quarles 144578.00457

[0130] After verifying the conjugation of oligo-barcodes to antibodies and IF validations (FIG. 8), the next step was the selection of the proper tissue processing for the BM. We tested perfusion, which is a common technique used to reduce autofluorescence from RBCs in the BM discussed in detail in the next section.

[0131] Perfusion vs non-perfusion approach to deplete RBCs from the BM cryosection

[0132] Autofluorescence from RBCs is a significant concern when it comes to imaging tissues that are rich in vasculature and have abundant RBCs. BM, being the site of hematopoiesis and being a vascular tissue, has abundant RBCs. Even though we selected channels that give the least autofluorescence from RBCs (550, 647, and 750 nm), RBCs still show some weak autofluorescence in all channels (FIGs. 3A-3C). Indeed, as shown in FIGs. 3A-3C, RBCs might interfere with image analysis by fluorescing in all the channels. This dilutes the specific signal from the actual cell markers, making it difficult to interpret the data.

[0133] One commonly used method to reduce the number of RBCs in tissues is to perfuse the animal with l x PBS, followed by a fixative such as 4% PFA to internally fix tissues. However, for Phenocycler® imaging of the BM, we used a different fixative (described above); therefore, the mice were perfused only with 1 x PBS. It is important to note that using Methanol, especially in the presence of water or a water-rich environment such as biological tissues, is exothermic, leading to excessive heat generation and potential tissue degradation.

[0134] Different speeds and durations of perfusion were tested to preserve BM architecture. However, even at reduced speed, perfusion damaged the marrow and vasculature, resulting in poor imaging quality for studying microarchitecture and vasculature. As shown in FIG. 3D-3E, we observed that perfusion, even at a reduced speed, destroys the marrow architecture and vasculature and, in turn, is not effective in getting rid of the RBCs to an extent to which they do not interfere with the specific cell marker signal. Importantly, perfused BM led to wide gaps in the marrow with a concomitant loss of vascular structures such as capillaries that can be seen in non-perfused BM. Therefore, our results suggest that the use of perfusion is likely to be associated with poor multiplex imaging quality of the BM, particularly as it relates to studying the microarchitecture and vasculature.

[0135] The workflow involving the selection of cell markers and extending to cryosectioningIU 2024-155-02 Quarles 144578.00457

[0136] A schematic of the workflow from designing and planning to tissue preparation for staining is described in FIG. 9. After the preparation of sections, a Phenocycler® run was executed. Mouse femur cryosections of 10 pm thickness were acquired from O. C. T. -embedded material on slides treated with silane or coated with Cell-Tak™. We compared both types of slides since we wanted to test which condition gives us the best possible result with respect to tissue adhesion as well as imaging. Sections were prepared following the protocol provided by the manufacturer, Akoya Biosciences, which was also described in detail by Goltsev et al.

[0022] , An antibody master mix described below was utilized. A pre- and post-stain fixation was utilized. Slides were mounted with a proprietary flow cell from Akoya Biosciences and imaged using the Phenocycler® 2.0 system. The workflow for the Phenocycler® staining and post-staining steps is shown in FIG. 10.

[0137] Reporter plate setup

[0138] The corresponding oligo-barcodes with reporter probes were added by the automated system of the Phenocycler® 2.0 during each cycle. The DNA-DNA bonds of reporter oligos were denatured at the end of each cycle to facilitate the subsequent addition of a new set of oligo-fluorophore reporters. The reporter probes were added to the wells of a black round-bottom low-binding 96-well plate. The reporter stock solution was added per well, minus the volume of the reporter probes. The workflow for setting up the reporter plate is shown below:

[0139] The reporter stock solution was prepared for the total number of cycles for each Phenocycler® run by utilizing the volumes given in Table 5.

[0140] Table 5: Volumes for the reagents to make the reporter stock solution.IU 2024-155-02Quarles 144578.00457

[0141] Each reporter plate was set up by adding 250 pl of reporter stock solution minus the volume of the oligo-barcode probes (5 pl per probe) added per well. For example: If well 1 has no probes, then the volume of reporter stock solution to be added to well 1 was 250 pl. If well 2 had 2 oligo-barcode probes, then the volume of reporter stock solution was 250- (5 * 2) pl = 240 pl. Since there are 3 channels available per cycle, the maximum number of probes that can be added per well is 3, hence, the total volume in the well would be 235 pl reporter stock solution + 15 pl total volume of probes (5 pl each). All wells containing reporter solution were sealed with adhesive aluminum strips to prevent evaporation before there are automatically dispensed to the stage by the Phenocycler® 2.0 fluidics handling system.

