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.