Expansion Microscopy Through Mechanical Force*

Interesting biophysical phenomena take place during the interaction between biomolecules at the nanoscale. Optical superresolution imaging techniques beat the diffraction limit enabling higher resolutions to study these phenomena. Expansion microscopy (ExM) is a sample preparation method that achieves superresolution imaging using osmotic forces to physically separate features smaller than the diffraction limit of light by 4.5X – 10X. Yet, sample preparation in ExM is slow, requires multiple chemical fixation and labeling steps, does not always achieve isotropic expansion, and fragments samples at high expansion factors. To overcome the fragmentation and time issues, we use a highly stretchable hydrogel and a mechanical stretcher device to achieve near 20X linear expansion faster. Since we do not rely on osmotic forces for the expansion, this method is compatible with other imaging techniques which are incompatible with excess water. We demonstrate our expansion microscopy method by imaging OVCAR cells at 500 nm resolution. We establish mechanical force ExM as a viable sample preparation method with the same advantages as ExM while being much faster, allowing researchers to study time-sensitive biophysical phenomena.