We thank the Hotchkiss Brain Institute Advanced Microscopy Platform for imaging infrastructure and expertise. RWJ was supported by postdoctoral fellowship funding from the University of Calgary Eyes High program and by a Multiple Sclerosis Society of Canada and Roche Canada unrestricted educational fellowship. VWY received salary support from the Canada Research Chair Tier 1 program. This work was supported by operating funds from the Canadian Institutes of Health Research Grant 1049959, the Multiple Sclerosis Society of Canada Grant 3236, and the US Department of Defense of the Congressionally Directed Multiple Sclerosis Research Program. Figure 1 is created with BioRender.com. The figures adapted in this publication were originally published in The Journal of Immunology. Rajiv W. Jain, David A. Elliott, and V. Wee Yong. 2023. Single Cell Analysis of High-Parameter Histology Images Using Histoflow Cytometry. J. Immunol. 210: 2038-2049. Copyright &#169; [2023]. The American Association of Immunologists, Inc.

This protocol does not cover sectioning tissues for histology; please see Jain et al.18 or19 for descriptions of how to section tissues for histology. This protocol can be used with any sectioned tissues on glass slides. This article uses inguinal lymph nodes isolated from an immunized animal as described previously18. The procedure and timeline for this protocol are summarized in Figure 1. The details of the reagents an...

Immunofluorescent Microscopy and Analysis for High-Parameter Tissue Imaging

<ol>
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W., Yong, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+in+central+nervous+system+disease:+Diversity,+locations+and+pathophysiology.">B cells in central nervous system disease: Diversity, locations and pathophysiology.</a> Nat Rev Immunol. 22 (8), 513-524 (2022).</li><li>Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis+pathology.">Multiple sclerosis pathology.</a> Cold Spring Harb Perspect Med. 8 (3), (2018).</li><li>Yong, V. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+single-cell+and+spatial+transcriptomics+to+elucidate+intercellular+tissue+dynamics.">Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics.</a> Nat Rev Genet. 22 (10), 627-644 (2021).</li><li>Ramaglia, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+imaging+of+immune+cells+in+staged+multiple+sclerosis+lesions+by+mass+cytometry.">Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry.</a> Elife. 8, (2019).</li><li>Bolognesi, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+staining+by+sequential+immunostaining+and+antibody+removal+on+routine+tissue+sections.">Multiplex staining by sequential immunostaining and antibody removal on routine tissue sections.</a> J Histochem Cytochem. 65 (8), 431-444 (2017).</li><li>Radtke, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IBEX:+An+iterative+immunolabeling+and+chemical+bleaching+method+for+high-content+imaging+of+diverse+tissues.">IBEX: An iterative immunolabeling and chemical bleaching method for high-content imaging of diverse tissues.</a> Nat Protoc. 17 (2), 378-401 (2022).</li><li>Radtke, A. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histo-cytometry:+A+method+for+highly+multiplex+quantitative+tissue+imaging+analysis+applied+to+dendritic+cell+subset+microanatomy+in+lymph+nodes.">Histo-cytometry: A method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes.</a> Immunity. 37 (2), 364-376 (2012).</li><li>Kotov, D. I., Pengo, T., Mitchell, J. S., Gastinger, M. J., Jenkins, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chrysalis:+A+new+method+for+high-throughput+histo-cytometry+analysis+of+images+and+movies.">Chrysalis: A new method for high-throughput histo-cytometry analysis of images and movies.</a> J Immunol. 202 (1), 300-308 (2019).</li><li>Jain, R. W., Elliott, D. A., Yong, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+of+high-parameter+histology+images+using+histoflow+cytometry.">Single-cell analysis of high-parameter histology images using histoflow cytometry.</a> J Immunol. 210 (12), 2038-2049 (2023).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Laboratory+Techniques.">General Laboratory Techniques.</a> Histological Sample Preparation for Light Microscopy. , (2023).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+An+open-source+platform+for+biological-image+analysis.">Fiji: An open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Berg, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ilastik:+Interactive+machine+learning+for+(bio)image+analysis.">ilastik: Interactive machine learning for (bio)image analysis.</a> Nat Methods. 16 (12), 1226-1232 (2019).</li><li>Reichard, A., Asosingh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practices+for+preparing+a+single+cell+suspension+from+solid+tissues+for+flow+cytometry.">Best practices for preparing a single cell suspension from solid tissues for flow cytometry.</a> Cytometry A. 95 (2), 219-226 (2019).</li><li>Ruhlandt, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+quantum+yield+measurements+of+fluorescent+proteins+using+a+plasmonic+nanocavity.">Absolute quantum yield measurements of fluorescent proteins using a plasmonic nanocavity.</a> Commun Biol. 3 (1), 627 (2020).</li><li>Combs, C. 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Here, the use of histoflow cytometry is described, a technique that has been validated previously18. It is demonstrated that when staining tissue sections with spectrally overlapping dyes, that bleed-through across channels can be removed using spectral compensation, resulting in a greater number of fluorescent parameters being clearly resolved than would normally be possible through conventional methods. As high-parameter histology images are difficult to analyze using conventional methods, an an...

