Imaging and analysis guidance was provided by the Research Resources Center &#8211; Research Histology and Tissue Imaging Core at the University of Illinois at Chicago established with the support from the office of the Vice Chancellor for Research. The work was supported by NIH/NCI RO1CA191317 to CLP, by NIH/NIAMS (SBDRC grant 1P30AR075049-01) to Dr. A. Paller, and by support of the Robert H. Lurie Comprehensive Cancer Center to the Immunotherapy Assessment Core at Northwestern University.

Mouse spleen and HLF16 mouse tumor tissues23 were obtained from our laboratory. Human tonsil tissue was purchased from a commercial vendor. Details are provided in the Table of Materials.
1. Tissue Embedding
<ol>
	<li>Embed fresh tissue in OCT (optimal cutting temperature) solution and snap freeze using either dry ice or liquid nitrogen.</li>
	<li>Store tissues at -80 &#176;C.</li>
</ol>
2. Cryosectioning
<ol>
	<li>Cut 8 &#95...

Frozen tissues have extensively been used for mIF imaging to traditionally detect three to four markers31 on a tissue using the direct and indirect method32. In the direct method, antibodies are conjugated to fluorescing dyes or quantum dots33 to label the tissue, whereas in the indirect method, an unconjugated primary antibody is used to label the tissue followed by a fluorophore-conjugated secondary antibody that specifically recognizes the primary...

Recent advances in microscopic imaging techniques have significantly improved our knowledge and understanding of biological processes and disease states. In situ detection of proteins in tissues via chromogenic immunohistochemistry (IHC) is routinely performed in pathology. However, detection of multiple markers using chromogenic IHC staining is challenging1 and newer methods to use multiplex immunofluorescence (mIF) staining approaches, wherein multiple biological markers are labeled on a single tissue sample, are being developed. The detection of multiple biological markers is useful, because information related to tissue architecture, spatial distribution of cells, and antigen co-expression are all captured in a single tissue sample2. The use of multispectral fluorescence imaging technology has made detection of multiple biological markers possible. In this technology, using specific optics the fluorescence spectra of each individual fluorophore can be separated or &#34;unmixed&#34;, enabling the detection of multiple markers without any spectral crosstalk3. Multispectral fluorescence imaging is becoming a critical approach in cell biology, preclinical drug development, clinical pathology, and tumor immune-profiling4,5,6. Importantly, the spacial distribution of immune cells (specifically CD8 T cells) can serve as a prognostic factor for patients with existing tumors7.
Various approaches to multiplex fluorescence staining have been developed and can be performed either simultaneously or sequentially. In the simultaneous staining method, all the antibodies are added together as a cocktail in a single step to label the tissue. UltraPlex technology uses a cocktail of hapten-conjugated primary antibodies followed by a cocktail of fluorophore-conjugated anti-hapten secondary antibodies. InSituPlex technology8 uses a cocktail of unique DNA-conjugated primary antibodies that are simultaneously added to the tissue followed by an amplification step and finally fluorophore-conjugated probes that are complementary to each unique DNA sequence on the primary antibody. Both of these technologies enable the detection of four markers plus 4&#8217;,6-diamino-2-phenylindole (DAPI) for nuclear staining. Two other approaches for simultaneous multiplex staining are based on secondary ion mass spectrometry9. The Hyperion Imaging System uses imaging mass cytometry10 to detect up to 