protein retention extension microscopy

Abstract

The present invention provides a method called protein retention ExM (proExM), in which cross-linking molecules are used to anchor proteins, rather than markers, to a swellable gel. This proExM strategy can be used to perform nanoscale imaging of immunostained cells and tissues as well as FP-expressing samples, since even when subjected to non-specific proteolytic digestion, genetically encoded fluorescent proteins and / or conventional fluorescent proteins anchored directly to the gel Fluorescent signals from labeled secondary antibodies and streptavidin were maintained.

Description

[0001] related application [0002] This application claims the benefit of priority to US Provisional Application Serial No. 62 / 202,423, filed August 7, 2015, the contents of which are incorporated herein by reference in their entirety. [0003] Government funding [0004] This invention was made with government support under NYSCF-R-NI10 awarded by the Hertz Foundation, NYSCF, NSF, and Rehabilitation Institute of Chicago, and 1-U01-MH106011 awarded by the NIH and Cargill Fund Bioengineering Fund. The government has certain rights in this invention. Background technique [0005] Extended microscopy (ExM) can image thick preserved specimens with ~70nm lateral resolution. With ExM, the optical diffraction limit is circumvented by physically expanding the biological sample prior to imaging, thereby bringing sub-diffraction-limited structures into a size range observable by conventional diffraction-limited microscopy. ExM can image biological specimens at the voxel rate of diff...

AI Technical Summary

Problems solved by technology

All chemicals required for ExM are commercially available except for the gel-anchorable marker, which requires custom synthesis and presents a barrier to researchers adopting the described method

Another disadvantage of the ExM protocol is that genetically encoded fluorophores cannot be imaged without antibody labeling

Additionally, ExM cannot maintain native proteins in gels and uses custom reagents that are not widely available

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