Expansion Pathology: A Method for High-resolution Optical Imaging of Clinical Tissue Samples

Expansion pathology, or ExPath, is a sample preparation technique for biological specimens, which allows conventional light microscopy to image nano-scale structures by expanding them using a polymer system. 

First, take a processed immunostained tissue specimen slide and cover it with an anchoring solution containing both protein and polymer-reactive groups. The protein-reactive group of the anchoring reagent binds to the amines of biomolecules within cells.

Next, add a suitable gelling solution over the specimen and incubate to allow its diffusion inside the tissue. The polymer-reactive group of the anchoring reagent binds to the free monomers within the gelling solution, allowing the biomolecules to anchor to the polymer as it gets polymerized.

Once the sample polymerization is complete, cover the slide with a proteinase solution. Proteinases digest cytoskeletal proteins to remove any structural resistance, allowing the cells to expand without rupturing. Upon enzymatic digestion, the gelled sample detaches from the slide and moves into suspension.

Now, transfer the specimen into an appropriate wash-buffer in a microscope-compatible container. Next, replace the buffer with water and incubate. The polymer absorbs an enormous amount of water relative to its mass and swells.

As the polymer expands, the biomolecules anchored to the polymer network move apart while retaining their spatial orientation. This allows for their easy visualization under a conventional light microscope.

Prepare anchoring solution according to manuscript instructions. Place the slides in a 100-millimeter Petri dish. Pipette the anchoring solution over the tissue and incubate them for at least 3 hours at room temperature. Then, prepare gelling solution according to manuscript directions.

Remove excess anchoring solution from the tissue section and place the slide back into the Petri dish. Add fresh cold gelling solution to the sample and incubate the mixture for 30 minutes at 4 degrees Celsius. To construct a chamber on the slide around the sample, create spacers by thinly cutting pieces of cover glass with a diamond knife. Secure the spacers on either side of the tissue with water and carefully place a cover glass lid over the slide, making sure to avoid trapping air bubbles over the tissue.

Then, incubate the sample at 37 degrees Celsius in a humidified environment for 2 hours. Remove the lid of the gelling chamber by gently sliding a razor blade under the coverslip and slowly lifting it off the gel surface. Trim the blank gel around the tissue to minimize the volume, making sure to cut the gel asymmetrically to track the orientation.

Dilute Proteinase K 1:200 in digestion buffer, making sure to prepare enough solution to completely submerge the gel. Then, incubate the sample with the solution in a closed container for 3 hours at 60 degrees Celsius. If the sample does not detach during digestion, use a razor blade to gently remove it.

Use a soft paintbrush to transfer the specimen into 1X PBS in a container compatible with the desired imaging system and large enough to accommodate the fully expanded gel. Wash the sample in PBS for 10 minutes, and if desired, restain it with 300-millimolar DAPI. Expand the samples by washing the sample with an excess volume of double distilled water 3 to 5 times for 10 minutes per wash. Then, perform fluorescence imaging.