[0142] Designing the Phenocycler® run and experimental setup on the instrument

[0143] Runs were configured as an experimental template in Akoya Phenocycler® Experiment Designer (Akoya PED™) software, and then this configuration was loaded at the start of the run. The template for the cycles with the pre-run and interleaved blanks is shown in FIG. 5. Akoya uses a blank at the beginning and a blank at the end to mathematically approximate the amount of autofluorescence in each round of imaging. We found that autofluorescence varied enough that this procedure gave inadequate results, and so we collected autofluorescence images every other round. To allow for the background subtraction for each marker, we set up the blanks corresponding to their respective markers set at exposure times that were the same as the markers. We also found that autofluorescence increased logarithmically as cycle number increased, an effect that could be minimized by imaging the unlabeled tissues several times before the addition of markers to reduce variation in autofluorescence during the portion of the run that introduces oligo-fluorophores. To accommodate this, we set up the pre-run blanks (pre-treat cycles) at exposure 150 ms for the autofluorescence to plateau the change in autofluorescence signal before the markers were added or blanks collected. Because of software constraints, we still needed to set a blank as the first and last cycles. These blanks are set up in wells Hl and 2 on a 96-well plate.

[0144] Multiplex data processing and background subtraction

[0145] Instead of utilizing the ‘.qptiff image files generated automatically by Akoya Biosciences®, a custom workflow was developed. Raw data was generated in a ‘temp’ folder during data acquisition. The ‘.raw.qptiff files were stitched using the Akoya Biosceinces® algorithm but do not have anyIU 2024-155-02 Quarles 144578.00457 background correction applied. The ‘raw.qptiff is then post-processed in ImageJ / FIJI (referred to as FIJI henceforth)

[0029] , as shown in FIGs. 4A-4B. Preceding each cycle, an empty cycle was run to generate a blank for each marker in the antibody panel, details of the setup are shown in FIG. 5. ‘.raw.qptiff files from each Phenocycler® 2.0 run were cropped on import at the highest resolution setting using FIJI Within FIJI, the Image Calculator Subtract function was used to remove the blank image associated with each marker. The resulting subtracted images for each individual marker were then imported into HALO® (Indica Labs) and fused with all other markers generating a multiplex ‘ .afi’ file. The fused images were used for segmentation for cell type identification. The High-Plex FL module was configured to identify critical phenotypes by establishing thresholds for positivity for each marker. Each phenotype is described in Table 1. Segmentation parameters and cut-off thresholds were iteratively analyzed in the real-time tuning window to lay a mask over the cells identified for the phenotype. These segmentation parameters were then applied equally to each tissue, and cell object data was exported to a table for quantification. MKs were segmented differently than the other cell types due to their size and polyploid nucleus.

[0146] Comparison of data from Phenocylcer 2.0 imaging and flow cytometry

[0147] BM region was first annotated using the annotation function in HALO®, and then this region was used to obtain the total number of nucleated cells. After applying segmentation parameters as described in the previous section, the number of cells for the main cell types of the BM were obtained. The cell percentages were calculated as:

[0148] (number of cells identified by segmentation parameters of that cell type in BM / total number of cells in BM) x 100. The cell percentages derived from the HALO® analysis were then compared to the flow cytometry cell percentages of that cell type. A regression analysis was performed to see the level of concordance between the cell percentages of the two modalities compared to each other (FIG. 11 A).

[0149] Results

[0150] Regression analysis for comparison of cell percentages from Phenocycler® 2.0 flow cytometryIU 2024-155-02Quarles 144578.00457