Inflammation driven by cells of the immune system and glial cells can contribute to chronic disorders of the CNS where each population can promote the activity of the other1,2,3. Understanding how the immune system interacts with these elements of the CNS to promote CNS inflammation is currently a major topic of interest and has been greatly facilitated by high-parameter techniques such as single-cell RNA sequencing. Through single-cell RNA sequencing, we have discovered that there is extensive communication occurring between glial cells and the immune system in several CNS disorders4,5,6. Understanding how these interactions are affecting these disorders will be crucial to elucidating the biology of these diseases.
One issue with single-cell sequencing analyses is that these techniques require that the tissue be disrupted to obtain single cells or nuclei, resulting in a complete loss of spatial information. Knowing where a cell exists in a tissue is critical for understanding the cell&#39;s role in driving inflammation. For example, immune cells such as B cells can concentrate in the CNS during neuroinflammation; however, they rarely enter the CNS parenchyma and instead concentrate in CNS barriers7. Given their localization, it is unlikely that these cells contribute to CNS inflammation by physically interacting with glial cells in the CNS parenchyma, suggesting any interactions they may be having with glial cells would occur through secreted factors. Additionally, the pathology occurring in CNS disorders often has structure8,9 such that a cell&#39;s localization in the tissue could critically determine whether it is actively contributing to the disorder or is a bystander. Thus, the usage of spatial orientation to evaluate a cell&#39;s role in pathology is essential.
Studying cells in tissue has typically been accomplished by using immunohistochemistry or immune fluorescence microscopy. An issue with these techniques is that they typically can only image up to four parameters simultaneously. This is a major limitation to these techniques, as we know from flow cytometry and single-cell RNA sequencing analyses that many cell populations require two or more parameters for their identification; also, the number of parameters required typically increases when looking for specific subsets of a cell type10. Thus, it is impractical to use standard imaging techniques for studying how subsets of cells may be interacting within a tissue.
This issue has been partially overcome through newer high-parameter methods that can retain spatial information, such as spatial RNA sequencing11 and imaging mass cytometry12. While these techniques are valuable, they do have several issues, such as not being widely available, reducing three-dimensional data into two dimensions, and requiring considerable expertise to execute. Another technique known as sequential staining, wherein tissues are stained with one set of antibodies followed by inactivation of the previous set of antibodies before staining with another set of antibodies, can achieve high-parameter histology without the need for specialized equipment or expertise13,14,15. However, sequential staining can be extremely labor intensive and requires a large amount of microscopy time, which may be impractical for laboratories that do not own a personal microscope. Thus, there is a need for techniques that can expand the number of fluorescent parameters that can be imaged at one time on microscopes that are widely available and in a timely fashion.
Once the high-parameter data has been acquired, another issue arises: conventional image analysis methods are unlikely to successfully analyze the data. Techniques such as manual counting or thresholding are only viable if the analysis consists of a single parameter or if multiple markers have the same localization where only overlapping signals are counted. This limitation makes traditional analysis inadequate to work with high-parameter datasets. Successful analysis of these datasets has been achieved by segmenting single cells from histology images and then conducting flow cytometry-like gating strategies to identify cell types16,17. However, another issue that affects these analyses is that they only work for datasets wherein all the cells of interest are physically separated from one another, as these techniques do not employ methods that can accurately separate cells that are in physical contact. Thus, a newer method is required that can conduct single-cell analyses on histology sections even if the cells are in physical contact.
In this article, a simple protocol called histoflow cytometry is described that has previously been introduced18 that expands the number of fluorescent parameters that can be imaged simultaneously using widely available microscopes. This protocol works by staining tissues with spectrally overlapping dyes and then using spectral compensation to remove bleed-through from overlapping channels to obtain clear single stains. To facilitate the analysis of high-parameter histology images, a detailed analysis pipeline is described that extracts single cells from tissue sections for the purpose of sorting cells into distinct populations using flow cytometry-like gating strategies. This protocol works in tissues where cells are diffusely present and in tissues where cells are closely compacted together, making this technique versatile for the study of tissues like the CNS in both homeostasis and neuroinflammation. Histoflow cytometry is, therefore, a useful technique for studying interactions between complex cell types that require multiple cell markers to define cells while maintaining spatial information.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100% Ethanol</td>
			<td>Sigma</td>
