Gel Expansion Microscopy Optimization on non-Membrane Bound Organelles

Abstract

Optical microscopes encounter a diffraction limit that prevents magnifications past a certain threshold. One method of resolving this issue is through the usage of gel expansion microscopy. In this procedure, monomers are crosslinked with charged monomers and the relevant signal. A 3D gel matrix is formed, the cell sample is mechanically homogenized (digested) and then the resulting gel is placed in a solution in which it expands. A methodology and optimization of a gel expansion microscopy protocol for the NPM1 protein and its relevant nucleoli structure, is outlined. The maximum average expansion procured is 4.3X +/- 0.6. This maximum is achieved by fixing at 3.2% paraformaldehyde and 0.1% glutaraldehyde for 10 minutes, using glutaraldehyde as a cross-linker, and digesting at 57*C for an hour. The maximum signal loss stems from digestion and expansion. To avoid digestion related artifacts, the digestion buffer must be pre-heated to digestion temperatures. A good way to retains signal is to use conjugated antibodies that fluoresce at similar wavelengths to the native signal. There remains the issue of gel expansion solvent as ddH2O does appear to dissolve native signal, while a buffered solution like PBS may not dissolve native signal but impedes significant expansion. Despite these issues, even for expansions of only 2X the original size, the resolution of an object is greatly improved, allowing for detail previously undetected.