[0151] Flow cytometry is a well-established technique and is used extensively to study the cell composition of the BM. To check the accuracy of our Phenocycler® 2.0 imaging and HALO® image analysis results, the cell percentages obtained from Phenocycler® 2.0 imaging and flow cytometry were compared. The process of obtaining cell percentages is described above. Markers that were selected for this comparison were CD45, Teri 19, B220, Grl, and CD41 since they cover the major cell types of the BM as shown in Table 1. Regression analysis showed that the correlation coefficient was 0.90 (FIG. 11 A). We observed the highest degree of concordance between the frequency of CD45, followed by Teri 19 and GR1 markers, utilizing flow cytometry and Phenocycler® 2.0. However, the concordance was lower for B220 and CD41 markers compared to CD45, Teri 19, and GR1. While the cell percentages compared relatively well in some cell types, certain cell types, such as CD41+ MKs, were challenging to segment due to the limitation of the HALO® analysis software to handle polyploid nucleated cells. The differences noted between the two modalities of measuring the cell percentages could be attributed to the techniques being inherently different (imaging a tissue section vs fluorophore detection on cells that are mostly in a single-cell suspension). Newer versions of the HALO® software might have better flexibility to detect unusually large or small cells that have different nuclear presentations. Even though some markers had lower concordance than others, the agreement between the two modalities of analysis was higher than the cell percentages obtained using HALO® from manufacturer built-in image processing. Using the manufacturer built-in image processing, some markers such as CD41, B220, and GR1 appeared over-saturated to the extent that the cells could not be segmented accurately in HALO® (FIGs. 1 IB-1 IE).

[0152] We were unable to directly compare our custom post-processing pipeline to the standard Akoya Biosciences® image processing due to several factors. Pixel intensity saturation was frequently observed confounding thresholding attempts. Segmentation attempts were further disrupted by the high level of background fluorescence observed.

[0153] Cell and structural markers identified

[0154] FIGs. 12A-12B show whole femur multiplex images along with the different individual cell and structural markers from a C57BL / 6J mouse femur. Similar results were seen in 4 runs for the C57BL / 6J mouse femur. FIG. 12A shows the whole femur, which was stained for CD45, Teri 19, Grl, SCA1, CD31, and Endomucin to demonstrate the overall structure of the murine BM on a Silane-IU 2024-155-02Quarles 144578.00457 treated slide. Inserts below show multiplex images at higher magnification. The higher magnification multiplex image on the left shows arterioles (CD31 and SCA1 positive, red) as well as MK (CD41, blue), and leukocytes (CD45, a pan-leukocyte marker, green). The higher-magnification multiplex image on the right shows the lineage markers, GR1 (orange) and Teri 19 (cyan), as well as MK (CD41, blue). We also observed that the lineage markers (GR1 and Teri 19) did not colocalize in tissues, confirming that the signal from these markers is specific and identifies the correct cell type. Individual cell and structural markers from the whole femur are shown at a higher magnification in the inserts. CD45 (pan-leukocyte marker, shown in green), along with immune cell markers such as B220 (red), GR1 (orange), and F4 / 80 (cyan), can be seen in the inserts below the whole femur. Erythroid cell markers, including Teri 19 (cyan) and CD71 (magenta) are also shown in the panel of inserts below the whole femur. As seen in these images, the labeling of these cell markers is bright and specific to the cell type. MKs are large cells and are identified by CD41 (blue) positive staining

[0030] , CD110 (red), thrombopoietin receptor, is critical for MK proliferation and is present on MKs as well as some other cells in BM such as stem and progenitor cells and platelets [31, 32], CD110 is shown colocalizing on MKs with CD41 in the merged image (red + blue).

[0155] Vascular markers such as Endomucin (red), which labels sinusoids [33, 34], and CD31 (red), along with SCA1 (red), which label different cells of arterioles such as endothelial cells and endothelial progenitors

[0035] , can be seen in FIG. 12A, highlighting the vascular structures. Fibulin2 (green), which is another structural marker found in the extracellular matrix (ECM) as well as the basement membrane of different cells in the BM

[0036] , can be seen colocalize with SCA1 (red) in arterioles, as seen in FIG. 12B insert (merged image (red + green)).

[0156] FIG. 12B also demonstrates the presence of additional BM-associated structural markers. Lumican (green) is secreted by osteoblasts in the BM and is found in the ECM of the BM, bone, and skeletal muscles abundantly [37, 38], Emilin2 (cyan) is secreted by mesenchymal stem cells (MSCs) in the ECM of BM [39, 40], EFEMP2 (green) is a secreted protein present in the basement membrane of cells of connective tissues such as the BM and skeletal muscle [41, 42], Fibromodulin (red) is expressed by BM stromal cells and osteoblasts and is found in the ECM of BM [43, 44], markers [40, 42, 44], We observed lumican (green), Emilin2 (cyan), EFEMP2 (green), and Fibromodulin (red) positive signals throughout the ECM of the BM. PEDF (cyan), a factor secreted by MSCs in the BM [45, 46, 47], was seen abundantly in the skeletal muscle attached to the bone and the BM. a-SMAIU 2024-155-02Quarles 144578.00457(green) is a cytoskeletal protein that is present in the cytoplasm of cells