			<td>676829-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaconda</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://www.anaconda.com/download</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A4503-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold fish stain gelatin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Collating multichannel data from Imaris.ipynb script</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://github.com/elliottcalgary/Histoflow-Cytometry-Analysis-</td>
		</tr>
		<tr>
			<td>Convert FlowJo output to txt file for Cell selection in Imaris.ipynb script</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://github.com/elliottcalgary/Histoflow-Cytometry-Analysis-</td>
		</tr>
		<tr>
			<td>Donkey anti-rat Alexa Fluor 647</td>
			<td>JacksonImmunoResearch</td>
			<td>712-605-153</td>
			<td>1:300 concentration</td>
		</tr>
		<tr>
			<td>Donkey anti-rat DyLight 405</td>
			<td>Jackson ImmunoResearch</td>
			<td>712-475-153</td>
			<td>1:200 concentration</td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>JacksonImmunoResearch</td>
			<td>017-000-001</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2-Goat anti-Mouse IgG PerCP-eFluor 710</td>
			<td>Thermofisher</td>
			<td>46-4010-82</td>
			<td>1:25 concentration</td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo LLC</td>
			<td></td>
			<td>Software 4</td>
		</tr>
		<tr>
			<td>Fluorescence spectraviewer</td>
			<td></td>
			<td></td>
			<td>https://www.thermofisher.com/order/fluorescence-spectraviewer/#!/</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fresh frozen human tonsil sections</td>
			<td>amsbio</td>
			<td>HF-707</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>VWR</td>
			<td>48393 106</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgA Alexa Fluor 488</td>
			<td>JacksonImmunoResearch</td>
			<td>109-546-011</td>
			<td>1:400 concentration</td>
		</tr>
		<tr>
			<td>Goat anti-human IgG Cy3</td>
			<td>JacksonImmunoResearch</td>
			<td>709-166-098</td>
			<td>1:400 concentration</td>
		</tr>
		<tr>
			<td>Goat anti-human IgM Dylight 405</td>
			<td>JacksonImmunoResearch</td>
			<td>109-476-129</td>
			<td>1:300 concentration</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit A546</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11035</td>
			<td>1:250 concentration</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG PE-Alexa Fluor 610</td>
			<td>Thermofisher</td>
			<td>A-20981</td>
			<td>1:250 concentration</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td></td>
		</tr>
		<tr>
			<td>Ilastik</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://www.ilastik.org/</td>
		</tr>
		<tr>
			<td>Ilastik FIJI plugin</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://www.ilastik.org/documentation/fiji_export/plugin</td>
		</tr>
		<tr>
			<td>Imaris File Converter</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Software 2</td>
		</tr>
		<tr>
			<td>Imaris with cell module</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>Software 3</td>
		</tr>
		<tr>
			<td>kimwipe</td>
			<td>Kimtech</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>LasX Life Science software</td>
			<td>Leica</td>
			<td></td>
			<td>Software 1</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD20</td>
			<td>VWR</td>
			<td>CA95024-322</td>
			<td>1:40 concentration</td>
		</tr>
		<tr>
			<td>Mouse anti-human CD38 APC-R700</td>
			<td>BD Biosciences</td>
			<td>564980</td>
			<td>1:20 concentration</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>JacksonImmunoResearch</td>
			<td>005-000-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Mouse Serum</td>
			<td>JacksonImmunoResearch</td>
			<td>015-000-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rabbit Serum</td>
			<td>JacksonImmunoResearch</td>
			<td>011-000-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rat Serum</td>
			<td>JacksonImmunoResearch</td>
			<td>012-000-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear Yellow</td>
			<td>Abcam</td>
			<td>ab138903</td>
			<td>Dissolve in DMSO at a concentration of 2 mg/ml and store at 4&#176;C in the dark</td>
		</tr>
		<tr>
			<td>PAP pen</td>
			<td>Cedarlane</td>
			<td>MU22</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human Ki67</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>1:500 concentration</td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse Iba1</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>1:500 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-human Blimp1</td>
			<td>Thermofisher</td>
			<td>14-5963-82</td>
			<td>1:40 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-mouse B220 Alexa Fluor 647</td>
			<td>BioLegend</td>
			<td>103226</td>
			<td>1:250 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD138</td>
			<td>Biolegend</td>
			<td>142502</td>
			<td>1:200 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD3 PE-eFluor 610</td>
			<td>Thermo Fisher Scientific</td>
			<td>61-0032-82</td>
			<td>1:40 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD4 Alexa Fluor 488</td>
			<td>BioLegend</td>
			<td>100529</td>
			<td>1:200 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 allophycocyanin-R700</td>
			<td>BD Biosciences</td>
			<td>565478</td>
			<td>1:50 concentration</td>
		</tr>
		<tr>
			<td>Rat anti-mouse IgD PerCP-eFluor 710</td>
			<td>Thermo Fisher Scientific</td>
			<td>46-5993-82</td>
			<td>1:50 concentration</td>
		</tr>
		<tr>
			<td>SP8 Confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Trueblack</td>
			<td>Biotium</td>
			<td>23007</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P7949-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracomp ebeads</td>
			<td>Thermofisher</td>
			<td>01-2222-42</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating and analyzing high-parameter histology images with histoflow cytometry

