Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain

Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain

Hannah Ampofo, Raymond Berry, Claire Spann, Ran Liu, Shaohua Yang

Pharmacology and Neuroscience Department, School of Biomedical Sciences, University of North Texas Health Science Center, Texas, United States.

Purpose-To establish Expansion microscopy (ExM) for super-resolution imaging of the rodent brain.

Background- ExM is a remarkable imaging technology that enables nanoscale resolution in three-dimensional (3-D) imaging of preserved cells and tissues. ExM, which was invented in 2015, physically expands specimens using a hydrogel, allowing high-resolution imaging to be done with conventional diffraction-limited microscopes. The basic idea is to attach anchors to biomolecules or labels chemically and link them to a hydrogel that is uniformly distributed throughout the material. This polymerization technique separates biomolecules while maintaining their spatial organization by enabling isotropic expansion. The procedure is similar to sketching an outline on an inflating object and blowing it up: the ink particles will move apart, but their relative organization remains the same. Traditional optical imaging is unable to resolve nanoscale structures with dimensions smaller than 200–300 nm due to the fundamental physical limitations imposed by diffraction. ExM offers faster imaging speeds as compared to super-resolution methods, and enhanced antibody efficiency due to the decrowding effect generated by expanding biomolecules. The original ExM could resolve the specimen at 70 nm, however, new variants such as iterative ExM, 10X ExM microscopy, and nine-fold microscopy can resolve down to 15 to 30 nm, comparable to super-resolution microscopes.

Method- The Paper-MAP version of expansion microscopy, a modified MAP method that allows immunostaining and expansion within two days was employed. The procedure involved staining floating mouse brain sections and incubating with a Paper-MAP cocktail (consisting of TEMED and sodium acrylate) and ammonium persulfate solution. The hydrogel matrix was created in situ through the crosslinking of sodium acrylate and bisacrylamide, forming a dense polyelectrolyte hydrogel. A denaturing solution was used to mechanically homogenize the sample and then expanded using deionized water. The pre and post-expanded sections were imaged using a Zeiss LSM 510 confocal microscope.

Results- Following the addition of the monomer solution, the expansion procedure produced a 2 fold increase in size. This was followed by an evident 4 to 5 fold increase after the expansion was completed. We compared the pre-expansion image to the post-expansion image and observed intricate and detailed structures with significantly enhanced resolution that were previously indistinguishable in the pre-expansion section using confocal microscope.

Conclusion- Expansion microscopy is a versatile and accessible imaging technique that resulted in significant improvements in imaging the microscopic configuration of the mouse brain. Its broad application offers a powerful tool for biological research in diverse organisms.