[0048] and is readily detected in almost all cells of the BM. Col la (green) is the most common type of collagen

[0049] in the body and is detected in abundance in the BM, as well as adjacent tissues such as skeletal muscle. CRTAP (green) is a protein that is associated with post-translational collagen modifications in articular cartilage and bone and is usually seen near the growth plates of long bones

[0050] , CRTAP was observed abundantly in the epiphyseal region of the femur. CGRP (green) is found in the BM, bone, and skeletal muscle

[0051] and was seen abundantly in the skeletal muscle and bone. FIG. 12B also shows other cell markers such as CD115 (magenta) and CD48 (red). CD115 (also known as colony stimulating factor 1 receptor) is a cell surface marker found on myeloid lineage cells such as monocytes, macrophages, and osteoclasts (which differentiate from monocyte lineage cells)

[0052] was observed on cells that were closer to endosteum (region of BM close to the bone). Even though the cells staining positive for CD115 were not abundant, the marker produced a bright signal on these cells. CD48 is a cell surface marker that is expressed by different progenitors such as myeloid-erythroid and B lineage progenitors, however, CD48 is not expressed by multipotent progenitors or primitive cells such as HSCs. Cells that were positive for CD48 (red) could be seen throughout the BM.

[0157] Comparison of tissue adhesives: Cell-Tak coated slides vs. Silane-treated slides

[0158] Tissue adhesion is of critical importance to the success of imaging using Phenocycler® 2.0 since the tissue is subjected to serial washes between imaging steps. Poor anchoring of the tissue to the slide during processing can severely compromise the image including resulting in tissue lifting off the slide, which can cause issues with focus. We found that both the tissue adhesives, Cell-Tak™ and Silane, provided sufficient tissue adhesion for BM imaging, although some differences were noted.

[0159] Cell-Tak™ is a tissue and cell adhesive that contains an adhesive protein from the common blue mussel (Mytihis edulis L.). Cell-Tak™ is used to increase adhesion for whole tissue sections on slides or cells in in vitro conditions [53, 54, 55], Silane-treated slides are routinely used for the adhesion of cells [56, 57, 58],

[0160] As shown in FIGs. 13A-13F, the lower and higher-magnification images highlight the differences between the images obtained using two tissue adhesives in a Phenocycler® 2.0 run. Cell and vascular markers such as CD31, SC Al, Endomucin (all in red), CD41 (blue), and CD45 (green) were selected to show the general architecture of the BM (FIGs. 13A-13F). The adjacent tissues, suchIU 2024-155-02Quarles 144578.00457 as skeletal muscle and bone, were shown by Col la (white) since Col la is abundant in these tissues (FIGs. 13E-13F).

[0161] The general architecture of the BM at lower magnification on a Cell-Tak™ coated slide (FIG. 13 A) and a Silane-coated slide (FIG. 13B). FIG. 13C shows the boxed area in FIG. 13 A at higher magnification. Similarly, FIG. 13D shows the boxed area in FIG. 13B at higher magnification. As seen in FIGs. 13A-13D; both the tissue adhesives show strong BM adherence to the slide as measured by the lack of tissue lifting and subsequent focus issues. The BM structurally in these images looks intact and remains on the slide surface. FIGs. 13C-13D also show the vascular structures shown by markers CD31, SCA1, and Endomucin in red along with MKs (CD41, blue) and BM cells positive for CD45 (green). However, as seen in FIGs. 13E-13F, Cell-Tak™ was better at preserving the adjacent tissue such as bone and skeletal muscle attached to the bone (seen labeled by Col la in white). FIG. 13F shows a Silane-treated slide with some skeletal muscle shown by the arrow and no bone. These observations are important to note since the choice of tissue adhesive would impact the Phenocycler® imaging for tissues of interest in a study. For example, Silane or Cell-Tak™ would both be suitable to image the BM cells and structures; however, only Cell-Tak™ would be suitable to study the BM adjacent tissues such as bone and skeletal muscles since Silane-treated slides did not adhere these tissues well.