37 markers. This technology uses a cocktail of metal-conjugated antibodies to stain the tissues, and specific areas of the tissues are ablated by a laser and transferred to a mass cytometer where the metal ions are detected. Another similar technology is the IONPath, which uses multiplexed ion beam imaging technology11. This technology uses a modified mass spectrometry instrument and an oxygen ion source instead of laser to ablate the metal-conjugated antibodies. While all these simultaneous multiplex staining approaches enable the detection of multiple markers, the costs involved for conjugating DNA, haptens, or metals to antibodies, the loss of tissue due to ablation, and the extensive image processing for unmixing cannot be underestimated. Moreover, kits and staining protocols are currently available only for FFPE tissues and developing custom panels entails additional time and expenditure.
The sequential multiplex staining method, in contrast, includes labeling the tissue with an antibody to one marker, stripping to remove the antibody, followed by sequential repeats of this process to label multiple markers12. The tyramide signal amplification (TSA) is the most frequently used sequential multiplexing method. Two other multiplexing technologies use a combination of simultaneous and sequential staining methods. The CODEX platform13 employs a cocktail of antibodies conjugated to unique DNA oligonucleotide sequences that are eventually labeled with a fluorophore using an indexed polymerization step followed by imaging, stripping, and repeating the process to detect up to 50 markers. The MultiOmyx multiplex staining approach14 is an iteration of staining with a cocktail of three to four fluorophore-conjugated antibodies, imaging, quenching the fluorophores, and repeating this cycle to detect up to 60 markers on a single section. Similar to the simultaneous multiplex staining method, while a broad range of markers can be detected, the time involved in staining, image acquisition, processing, and analysis is extensive. The stripping/quenching step involves heating and/or bleaching the tissue sample and thus, the sequential multiplex staining approach is commonly performed on FFPE tissues that maintain tissue integrity upon heating or bleaching.
Formalin fixation and subsequent paraffin embedding is readily performed in a clinical setting, tissue blocks are easy to store, and several multiplex staining protocols are available. However, the processing, embedding, and deparaffinization of FFPE tissues, as well as antigen retrieval15, a process by which antibodies can better access epitopes, is time-consuming. Furthermore, the processing involved in FFPE tissues contributes to autofluorescence16 and masks target epitopes, resulting in the variability and lack of antibody clone available to detect antigens in FFPE tissues17,18,19. An example is the human leukocyte antigen (HLA) class I alleles20. In contrast, snap freezing of tissues does not involve extensive processing steps prior to or after fixing, circumventing the need for antigen retrieval21,22, and making it beneficial for detecting a wider range of targets. Therefore, using frozen tissues for multispectral fluorescence imaging can be valuable to detect targets for preclinical and clinical studies.
Given the above mentioned limitations when using FFPE tissues, we asked whether multispectral fluorescence imaging can be performed on frozen tissues. To address this question, we tested a simultaneous multiplex staining method using a panel of fluorophore-conjugated antibodies to detect multiple antigens and analyzed the staining using a semiautomated multispectral imaging system. We were able to simultaneously stain up to six markers in a single tissue section within 90 min.

