A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Summary

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

Abstract

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.

Introduction

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 "unmixed", 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-profiling^4,5,6. 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’,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 tissues^17,18,19. 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 retrieval^21,22, 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.

Protocol

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

1. Embed fresh tissue in OCT (optimal cutting temperature) solution and snap freeze using either dry ice or liquid nitrogen.
2. Store tissues at -80 °C.

2. Cryosectioning

1. Cut 8 μm sections in a cryostat with temperatures set at -25 °C.
   NOTE: The preferred section thickness can be adjusted to generate crisper images.
2. Place sections on charged glass slides.
3. Air-dry the sections for 1 h at room temperature (RT) prior to fixing in histology grade ice-cold acetone for 10 min.
   NOTE: Acetone causes coagulation of water-soluble proteins and extracts lipids but does not impact carbohydrate-containing components. In contrast, formalin preserves most lipids and has little impact on carbohydrates²⁴. The choice of fixative is important depending on the choice of marker being detected.
4. Store the slides at -20 °C.

3. Selection of Antibodies and Fluorophores

NOTE: Before tissue staining, antibody clones that will robustly and specifically stain their antigens of interest within sequential sections from acetone fixed tissue must be validated. Some antibodies may require a different fixative, and their compatibility with other antibodies in the panel will also have to be empirically determined. The goal is also to identify fluorophores with minimal overlap that can be detected with the epifluorescence filters for DAPI, FITC, Cy3, Texas Red, and Cy5.

1. Confirm staining by conventional IHC or immunofluorescence (IF) detection in tissue sections with known expression target antigen.
2. Using the excitation and emission filter sets available on the semiautomated imaging system and after testing various combinations of fluorophore-conjugated primary antibodies, prepare fluorophores to be used that have minimal spectral overlap (e.g., see Table 1).

4. Staining

NOTE: The tissue rehydration and slide washes were performed in a Coplin jar. The blocking and antibody incubation steps were performed in a humidified slide box.

1. Allow the slides to warm to RT for 5–10 min.
2. Rehydrate in phosphate buffered saline (PBS) for 5 min.
3. Perform a blocking step prior to staining tissues with antibodies. For mouse sections, use specialized blocking solution (see Table of Materials) for 10 min at RT. For human sections, use 10% normal pooled human serum (NHS) diluted in PBS for 15 min at RT.
   NOTE: Different blocking buffers may be tested as needed to preserve the specific properties of the sections depending on the follow-up procedures to be used.
4. Wash the slides for 5 min in PBS after blocking.
5. For multiplex staining, prepare a cocktail of antibodies with compatible fluorophores at predetermined optimal dilutions.
6. Add the cocktail of fluorophore-conjugated antibodies to the slides. For single-marker staining, add only the primary-conjugated antibody to the slide.
7. Include a control unstained slide that undergoes the same staining procedure without the addition of any primary-conjugated antibody.
8. Incubate the slides for 1 h at RT in the dark and then wash the slides 2x with PBS for 5 min each. From here on, the resulting slide is referred to as multiplex-stained.
9. To counterstain, add DAPI to the multiplex-stained slide, incubate for 7 min in the dark at RT, and wash the slides 2x with PBS for 5 min each. Do not counterstain single-stained and unstained slides.
10. To coverslip, add a drop of the mounting medium, and gently place the glass coverslip over the tissue.

5. Preparing a Spectral Library

1. Image acquisition
   1. Set the lamp power to 100%. Usually the power is set to 10% because fluorescence detection on FFPE tissues includes a signal amplification step.
   2. Begin by opening the microscope operating software (see Table of Materials).
   3. Select Edit Protocol and then New Protocol.
   4. Provide a "Protocol Name", and select Fluorescence under Imaging Mode, and provide a "Study Name".
   5. Place the single-stained slides on the stage and examine each marker in its corresponding fluorescence channel to ensure staining. Choose a region on the tissue expressing the strongest signal for the marker.
   6. Adjust the exposure times using the Autofocus and Autoexpose options.
   7. Acquire snapshots for the single-stained and unstained slides and save the protocol.
      NOTE: The following steps are performed in machine learning software (see Table of Materials), using the single stained and unstained slides to verify specific staining as well as to determine antibody cross talk.
   8. Under the Build Libraries tab in the software, load each single-stained slide image, choose the appropriate Fluor, and click Extract. The software will automatically extract the fluorescence signal chosen in the Fluor.
   9. To save the extracted color, click on Save to Store. A "New Group" may be created or the extracted color can be stored to an existing group.
2. Verifying the spectral library
   1. Check the emission spectral curve window, located to the right of the extracted image, for each filter set.
      NOTE: The extracted signal is correct if the spectral curve is observed only in the filter sets where the fluorophore is detected. If a spectral curve is observed in the wrong filter set, it can mean that either the primary signal expected in the filter set is not strong enough, or the software is detecting another signal that is too high, possibly due to spectral overlap. In this case, first try using the Draw Processing Regions tool to draw regions around areas expressing the fluorescent marker and autofluorescence in the image. This trains the software to detect the true signal and remove any interfering signals. If this does not work, repeat the staining process for the single-stained slide to test different antibody titrations.