[0162] Phenocycler® 2.0 imaging of murine wild type and MK ablation model femurs

[0163] FIGS. 14A-14D shows the differences between the MKs in male C57BL / 6J (wild type, WT) femur and Diphtheria toxin (DT) injected PF4 (platelet factor 4) Cre; iDTR (inducible diphtheria toxin receptor) mouse femur. PF4Cre; iDTR mouse is an inducible mouse model to ablate MKs and platelets when DT is injected

[0059] , This mouse is generated by crossing PF4Cre mice with iDTR homozygous mice. When injected with DT, the DT binds specifically to the induced receptors on MKs in the PF4Cre; iDTR mice and ablate the MK populations [59, 60], This mouse model is used to study the effects of MKs and platelets in BM microenvironments or to study the effects of different treatments in MK ablated conditions [59, 61], The PF4Cre; iDTR mice for the Phenocycler® 2.0 imaging experiment were injected with a higher dose of DT (100 ng / ml; 2x weekly) to show the drastic depletion of the MKs and, in turn, CD41, which is an MK marker. FIGs. 14A-14D highlight the differences between the WT (FIG. 14A) and DT-injected MK ablated femur (FIG. 14B). CD41 (blue)IU 2024-155-02Quarles 144578.00457 and SCA1 (red) were the only 2 markers selected to show the drastic differences in the loss of MKs upon DT treatment of this mouse model. Even though rare (0.01-0.02% of BM), due to their size, MKs appear as large cells in the WT femur at lower magnification. CD41 is also a marker for platelets. Platelets can be found closer to the arterioles when the MKs release them. Together, MKs and platelets are seen throughout the WT femur and appear to dominate the tissue. Higher-magnification image of the WT femur (FIG. 14C) shows the CD41 stained MKs and adjacent SCA1 stained arterioles. FIG. 14D shows no CD41 staining and only SCA1 positive staining for arterioles, suggesting MK ablation. It is important to note that both the femur sections (WT and DT-injected PF4Cre; iDTR femurs) were imaged on the same slide and were stained and imaged on Phenocycler® 2.0 at the same time. This eliminates the possibility of experimental variation as the cause of the depletion.

[0164] Phenocycler® 2.0 imaging of primitive and more committed progenitor cells in a murine wild-type femur

[0165] We next attempted to identify more primitive stem and progenitor cells of the BM. Lineage negative (Lin-) is defined as negative for B220, GR1, and Teri 19. As seen in FIG. 15A, we readily identified Lin-, SCA1+, and CD117+ also known as LSK cells of the BM. These are a population of BM cells that contain the most primitive fraction of long-term initiating stem cells. FIG. 15B shows the presence of Lin-, SCA1-, and CD117+ cells, and FIG. 15C demonstrates the presence of Lin-, SCA1 + , CD1 17- fraction of more primitive cells. A CMP cell is a multipotent cell that can differentiate into MK-erythrocyte progenitor (MEP) or granulocyte-monocyte progenitor (GMP) [10, 62, 63], As shown in FIG. 15B, CMP cells were identified as Lin-, SCA1-, CD117+ cells. A CLP cell can differentiate to form lymphocytes such as T, B, and natural killer cells [9, 63, 64], As shown in FIG. 15C, CLP cells were identified as Lin-, CD117-, SCA1+. LSK cells are rare multipotent cells or primitive progenitors that can differentiate into all types of blood cells, such as CMP and CLP as well as consist of self-renewing stem cells (primitive progenitors)

[0065] ,

[0166] Limitations

[0167] Phenocycler® 2.0 is a useful tool for achieving multiplexed imaging of tissues that need modalities that can surpass the limited number of markers available traditionally. However, it is also complex due to the inclusion of multiple markers in a panel that have differential expressions in the tissue of interest. For example, some antibodies (either conjugated by the user or purchasedIU 2024-155-02Quarles 144578.00457 commercially) might generate a very bright signal that is well above the noise from the tissue autofluorescence. However, some antibodies might be dim due to either the nature of the epitopes or the rarity of the markers themselves. This is usually not the limitation of traditional techniques, such as IF, which can image ~3+ markers in one imaging run. In IF, the conditions needed to obtain the optimal signal from an individual marker can be applied to the tissue and can be detected without any need for special software.

[0168] The addition of “pre-treat” and interleaved blanks for individual markers added significantly to the run time. With the expansion of the panel to include more markers, the number of interleaved blanks will also increase, in turn leading to longer run time on the Phenocycler® 2.0 instrument. The manual background subtraction using \ raw. qp tiff files and FIJI is also time-consuming compared to the built-in image processing from the manufacturer using Phenochart 2.0™ to visualize the image. While longer run time on the instrument is a significant limitation, the images that were obtained were more accurate than the built-in software generated QPTiffs, especially in visual representations of some markers such as CD117, CD41, a-SMA, CD48, Endomucin, and many of the ECM markers.

