Highly-Multiplexed Tissue Imaging with Raman Dyes

Highly-multiplexed vibrational imaging provides a one-shot optical approach to interrogate multiple protein markers in tissues with subcellular resolution. This platform provides a comprehensive picture of protein interactions of biological systems. The main advantage of this technique is its cycle-free multiplexity.

This technique is particularly suitable for applications where cycling strategies do not function well, such as in thick tissue sections. This method enables individual cell characterization in situ for understanding structures of the complex system, such as building tissue atlas, phenotyping tumor micro environments, and the profiling brain circuit. To begin, prepare approximately 0.1 molar sodium bicarbonate in PBS buffer at PHA 0.3 to be used as conjugation buffer and store it at four degrees Celsius.

Then prepare three millimolar n-hydroxysuccinimide ester functionated MARS probe solution in anhydrous dimethyl sulfoxide. Dissolve the antibody solids in the conjugation buffer to a concentration of two milligrams per milliliter. For antibodies dissolved in other buffers, concentrate them in a centrifugal filter and then add into the conjugation buffer to a concentration of one to two milligrams per milliliter.

To perform the conjugation reaction for secondary antibodies, add 15-fold molar excess of dye solution to the antibodies solution in a glass vial slowly with stirring and incubate the reaction mixture at room temperature with stirring for one hour. To perform the purification, prepare the slurry by adding 10 milliliters of gel filtration resin powder into 40 milliliters of PBS buffer in a 50 milliliter tube. Keep the solution in a water bath at 90 degrees Celsius for one hour.

Then decant the supernatant, add the PBS up to 40 milliliters, and store the slurry at four degrees Celsius. Pack the size exclusion column with the slurry solution to the height of 10 to 15 centimeters, then rinse and wash the column with approximately 10 milliliters of PBS to further pack the resin. Subsequently, pipette the conjugation reaction mixture to the column.

After loading the reaction mixture, immediately add one milliliter of PBS as the elution buffer. Afterward, collect the eluent of the conjugate solution by looking at the color on the column or by measuring the absorbance at 280 nanometers. Using the centrifugal filter, concentrate the collected solution to one to two milligrams per milliliter.

Using a hydrophobic pen, draw a boundary around the tissue sections on the slide. Then incubate the tissues with 0.3 to 0.5%PBST for 10 minutes and a blocking buffer for 30 minutes. To prepare the primary staining solution, add all primary antibodies to 200 to 500 microliters of staining buffer at desired concentrations.

Centrifuge the primary staining solution at 13, 000 times G for five minutes and use the supernatant if precipitate appears. Then incubate the tissue in the primary antibody solution at four degrees Celsius for one to two days. Wash the slides thrice with 0.3 to 0.5%PBST for five minutes at room temperature in a slide standing jar and make sure that the tissues are immersed in the solution.

Later, incubate the tissue in 200 to 500 microliters of blocking buffer for 30 minutes. To prepare the secondary staining solution, add all secondary antibodies to 200 to 500 milliliters of staining buffer with desired concentrations. Then centrifuge the secondary staining solution at 13, 000 times G for five minutes and use the supernatant if precipitate appears.

Next, incubate the tissues in 200 to 500 microliters of secondary antibody solution at four degrees Celsius for one to two days. Then wash the slides twice with 0.3 to 0.5%PBST at room temperature for five minutes each. Further, incubate with 200 to 500 microliters of DAPI solution for 30 minutes.

Again, wash the slides thrice with PBS at room temperature for five minutes each and transfer the floating tissue sections to the glass slides with the glass dropping pipette. Spread the tissue with a tissue brush and clean the surrounding with wipes if needed. Subsequently, mount the tissue in a drop of anti-fade reagents with a glass coverslip and secure it with nail polish.

To perform multichannel-eprSRS imaging, set the laser power of the pump beam to 10 to 40 milliwatt and the stokes beam to 40 to 80 milliwatt on the laser control panel. Then set the pixel dwell time to two to four microseconds and use multiple frames averaging typically 10 to 20 frames on the microscopy software. Further, set the time constants of the lock-in amplifier to half of the pixel dwell time.

Raman dye imaging of distinct protein markers and HeLa cells paraformaldehyde-fixed mouse brain cortex and humid kidney FFPE tissue were performed through immuno labeling. The immuno-eprSRS imaging of HeLa cells with an alpha-tubulin revealed fine subcellular structures such as microtubules. The eprSRS imaging of MARS 2145 stained astrocytes and MARS 2228 stained cerebellar granule neurons in mouse brain tissue demonstrated three-dimensional patterns with subcellular resolution.

The seven-color SRS fluorescence tandem imaging of hormones and transcription factors on frozen mouse islet tissue was performed. The obtained images revealed a good contrast and correct patterns. The eight-color SRS fluorescence tandem imaging of cell type markers on paraformaldehyde-fixed mouse cerebellum tissues was performed.

Different types of cells were identified such as cerebellar granule neurons, Purkinje neurons, astrocytes, oligodendrocytes, and GABAergic neurons. This procedure can be further combined with tissue clearing to perform highly multiplexed protein imaging in thick intact tissues. Our group has developed a Raman-dye-tailored tissue clearing protocol for it.