6. Multispectral Imaging

NOTE: Once the spectral library is created and verified, perform the following steps for the multiplex-stained slide.

1. Whole slide scan
   1. Adjust focus and exposure times on the multiplex-stained slide as mentioned under in section 5.1.
   2. Under Scan Slides, create a New Task and choose the protocol saved above.
   3. Perform a whole slide scan on the multiplex-stained slide.
   4. Using the whole slide scan software, open the whole slide scan image. This image has not been spectrally unmixed.
   5. Select regions of interest (ROI) across the whole slide scan image using the Stamp or ROI tool. These ROIs will be scanned using the exposure times set in 6.1.1 to be used for spectral unmixing and analysis.
   6. Click Process Slide to acquire ROIs at 20x magnification
2. Spectral unmixing
   1. Once the ROIs have been acquired, in the machine learning software, under the Manual Analysis tab, load the multiplex stained images by clicking Open under File.
   2. In the Spectral Library Source dropdown menu click Select Fluors.
   3. A new window will open. Here, choose the spectral library or group created above.
   4. Load the unstained slide image. Click the AF ink marker icon located above the selected spectral library and draw a line or region on the unstained slide to identify autofluorescence in the tissue.
   5. Under the Edit Markers and Colors tab, assign names for each marker. Pseudo colors can be assigned at this step.
      NOTE: The color for the Autofluorescence image defaults to DarkSlateGray. Change this to Black.
   6. Click Prepare All.
3. Verifying spectrally unmixed images
   NOTE: When the spectral unmixing step is completed, a composite image consisting of all the colors is created.
   1. Click the Edit the View eye icon. Here, each color in the "Component Display" can be turned off or on to view the staining of each individual marker.
   2. Visually inspect the staining and the morphology of the cells to ensure that there is no overlapping of marker, unless it is biologically relevant. A pathologist can help verify the staining as well.
      NOTE: The staining on the multiplexed slide should be validated by leaving out one fluorophore at a time and reviewing the staining pattern. In addition, the validation will also help to identify strong fluorophores that appear in adjacent spectra due to antibody cross talk or bleed-through.
   3. For Pathology Views, which simulate brightfield images for each fluorescent marker, click the Select a Component Image button. Here, choose a marker to view a simulated brightfield image.

7. Analyzing Multispectral Images via Cell Segmentation and Phenotyping

NOTE: After verifying the spectrally unmixed image, cell segmentation can be performed using the machine learning software, which will provide step-by-step instructions. Tissue segmentation was not performed here. If the panel includes one or more tissue specific marker and especially if the tissue is messy, tissue segmentation should be performed.

1. Select "Cytoplasm" and "Membrane" under the 'Segment' option.
   NOTE: "Nuclei" is chosen by default.
2. Select a marker from the panel. Configure the marker to detect either nuclei, cytoplasm, or membrane. For example, DAPI can be selected to detect nuclei and CD3 for membrane.
3. Click on the ellipsis button ('...') to select an option under "use this signal to find". For example, 'nuclei' for DAPI. Multiple markers from the panel can be selected for segmentation.
   NOTE: For cytoplasmic or membrane markers, select the "Use this signal to assist in nuclear segmentation" option.
4. The software automatically detects and creates a mask each for the nucleus, cytoplasm, and membrane in the image.
5. Ensure all the cells are 'masked' for segmentation. To adjust, switch to the Pathology View for the specific marker chosen in 7.3. and use the configuration options in the software.
6. Click "Segment All" to segment cells.
7. After cell segmentation, proceed with phenotyping cells. In this step, choose the markers needed for phenotyping and manually select at least five cells that are brightly stained with the chosen marker. This trains the software to then automatically detect all cells stained with the chosen marker in the image.
8. A phenotype map is created. Analyze to ensure that the cell stained with the marker is correctly phenotyped.
   NOTE: Cell phenotyping can be an iterative process. If the software is unable to phenotype the cells correctly, it means that the training is inadequate or incorrect. In this case, the user has to manually select more cells and retrain the software and repeat this step until the user is satisfied with the training.
9. Create a group named "Others" and include cells that are not stained for any of the markers.
   NOTE: This step is important to train the software to exclude all the unstained cells from phenotyping.