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically limited by the number of fluorescent parameters that can be imaged at a single time. Most immune cell subsets have been defined using flow cytometry by using complex combinations of protein markers, often requiring four or more parameters to conclusively identify, which is beyond the capabilities of most conventional microscopes. As flow cytometry dissociates tissues and loses spatial information, there is a need for techniques that can retain spatial information while interrogating the roles of complex cell types. These issues are addressed here by creating a method for expanding the number of fluorescent parameters that can be imaged by collecting the signals of spectrally overlapping fluorophores and using spectral unmixing to separate the signals of each individual fluorophore. These images are then processed using an analysis pipeline to take high-parameter histology images and extract single cells from these images so that the unique fluorescent properties of each cell can be analyzed at a single-cell level. Using flow cytometry-like gating strategies, cells can then be profiled into subsets and mapped back onto the histology sections to not only quantify their abundance, but also establish how they interact with the tissue environment. Overall, the simplicity and potential of using histoflow cytometry to study complex immune populations in histology sections is demonstrated.

Author Spotlight: Enhanced Multiplex Immunofluorescent Microscopy Protocol for Neuroscience Research

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry

Our overarching goal is to study how cells of the immune system and glial cells within the central nervous system contribute to the development and progression of neurological disorders such as multiple sclerosis. Immunofluorescent microscopy is a standard and widely used technique across biological disciplines and is essential to neuroscience and immunology research. One of the main issues that plague researchers is that immunofluorescent microscopy is typically limited in the number of probes that can be imaged at a single time due to the limitations of conventional microscopes.In this protocol, we describe a method to expand the number of probes that many microscopes can image at a single time, and provide an analysis pipeline to process information-dense images. The advantage of this protocol is that it can be adapted to many widely available microscopes and is easy to execute, which would make multiplex histology analysis available to a greater number of labs.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66889/66889fig01.jpg" /> 
Figure 1: Histoflow cytometry workflow. Tissue sections are stained with spectrally overlapping dyes (step 1). Images are collected across individual excitation lasers paired with tunable bandpass filters to minimize spectral bleed-through between fluorophores (step 2). Spectral bleed-through between channels is corrected based on a compensatio...

Described here is a method that can be used to image five or more fluorescent parameters by immunofluorescent microscopy. An analysis pipeline for extracting single cells from these images and conducting single-cell analysis through flow cytometry-like gating strategies is outlined, which can identify cell subsets in tissue sections.

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically ...

generating-analyzing-high-parameter-histology-images-with-histoflow