[0169] Another limitation is the presence of autofluorescent particles or bodies that are not cells (these are enucleated) and are ubiquitous in the BM. We were not able to get rid of these autofluorescent particles by either photobleaching or by treating the sections with bleaching agents such as hydrogen peroxide at different concentrations (data not shown). Even though these particles do not affect the cell segmentation and identification as they can be eliminated from the HALO® analysis, these can be very distracting, especially to the human eye when identifying different cells close to these autofluorescent particles. A recent study done by Bandyopadhyay et al. also observed similar autofluorescent particles in the BM of humans

[0021] , The authors labeled these particles with human anti-mast cell tryptase for identification since these particles stained positive for almost all the markers. Further studies need to be done to identify the true nature of these autofluorescent particles as well as to either dampen their signal or eliminate them from the images completely.

[0170] We have used cryosectioned femurs as our tissues to stain for the Phenocycler® 2.0 imaging of the murine BM. However, for this tool to be widely used for studying and interrogating different disease states and physiological conditions, it needs to be adapted for formalin or PFA-fixed murine tissues with higher autofluorescence. High autofluorescence in formalin or PFA-fixed tissue developsIU 2024-155-02 Quarles 144578.00457 due to the crosslinks that form in proteins post-fixation in aldehyde-based fixatives. Our observation of some of the markers used to identify the progenitors and main cell types of murine BM shows that some of these markers produce dim signals (such as CD117). These markers might be challenging to adapt to the fixed and paraffin-embedded tissues without major modifications to the protocol we recommend. We have used young male mice (12-15 weeks old) for our protocol. It would be particularly interesting to see if this protocol can also be used to image the cryosections from older mice since older tissues often have higher autofluorescence due to the accumulation of lipofuscin [66,67,68],

[0171] Despite the above-mentioned limitations, Phenocycler® 2.0 is still a desirable multiplex imaging tool that can not only image rare cell types and give users a means for conducting in-depth cellular analyses, but it is also the only multiplex imaging platform that can recover the tissue at the end. The recovered tissue can be re-run without the need to be stained again, or other histochemical analyses can be performed, such as H&E staining. Our protocol covers a broad panel of antibodies that cover several markers for immune cells, hematopoiesis, vasculature, and ECM. However, this panel can be expanded by other researchers using the information in our study. One of the biggest advantages of using Phenocycler® 2.0 is that markers that have commercially available antibodies can be custom-conjugated and used in conjunction with either the existing pre-conjugated commercial antibodies or other user-developed panels (such as this protocol).

[0172] Conclusion

[0173] Phenocycler® 2.0 can be a particularly useful tool to study unperturbed BM with the cell neighborhoods and structures intact. It can be a powerful analytical tool when combined with an informatics tool such as HALO® image analysis software to study cellular interactions and how they change in the knock-out or knock-in murine models, which can help develop therapies for different diseases. However, the accuracy of the visual representation of the markers needs to be scrutinized and cross-checked with established techniques such as IF. We were able to develop a protocol from tissue processing steps to image processing and analysis, which was able to provide accurate visualizations of the cell and structural markers.

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[0242] The existing software can be altered to incorporate the inventors’ changes without any change to the hardware or general workflow of the system. This idea can be immediately implemented with the current equipment by making a change to the imaging routine. Essentially, instruct the software to capture a blank automatically during the rinse of the stripping solution. It would add additional time to the acquisition, but would result in better blank generation, ultimately resulting in a more accurate final output.

[0243] The current standard workflow, to the best of our knowledge is as follows:

[0244] Step 1. Tissue in ‘Low DMSO Buffer’IU 2024-155-02 Quarles 144578.00457

[0245] Step 2. Oligo-barcode fluorescent reporters added (or in the case of a blank, no oligos are added - a mock buffer is added)

[0246] Step 3. A ‘Low DMSO buffer’ rinse step to remove any non-specific binding

[0247] Step 4. Automated imaging with microscope

[0248] Step 5. A ‘High DMSO buffer’ / ‘Stripping Buffer’ to remove / wash the oligo-barcode fluorescent reporters away

[0249] Step 6. A rinse to remove the ‘High DMSO buffer’ / ‘Stripping Buffer’ and return it to step 1.

