Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

We are presenting expansion pathology, or ExPath for short, a low cost measure to image biomolecule of interest in the standard clinical tissue sections, with nanoscale precision. ExPath circumvents the diffraction limit of conventional light microscopy by chemically infusing the clinical tissue section with a water swellable hydrogel, and then physically and evenly expanding the treated samples by about a hundred fold, allowing previously overlapped fluorescently labeled biomolecules to be separated and observed with a conventional optical microscope. ExPath allows users to study nano scale configurations of disease-related biomolecules in tissue samples, without needing to invest in new imaging hardware.

Thus, enabling new insights of the disease and fostering new diagnosis. This method can be applied to a broad array of tissue types and be used to provide new insight into the pathogenesis of various complex diseases, such as cancer, brain disease, auto-immune disease, and others. Visual demonstration is important to illustrate how to do the critical steps properly, such as construction of the geling chamber and handling the gelled sample before and after Proteinase K digestion.

Start by converting the tissue of interest into an ExPath compatible format. If working with FFPE clinical samples, place the slide with the sample in a 50ml conical tube and add 15ml of Zylene. Cap the tube and place it horizontally on an orbital shaker at approximately 60rpm for three minutes.

Repeat the wash with another 15 ml of Zylene. Then wash the slide in a series of Ethanol dilutions, according to manuscript directions. If working with stained and mounted permanent slides, place each slide in a 50ml conical tube, then cover it with Zylene.

Carefully remove the cover slip with a razor blade. Then, process the slide in the same way as the FFPE clinical samples. If working with unfixed frozen tissue slides in OCT solution, fix the tissue in acetone at 20 degrees Celsius for ten minutes, then wash the samples with 1X PBS solution three times for ten minutes per wash.

If processing previously fixed and frozen clinical samples, leave the slides for two minutes at room temperature to melt the OTC solution, then wash the sample with 1X PBS three times, for five minutes per wash. After format conversion, perform antigen retrieval on all samples. Heat 20 millimolar Citrate solution to 100 degrees Celsius in a microwave and place the slide in the solution.

Immediately transfer the container into an incubation chamber and incubate it at 60 degrees Celsius for thirty minutes. To stain the sample, use a hydrophobic pen to draw a boundary around the tissue section on the slide. Place the slide in a petri dish, then incubate the tissue with blocking buffer for one hour at 30 degrees Celsius.

Then, incubate the tissues with the primary antibody solution for at least three hours at room temperature or 37 degrees Celsius. After the incubation, wash the tissue three times with blocking buffer, and incubate it in secondary antibody solution for at least one hour at room temperature, or 37 degrees Celsius. Repeat the washes with the blocking buffer, then perform fluorescent imaging, using a conventional wide-field microscope or other imaging system of choice.

Prepare anchoring solution according to manuscript instructions. Place the slides in a 100 ml petri dish, pipet the anchoring solution over the tissue, and incubate them for at least three hours at room temperature. Then, prepare geling 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 geling solution to the sample and incubate the mixture for thirty minutes, at four 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 coverglass 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 two hours. Remove the lid of the geling 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 one to two hundred 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 three hours at 60 degrees Celsius.

If the sample does not detach during digestion, use a razor blade to gently remove it. Use a soft paint brush 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 ten 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 three to five times for ten minutes per wash. Then, perform fluorescence imaging. If this protocol is performed successfully, samples appear as a flat and transparent gel after mechanical homogenization and can expand by a factor of 3 to 4.5 in water.

A five micrometer thick FFPE kidney sample was expanded 4.5 times, which resulted in a resolution of 63 nanometers using a 0.95 NA objective. The tissue was then converted into an expansion pathology compatible format and stained with antibodies for Alpha Actinin 4 and Vimentin, DAPI to visualize nuclear DNA, and Wheat Germa Glutenin to label carbohydrates. Spinning disc confocal microscopy was then used to image the specimen.

The kidney tissue was then fully expanded in water and imaged again. Cracking, distortions, and loss of labeled targets can be the result of inadequate anchoring or homogenization. Hematoxylin and eosin stained normal breast tissue was treated as an FFPE sample, expanded, labeled with DAPI, and imaged on a spinning disc confocal microscope.

Unfixed and frozen kidney slices were also processed using this protocol. The tissue was fixed in cold acetone and stained with Alpha Actinin 4, Vimentin, DAPI, and Wheat Germa Glutenin. When performing this technique, it is important to remember the timing of the gelation step is critical.

Premature gelation can cause distortions, limit expansion and result in loss of target molecules. Following this procedure, fluorescent imaging can be performed on a microscope after digestion and expansion of the sample. ExPath can be applied broadly in both pathologies and beyond.

As it enables researchers to visualize subtle details in the tissue samples of interest. Therefore, it may help researchers to gain new insights in the pathogenesis of diseases or mechanisms of biological processes.