<table>
	<tbody>
		<tr>
			<td>Acetone (histological grade)</td>
			<td>Fisher Scientific</td>
			<td>A16F-1GAL</td>
			<td>Fixing tissues</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 anti-mouse CD3</td>
			<td>BioLegend</td>
			<td>100212</td>
			<td>Clone - 17A2; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488, eBioscience anti-human CD20</td>
			<td>ThermoFisher Scientific</td>
			<td>53-0202-82</td>
			<td>Clone - L26; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Mouse anti-Ki-67</td>
			<td>BD Biosciences</td>
			<td>558617</td>
			<td>Primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-human CD3</td>
			<td>BioLegend</td>
			<td>300446</td>
			<td>Clone - UCHT1; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 anti-mouse CD8a</td>
			<td>BioLegend</td>
			<td>100758</td>
			<td>Clone - 53-6.7; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-human CD8a</td>
			<td>BioLegend</td>
			<td>372906</td>
			<td>Clone - C8/144B; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD206 (MMR)</td>
			<td>BioLegend</td>
			<td>141711</td>
			<td>Clone - C068C2; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 anti-mouse CD4 Antibody</td>
			<td>BioLegend</td>
			<td>100426</td>
			<td>Clone - GK1.5; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>C57BL/6 Mouse</td>
			<td>Charles River Laboratories</td>
			<td>27</td>
			<td>Mouse frozen tissues used for multispectral training</td>
		</tr>
		<tr>
			<td>Coplin Jar</td>
			<td>Sigma Aldrich</td>
			<td>S6016-6EA</td>
			<td>Rehydrating and washing slides</td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>BD Biosciences</td>
			<td>564907</td>
			<td>Nucleic Acid stain</td>
		</tr>
		<tr>
			<td>Diamond White Glass Charged Slides</td>
			<td>DOT Scientific</td>
			<td>DW7590W</td>
			<td>Adhering tissue sections</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline 1x (without Ca and Mg)</td>
			<td>Fisher Scientific</td>
			<td>MT21031CV</td>
			<td>Washing and diluent</td>
		</tr>
		<tr>
			<td>Gold Seal Cover Slips</td>
			<td>ThermoFisher Scientific</td>
			<td>3306</td>
			<td>Protecting stained tissues</td>
		</tr>
		<tr>
			<td>Human Normal Tonsil OCT frozen tissue block</td>
			<td>AMSBio</td>
			<td>AMS6023</td>
			<td>Human frozen tissue used for multispectral staining</td>
		</tr>
		<tr>
			<td>Human Serum 1X</td>
			<td>Gemini Bio-Products</td>
			<td>100-512</td>
			<td>Blocking and diluent for human tissues</td>
		</tr>
		<tr>
			<td>inForm</td>
			<td>Akoya Biosciences</td>
			<td>Version 2.4.1</td>
			<td>Machine learning software</td>
		</tr>
		<tr>
			<td>PerCP/Cyanine5.5 anti-human CD4</td>
			<td>BioLegend</td>
			<td>300529</td>
			<td>Clone - RPA-T4; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>PerCP-Cy 5.5 Rat Anti-CD11b</td>
			<td>BD Biosciences</td>
			<td>550993</td>
			<td>Clone - M1/70; primary conjugated antibody</td>
		</tr>
		<tr>
			<td>Phenochart</td>
			<td>Akoya Biosciences</td>
			<td>Version 1.0.8</td>
			<td>Whole slide scan software</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36965</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Research Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050 S</td>
			<td>Sectioning tissues</td>
		</tr>
		<tr>
			<td>Superblock 1X</td>
			<td>ThermoFisher Scientific</td>
			<td>37515</td>
			<td>Blocking mouse tissues</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T Solution</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td>Embedding tissues</td>
		</tr>
		<tr>
			<td>Vectra 3.0 Automated Quantitative Pathology Imaging System, 6 Slide</td>
			<td>Akoya Biosciences</td>
			<td>CLS142568</td>
			<td>Semi-automated multispectral imaging system</td>
		</tr>
		<tr>
			<td>Vectra Software</td>
			<td>Akoya Biosciences</td>
			<td>Version 3.0.5</td>
			<td>Software to operate microscope</td>
		</tr>
	</tbody>
</table>

