High-plex Imaging using Spectral Confocal Microscopy to Minimize Non-specific Tissue Fluorescence

We optimize an IBEX workflow that combines heparin blocking and spectral detection to suppress the background in eosinophil-rich human tissues. Auto fluorescence and eosinophil granule binding obscure true signal. We target both to enable clean high plex maps.

To begin, draw a circle on the glass slide around the mounted tissue using a pap pen to create a hydrophobic barrier. Add approximately 100 microliters of PBS to each tissue to rehydrate them, and incubate the slide for about two minutes at room temperature to wash off the optimal cutting temperature compound. Aspirate the PBS from around the tissue using a suction glass Pasteur pipette.

Then add 100 microliters of blocking buffer to each tissue, and incubate at room temperature in a humidity chamber for one hour. Now aspirate the blocking buffer using a pipette. add 100 microliters of staining mixed to each tissue and incubate the slide in a covered humidity chamber inside a refrigerator at four degrees Celsius overnight.

Similarly, prepare section stained with only one fluorophore and a negative control without any stain to define the fluorophore spectrum and tissue autofluorescence. After aspirating the staining mix, wash the tissue three times with PBS, aspirate the liquid after each wash. Mount the sample on the microscope for imaging.

Focus on the sample using brightfield or differential interference contrast mode. Then switch to fluorescence mode and configure the spectral acquisition parameters according to the experimental requirements. Assign the appropriate lasers for the fluorophores used in the experiment.

Select a beam splitter that corresponds to the chosen laser lines. Enable all detectors to capture the full emission spectrum, and adjust the image acquisition resolution to one of the predefined resolutions. Or choose the maximum resolution.

Adjust the pinhole size to one area unit for optimal optical sectioning. Optimize the laser settings before acquisition. Using a section stained with all markers, start continuous scanning mode at low laser power.

Adjust each detector's master gain as well as laser intensities until all markers are identifiable with a good signal to background ratio. Fine tune laser intensities and iteratively alternate between single stain and multi-stain sections until all fluorophore signals are visible without oversaturation. Enable the tiles feature in the acquisition software and define the tile acquisition region around the tissue to ensure full image coverage.

Optimize the focus for the tile acquisition region at the center of the tissue. Identify a unique structural feature such as a distinct set of cells in a recognizable arrangement to serve as a reference for alignment in later imaging rounds. Now enable the Z-stack feature and navigate to the Z-stack tab.

under the center tab, set the number of slices to at least six with an interval of no less than one micrometer between slices. Click the center button to set the current focus point determined in the previous step as the middle slice of the stack. Click the start experiment button to initiate image acquisition.

Once acquisition is complete, right click on the image thumbnail, select Save As.Choose a file location and name the image. Back up the image data to secure storage. Un-mix the image using the same optimized settings applied during tiled region acquisition.

Capture a snap from each single-stained control to obtain the true signal spectrum and from the unstained control, to capture background or autofluorescence. use these images exclusively for spectral unmixing. Confirm that the selected region represents a true signal by checking that the intensity and morphology are absent or equivalent to background levels in the unstained control and that the markers localization matches its expected cellular pattern such as membrane, nuclear, cytoplasmic, or secreted distribution.

Open the snap image in the software. Click on the unmixing tab and select one of the region selection tools under the unmixed tools tab, draw a region of interest or ROI around the positive signal to generate the spectrum of that region. Compare each generated spectrum to the theoretical emissions spectrum of the corresponding fluorophore.

Ensure that the region accurately represents the true signal. Name each spectrum according to the fluorophore identity, and save it to the spectral database. Next, load the tiled image and open the unmixing tool.

Use the plus button to add the reference spectra for each fluorophore to the unmixing list. Click the linear unmixing button to separate the spectral signals and save the unmixed image. Prepare the bleaching solution in a fume hood.

Using a micro spatula, weigh 10 milligrams of lithium borohydride directly into a dry beaker. Carefully add 10 milliliters of ultrapure water into the beaker and mix until the compound is fully dissolved. Add approximately 100 microliters of the freshly prepared bleaching solution to each tissue section using a glass Pasteur pipette and incubate the slides at room temperature for 15 minutes inside the fume hood.

If bleaching brilliant violet dies, carefully transfer the slide to an epifluorescent microscope and select the dappy filter. Focus the sample and illuminate it at full power. Now use a 5X objective to achieve a larger field of illumination and lower the stage completely to prevent any contact between the slide and the objective or housing.

Finally, decant the lithium borohydride solution from each well using a glass Pasteur pipette and return it to the original beaker of bleaching solution. Wash the slides three times with dilution buffer and incubate for approximately three minutes during each wash. Add the antibody mix and stain the sample as demonstrated earlier.

In untreated nasal polyp tissue staining with CD3 CD4 and CD20 showed intense non-specific granular fluorescence in eosinophil rich regions. While true membrane localized signal was present at lower intensity. Pre-treatment with 5%skim milk failed to completely block non-specific fluorescence, and also reduced signal intensity for fluorophore such as AF 532.

Dab pre-treatment reduced non-specific fluorescence from CD20, CD3 and CD4 but also decreased true signal intensity across multiple channels including DAPI and AF 532. Pretreatment with heparin effectively blocked non-specific binding while maintaining high signal intensity for CD3, CD4, CD20, and DAPI. Heparin blocked non-specific signal in a concentration dependent manner with complete suppression achieved at 10 units and no detrimental effects observed at higher concentrations.

A three track imaging setup reduced acquisition time, but introduced crosstalk including bleed through of AF 594 into the PE channel, and enhanced tissue autofluorescence due to multiple laser excitations. Spectral imaging eliminated nearly all background and crosstalk while also reducing acquisition time and improving signal to background ratio. Spectral IBEX imaging of a human nasal polyp revealed epithelial structures marked by EpCAM.

immune cells such as CD3 positive and CD20 positive lymphocytes, and vessels expressing CD31 and CD34. Heparin treatment eliminated granule binding without reducing the signal, maintaining strong background suppression across IBEX cycles. Spectral unmixing minimize bleed through, and autofluorescence increasing signal to background, and significantly speeding multi-panel acquisition versus multi-track confocal.

This enables robust spatial proteomics and challenging tissues supporting accurate cell state mapping and neighborhood level analysis.