[0250] The first change is the pre-treatment of tissue at the beginning of the experiment. For these pre-treatment steps we are essentially equilibrating the tissue to the DMSO / buffer mixture through additional rinses. These steps do not need to be imaged. The inventors propose to simply allow users the option to turn off the imaging step or skip imaging (step 4) during these initial cycles.

[0251] The second change is to automate the capture of the more accurate blank background image. Right now, because we are limited by the software, we run through steps 1-6 for a blank, then run steps 1-6 for the experimental acquisition of our marker and repeat until all markers are acquired. This takes time and is wasteful of expensive buffer. Rather than have an entire cycle dedicated to acquiring a blank image, one could capture a blank after step 6, either after or during the re-equilibration of the tissue for the next cycle.

[0252] Step 7. Automated imaging with microscope

[0253] This would allow for a blank that is dedicated to each marker that was just imaged and be ever more temporally linked to the proper experimental channel it will be subtracted from. Depending on how much DAPI is rinsed out of tissue with the ‘High DMSO buffer’ / ’ Stripping Buffer’, the system may need to re-stain with a DAPI solution before this step. If that is true, one might be able to omit DAPI from the other reporter wells.

[0254] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may beIU 2024-155-02Quarles 144578.00457 practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0255] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

1. IU 2024-155-02Quarles 144578.00457CLAIMS1. A method of generating a corrected multiplex image of a sample, the method comprising:(A) contacting the sample with a composition comprising a first set of target specific markers and acquiring a first blank image for a first set of target specific markers over a first duration of time;(C) contacting the sample with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of target specific markers, wherein each of the first set of target specific markers comprise a first polynucleotide and the first set of reporter molecules each comprise a first hybridizing polynucleotide that is complementary to the first polynucleotide;(D) acquiring a first sample image of a sample comprising the first set of target specific markers for the first duration of time;(E) subtracting the first blank image from the first sample image to generate a first corrected image;(F) removing the first set of reporter molecules and / or removing a signal from the first set of reporter molecules from the sample;(G) contacting the samples with an hf11set of target specific markers and acquiring an N111blank image for an Nlhset of target specific markers over an Nlhduration of time, wherein N is a positive integer;(H) contacting the sample with an N* reporter solution, wherein the N* reporter solution comprises an N111set of reporter molecules that are specific for the N* set of target specific markers, wherein each of the N11' set of target specific markers comprise an N11' polynucleotide and the N* set of reporter molecules each comprise an N* hybridizing polynucleotide that is complementary to the first polynucleotide;(I) acquiring an N* sample image of the sample comprising the first set of target specific markers;(J) subtracting the Nthblank image from the Nthsample image to generate an N* corrected image; andIU 2024-155-02 Quarles 144578.00457(K) combining the first corrected image and the N* corrected image to generate a corrected multiplex image of the sample, wherein acquiring comprises acquiring an image using a digital microscope with automated liquid handling.

2. The method of claim 1, wherein the method further comprises acquiring a pre-run blank of the sample prior to contacting the sample with the composition comprising the first set of target specific markers.

3. The method of claim 2, wherein the pre-run blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the composition comprising the first set of target specific markers, and the first blank comprises the pre-determined number of acquisitions of the sample before contacting the sample with the first reporter solution.

4. The method of claim 1, wherein the sample is a demineralized bone sample comprising bone marrow.

5. A method comprising:(A) acquiring a first blank image of a sample comprising a first set of target specific markers wherein the first blank image is acquired for a first duration of time;(B) contacting the sample of (a) with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of markers;(C) acquiring a first sample image of the sample of (B) for the first duration of time.

6. The method of claim 5, further comprising subtracting the first blank image from the first sample image to generate a first corrected image.

7. The method of claim 5 or 6, wherein the first duration of time is pre-determined based on an optimized duration of exposure of the first set of reporter molecules bound to the first set of target specific markers.IU 2024-155-02Quarles 144578.004578. The method of claim 5, further comprising:(D) removing the first set of reporter molecules and / or removing a signal from the first set of reporter molecules from the sample;(F) acquiring an Nthblank image of the sample for an N111set of target specific markers over an N* duration of time, wherein N is a positive integer;(G) contacting the sample of (F) with an N111reporter solution, wherein the Nthreporter solution comprises an N* set of reporter molecules that are specific for the N* set of target specific markers; and(H) acquiring an N111sample image of the sample of (G).