a rapid method for multispectral fluorescence imaging of frozen tissue sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

Detection of single-stained markers on frozen spleen sections 
As the semiautomated imaging system uses a liquid crystal tunable filter (LCTF) system that allows for a wider range of wavelength detection25, and because no signal amplification steps were performed here, we first optimized the detection of our primary-conjugated antibodies for each marker on the microscope. An example is shown in Figure 1, where each single-stained marker is pseudo...

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A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

This method is helpful to detect, and co-localize multiple biomarkers in a single cryosection including markers that are not normally detectable in paraffin sections. Using this experimental staining method, the staining time is significantly reduced and the method can be performed in any laboratory. Demonstrating this method will be Dinesh Jaishankar.He is a skilled postdoc from the lab, and also the Immunotherapy Assessment Core, Facilities Manager. When selecting antibodies for the analysis, consider the excitation and emission filter sets available on the semi-automated imaging system. And after testing various combinations of fluorophore conjugated primary antibodies, prepare fluorophores to be used so that you have minimal spectral overlap as suggested in the table.For multiplex staining, prepare a cocktail of antibodies with compatible fluorophores at predetermined optimal delusions, and add the cocktail to the tissue sections of interest. For single marker staining, add the primary conjugated antibody only to the slide. For all stainings, include a control unstained slide that undergoes the same staining procedure without the addition of a primary conjugated antibody.After a one-hour incubation at room temperature protected from light, wash the slides two times with PBS for five minutes per wash. After the second wash, counter stain the multiplex stained slides with DAPI for seven minutes at room temperature protected from light, followed by two five-minute washes in PBS. To create a spectral library, set the lamp power of the multispectral imaging system to 100%and open the microscope operating software.Select Edit Protocol and new protocol. Enter a protocol name and select Fluorescence under imaging mode. Then provide a study name, and load the single stained slides onto the stage.Click Edit Exposure, and use the auto-focus and auto-expose options to adjust the exposure times and acquire snapshots for the single stained slides. After imaging the single stained slides, save the protocol. And in the machine learning software under the Build Library's tab, load each single stained image.Select the appropriate floor and click Extract. The software will automatically extract the fluorescent signal selected in the floor. To save the extracted color, click Save to store.A new group may be created or the extracted color can be stored to an existing group. For multispectral imaging, once the spectral library has been created and verified, adjust the focus and exposure times on the multiplex stained slide as demonstrated, and opens scanned slides to create a new task. Provide a slide ID and select a protocol.Under task, select whole slide scan to perform a whole slide scan on the multiplex stained slide. In the whole slide scan image, select regions of interest across the image. These regions will be scanned using the previously set exposure times on the multiplex slide to be used for spectral unmixing and analysis.In the microscope operating software, select acquire MSI fields under task, and then click Process Slide to acquire regions of interest at a 20x magnification. Once the regions of interest have been acquired, in the machine learning software, click File and Open under the manual analysis tab to load the multiplex stained images. Select the spectral library source, and click Select Floors to choose the previously saved spectral library.Click File and Open to load the unstained image. Then click the autofluorescence ink marker icon and draw a line or region on the unstained slide to identify autofluorescence within the tissue. Then under the Edit Markers and Colors tab, assign names for each marker and click Prepare All.After verifying the spectrally unmixed image, in the machine learning software, select the cytoplasm membrane and marker options from the panel. And using the ellipsis button, select use this signal to find, to configure the marker to detect either the nuclei, cytoplasm or membrane. when a cytoplasmic or membrane marker is chosen, select the Use the Signal to assist in nuclear splitting option.The software will automatically detect and create individual masks for the nucleus, cytoplasm and membrane. Use the pathology view for a specific marker and the configuration options in the software to adjust the masks, and click Segment All to create a cell segmentation map. After cell segmentation, select the markers to phenotype the positive cells.Additionally, select at least five cells that are not stained with the chosen marker to phenotype the negative cells. Then click Train Classifier to train the software to automatically detect all of the cells stained with the selected markers within the image. A phenotype map will be created.As these images demonstrate, antibodies validated for flow cytometry could be used to visualize tissue staining using the liquid crystal tunable filter in the semi-automated imaging system. Here a spectrally unmixed image of frozen human tonsil can be observed that includes labeling of B-cells and Proliferating cells within the follicular germinal center, and the inter follicular T-cell region. The pathology views option allows visualization of the individual staining pattern for each marker within the tissue of interest, suggesting that the multiplex staining method and spectral imaging work on frozen tissue.As demonstrated on a frozen mouse tumor section, multispectral imaging can also be used to visualize tumor infiltrating T-cells, and other myeloid cell lineages including tumor associated macrophages. Cell segmentation and phenotype maps, and the number of stained cells for each marker analyzed by the software can be generated, demonstrating that the software can be used for quantification of the multispectral staining on frozen tissues. For someone new 6to the technique, it's important to get to know the specific features of your fluorochromes, and prepare quality library slides for spectral unmixing with minimal spectral overlap.It's also important to perform antibody titration. In our opinion, this method can provide bedside information about the efficacy of immunotherapy for cancer and other therapeutic applications, particularly when timing is of the essence.

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Abstract