Universal Molecular Retention with 11-Fold Expansion Microscopy

Presented here is a new version of expansion microscopy (ExM), Magnify, that is modified for up to 11-fold expansion, conserving a comprehensive array of biomolecule classes, and is compatible with a broad range of tissue types. It enables the interrogation of the nanoscale configuration of biomolecules using conventional diffraction-limited microscopes.

We're trying to find easy and low cost ways for researchers to image their biological specimens at resolutions below the diffraction limit, particularly in the areas of neuroscience and clinical pathology. Many recent expansion microscopy developments involve modifying gel chemistry to expand samples further, changing the anchoring molecule to visualize different targets, and utilizing heating denaturation to preserve protein epitopes. With innovated Magnify, a robust expansion microscopy method that allows nanoscale imaging of various biological or clinical specimens.

It simplifies super resolution microscopy by eliminating specialized equipment needs, while preserving intricate biological features with up to 15 nanometers resolution, hence, increasing accessibility and utility in labs worldwide. Previously, users had to experiment with different expansion microscopy techniques depending on the tissue and biomolecules they wanted to image. Magnify enables high resolution imaging across various biomolecules and tissue types without requiring specific anchoring steps or specialized equipment, thus, bridging a significant gap in nanoscale imaging.

Magnify expands tougher clinical samples over fourfold while preserving protein epitopes, enabling more accurate investigation of human disease. Its chemical anchor also preserves a wide range of biomolecules, making it useful for basic research in analog models. Magnify symbolizes nanoscale imaging of biomolecules and intact specimens without complex optics or specialized equipment.

It's adaptable across various imaging modalities and expansion microscopy strategies, allowing accessible cost-effective nanoscale imaging that may accelerate drug treatments and disease studies, while potentially transforming tissue atlas generation. Magnify offers endless possibilities for research. Current focus areas include expanding tissue sites, investigating pathological human samples at the nanoscale, and studying the smallest scale changes on brain during learning and disease.

Begin by taking the prepared archived fresh tissue or paraformaldehyde-fixed mouse brain tissue slides. Cut thin pieces of cover glass with a diamond-tipped pen to make spacers. Then secure them to the slide with small amounts of water, PBS, or super glue.

Gently place the spacers on either side of the tissue. Prepare the final gelling solution by combining the reagents immediately before use. Place the slide in a Petri dish after removing the excess solution from the tissue section.

Add the freshly prepared cold gelling solution to the sample and incubate the mixture on the tissue for 30 minutes at four degrees Celsius to allow diffusion into the tissue. Carefully, place a second uncoated glass slide over the tissue and gel solution, avoiding air bubbles. Allow the samples to polymerize by incubating overnight at 37 degrees Celsius in a humidified environment.

To begin, take a slide with polymerized archived fresh tissue or paraformaldehyde-fixed mouse brain tissue. Wearing eye protection, use a razor blade to separate the two glass slides of the gelling chamber. Trim the blank gel around the tissue to minimize the volume and ensure to cut asymmetrically to track the orientation of the gel after homogenization.

Gently lift the tissue containing gel from the slide with a razor blade. Transfer it into a two-milliliter centrifuge tube. Fill the tube to the top with homogenization buffer preheated to 80 degrees Celsius.

Then incubate the sample with shaking at 80 degrees Celsius for eight to 60 hours. Pour the contents of the centrifuge tube into a single well of a six-well plastic cell culture plate and remove the denaturation buffer with a transfer pipette. To completely remove the remaining SDS from the hydrogel, wash the sample in a 1%non-ionic surfactant solution.

Finally, store the sample in PBS and 0.02%sodium azide at four degrees Celsius. To begin, take the digested and expanded archived or fresh clinical tissue sample and proceed with antibody staining by placing it in a single well of a six-well cell culture plate. Wash the sample three times with PBS for 10 minutes each at room temperature.

Depending on the sample size, add 0.5 to two milliliters of the primary antibodies in the staining buffer to cover the gel adequately. Incubate overnight at 37 degrees Celsius. Then wash the sample three times with PBS for 10 minutes each at room temperature.

Add the corresponding secondary antibodies, and incubate for three hours at 37 degrees Celsius in the staining buffer of choice. Rewash the sample as demonstrated earlier and add one to two milliliters of polyethylene glycol onto the sample in a six-well plate. Stain the sample with fluorophore-conjugated NHS ester for three hours at room temperature.

At the end of the incubation, wash the sample three times with PBS. Next, to perform lipid staining, take the expanded tissue sample washed three times in PBS. Apply a conventional lipophilic dye diluted 200-fold in two milliliters of 0.1%Triton X-100 or PBS for 72 to 96 hours at room temperature and wash at least three times with PBS.

To perform FISH, prepare the hybridization buffer by combining 2x SSC, 10%dextran sulfate, 20%ethylene carbonate, and 0.1%Tween 20. Place homogenized gel samples in a hybridization buffer preheated at 60 degrees Celsius for 30 minutes. Then incubate the gel samples with a hybridization buffer containing 10 picomolar FISH probes against the target gene overnight at 45 degrees Celsius.

Wash the samples with stringency wash buffer twice for 15 minutes each at 45 degrees Celsius and twice for 10 minutes at 37 degrees Celsius. Wash the sample three times with PBS for 10 minutes and proceed with imaging or storing the sample in PBS plus 0.02%sodium azide at four degrees Celsius. To fully expand and image the tissue, move the samples expanded fourfold in PBS to a glass bottom six-well imaging plate.

If the sample has been expanded further, cut it into smaller pieces. Apply 0.1%poly-L-lysine to the glass surface. Place the sample on the slide and remove any excess liquid around the gel with a paper laboratory cloth to prevent gel movement during imaging.

Afterward, perform fluorescence imaging using a conventional wide field microscope, a confocal microscope, or another imaging system of choice. After imaging, shrink the samples in PBS with at least three 10-minute washes as long-term storage in deionized water leads to degradation of the fluorescent signal. Finally, store the samples in PBS with 0.02%sodium azide at four degrees Celsius.

Confocal images of fully expanded mouse brain tissue allowed the visualization of proteins and cell and mitochondrial membrane structures. The nucleic acids of formal and fixed paraffin-embedded human lymph node tissue were also identified. After a 3.5-fold expansion of the kidney section, the crack-free expanded glomerulus was seen.

However, inadequate anchoring or homogenization resulted in cracking, distortions, and the loss of labeled targets in another kidney section.