Mechanical expansion microscopy

Abstract

This chapter describes two mechanical expansion microscopy methods with accompanying step-by-step protocols. The first method, mechanically resolved expansion microscopy, uses non-uniform expansion of partially digested samples to provide the imaging contrast that resolves local mechanical properties. Examining bacterial cell wall with this method, we are able to distinguish bacterial species in mixed populations based on their distinct cell wall rigidity and detect cell wall damage caused by various physiological and chemical perturbations. The second method is mechanically locked expansion microscopy, in which we use a mechanically stable gel network to prevent the original polyacrylate network from shrinking in ionic buffers. This method allows us to use anti-photobleaching buffers in expansion microscopy, enabling detection of novel ultra-structures under the optical diffraction limit through super-resolution single molecule localization microscopy on bacterial cells and whole-mount immunofluorescence imaging in thick animal tissues. We also discuss potential applications and assess future directions.

Keywords: Anti-photobleaching; Bacteria; Cell wall; Immunofluorescence; Interpenetrating polymer networks; Mechanically locked expansion microscopy; Mechanically resolved expansion microscopy; Nervous system; Planarian flatworm; Super-resolution fluorescence imaging.