Abstract
Since Antonie van Leeuwenhoek used a microscope to view microorganisms for the first time, biologists have been attempting to image specimens at increasingly high levels of resolution. The diffraction limit of visible light limits the resolution of conventional optical microscopy to around two hundred nanometers, which has led to the development of super-resolution microscopy. Expansion microscopy is a super-resolution technique that allows biological specimens to be imaged at a higher resolution than allowed by the diffraction limit of visible light. Expansion microscopy achieves this purpose by labeling a specimen with fluorophores, anchoring it to a polyelectrolyte gel, digesting the sample with enzymatic activity, and expanding the gel, which retains the fluorophores from labeling in the same spatial relationships as they were position in the sample. Since its introduction in 2015, expansion microscopy has undergone several improvements, one of which was the advent of X10 expansion microscopy. This protocol currently achieves the greatest expansion factor with one gel synthesis due to its specific gel solution. In this project, X10 expansion microscopy was used in an attempt to image Escherichia coli stained with antibodies for lipopolysaccharides. It was also used to image samples of mammalian neural tissue from squirrels stained with DAPI for DNA. The expansion factor was measured by calculating the average cell sizes of samples before and after expansion. Results show that X10 expansion microscopy is compatible with mammalian neural tissue, but the protocol must be further improved for the imaging of E. coli.