8. Exporting Images and Analysis Tables

1. Click the Export button to view the Export Settings panel.
2. In the "Export Directory", click Browse to select a location to export the images.
3. In the "Image Export Options", choose the Image Output Format.
4. In the "Images to Export" list, select the images to be exported. "Composite image" is the final pseudocolored unmixed image. "Pathology Views" are the individual simulated brightfield images and the "Component Images (multi-image TIFF)" is a multi-image TIFF file of component data that can be used by third party analysis software.
5. Click "Export for All" button to export the images.
   NOTE: Tables from analysis can also be selected and exported at this step.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Acetone (histological grade)	Fisher Scientific	A16F-1GAL	Fixing tissues
Alexa Fluor 488 anti-mouse CD3	BioLegend	100212	Clone - 17A2; primary conjugated antibody
Alexa Fluor 488, eBioscience anti-human CD20	ThermoFisher Scientific	53-0202-82	Clone - L26; primary conjugated antibody
Alexa Fluor 555 Mouse anti-Ki-67	BD Biosciences	558617	Primary conjugated antibody
Alexa Fluor 594 anti-human CD3	BioLegend	300446	Clone - UCHT1; primary conjugated antibody
Alexa Fluor 594 anti-mouse CD8a	BioLegend	100758	Clone - 53-6.7; primary conjugated antibody
Alexa Fluor 647 anti-human CD8a	BioLegend	372906	Clone - C8/144B; primary conjugated antibody
Alexa Fluor 647 anti-mouse CD206 (MMR)	BioLegend	141711	Clone - C068C2; primary conjugated antibody
Alexa Fluor 647 anti-mouse CD4 Antibody	BioLegend	100426	Clone - GK1.5; primary conjugated antibody
C57BL/6 Mouse	Charles River Laboratories	27	Mouse frozen tissues used for multispectral training
Coplin Jar	Sigma Aldrich	S6016-6EA	Rehydrating and washing slides
DAPI Solution	BD Biosciences	564907	Nucleic Acid stain
Diamond White Glass Charged Slides	DOT Scientific	DW7590W	Adhering tissue sections
Dulbecco's Phosphate Buffered Saline 1x (without Ca and Mg)	Fisher Scientific	MT21031CV	Washing and diluent
Gold Seal Cover Slips	ThermoFisher Scientific	3306	Protecting stained tissues
Human Normal Tonsil OCT frozen tissue block	AMSBio	AMS6023	Human frozen tissue used for multispectral staining
Human Serum 1X	Gemini Bio-Products	100-512	Blocking and diluent for human tissues
inForm	Akoya Biosciences	Version 2.4.1	Machine learning software
PerCP/Cyanine5.5 anti-human CD4	BioLegend	300529	Clone - RPA-T4; primary conjugated antibody
PerCP-Cy 5.5 Rat Anti-CD11b	BD Biosciences	550993	Clone - M1/70; primary conjugated antibody
Phenochart	Akoya Biosciences	Version 1.0.8	Whole slide scan software
ProLong Diamond Antifade Mountant	ThermoFisher Scientific	P36965	Mounting medium
Research Cryostat	Leica Biosystems	CM3050 S	Sectioning tissues
Superblock 1X	ThermoFisher Scientific	37515	Blocking mouse tissues
Tissue-Tek O.C.T Solution	Sakura Finetek	4583	Embedding tissues
Vectra 3.0 Automated Quantitative Pathology Imaging System, 6 Slide	Akoya Biosciences	CLS142568	Semi-automated multispectral imaging system
Vectra Software	Akoya Biosciences	Version 3.0.5	Software to operate microscope