9. The method of claim 8, further comprising subtracting the N111blank image from the N* sample image to generate an N11' corrected image.

10. The method of claim 9, further comprising combining one or more of the first corrected image and the N* corrected image to generate a corrected multiplex image of the sample.

11. The method of claim 5, wherein the pre-run blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the composition comprising the first set of target specific markers, and the first blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the first reporter solution.

12. A method of acquiring an image of a sample, the method comprising:(A) acquiring a pre-run blank of the sample;(B) contacting the sample with a composition comprising a first set of target specific markers and acquiring a first blank image of the sample for a first set of target specific markers over a first duration of time;(C) contacting the sample of (B) with a first reporter solution, wherein the first reporter solution comprises a first set of reporter molecules that are specific for the first set of target specific markers; and(D) acquiring a first sample image of a sample of (C) comprising the first set of target specific markers for the first duration of time;IU 2024-155-02Quarles 144578.00457 thereby acquiring an image of the sample.

13. The method of claim 12, further comprising subtracting the first blank image from the first sample image to generate a first corrected image.

14. The method of claim 12, wherein the first duration of time is pre-determined based on an optimized duration of exposure of the first set of reporter molecules.

15. The method of claim 12, further comprising:(E) removing the first set of reporter molecules and / or removing a signal from the first set of reporter molecules from the sample;(F) acquiring an Nthblank image of the sample for an N* set of target specific markers over an N111duration of time, wherein N is a positive integer;(G) contacting the sample of (F) with an N111reporter solution, wherein the N111reporter solution comprises an N111set of reporter molecules that are specific for the Nthset of target specific markers; and(H) acquiring an N111sample image of the sample of (G) comprising the first set of target specific markers; thereby acquiring an N* image of the sample.

16. The method of claim 15, further comprising subtracting the Nthblank image from the Nthsample image to generate an N111corrected image.

17. The method of claim 16, further comprising combining the first corrected image and the N111corrected image to generate a corrected multiplex image of the sample.

18. The method of claim 15, wherein the pre-run blank comprises a pre-determined number of acquisitions of the sample before contacting the sample with the composition comprising the first set of target specific markers, and the first blank comprises the pre-determined number of acquisitions of the sample before contacting the sample with the first reporter solution.IU 2024-155-02Quarles 144578.0045719. The method of claim 15, wherein the N* blank image comprising the pre-determined number of acquisitions of the sample before contacting the sample with the N* reporter solution.

20. The method of claim 19, wherein the pre-determined number of acquisitions is an integer from 1 to 100.

21. The method of claim 19, wherein the pre-determined number of acquisitions is an integer from 1 to 20.

22. The method of claim 19, wherein the pre-determined number of acquisitions is an integer from 5 to 10.

23. The method of any one of claim 19, wherein the pre-determined number of acquisitions is 7.

24. The method of any one of claims 1-23, wherein the each of the first set of target specific markers comprise a first polynucleotide and the first set of reporter molecules each comprise a first hybridizing polynucleotide that is complementary to the first polynucleotide.

25. The method of any one of claims 1, 8, or 15, wherein each of the N* set of target specific markers comprise an Nthpolynucleotide and the Nthset of reporter molecules each comprise an N11' hybridizing polynucleotide that is complementary to the first polynucleotide.

26. The method of any one of claims 1-25, wherein the sample is a fixed sample.

27. The method of any one of claims 1-26, wherein the sample is a tissue sample.

28. The method of any one of claims 1-27, wherein the sample is a demineralized bone sample comprising bone marrow.IU 2024-155-02 Quarles 144578.0045729. The method of any one of claims 1-28, wherein the sample is a sectioned frozen sample embedded in tissue freezing medium.

30. The method of any one of claims 1-29, wherein acquiring comprises acquiring an image using a digital microscope with automated liquid handling.

31. The method of any one of claims 1-30, wherein at least one of the target specific markers in the first set to the Nthset of target specific markers comprises a marker specific for any one of TERI 19, CD41, CD 117, CD 150, Grl, CD48, endomucin B, alpha actinin, B220, fibromodulin, emilin2, CD45, EFEMP2, CRTAP, LUMICAN, CD31, PEDF, CD110, CD115, fibulin2, CollA, F4 / 80, CFGRP, CD71, CD61, and Sca-1.

Images