Highly Multiplexed in Situ Protein Imaging Using DNA-Exchange-Imaging and Immuno-SABER

Abstract

Mapping the molecular composition of individual cells in their native environment is critical for understanding the cellular and subcellular organization of healthy and diseased tissues. Immunofluorescence (IF) imaging using labeling probes such as antibodies provides an important tool to uncover the spatial distribution of proteins in biological samples. Traditional IF imaging methods suffer from one or more following problems: low multiplexing capability, poor detection sensitivity and limited optical resolution. In this dissertation, I presented my work, in collaboration with others, to develop highly multiplexed in situ protein imaging methods that have superior detection sensitivity and are able to perform high/super-resolution imaging. I first developed DNA-antibody conjugation and purification methods to obtain high quality labeling probes. I then developed DNA-Exchange-Imaging, a technique based on Exchange-PAINT, to apply the DNA-Exchange concept to various imaging platforms for highly multiplexed diffraction-limited confocal imaging and super-resolution structure-illumination, STED and PAINT imaging. This technique was further developed to integrate signal amplification capability, by combining with Hybridization Chain Reaction (HCR) and Signal Amplification By Exchange Reaction (SABER), to enable highly multiplexed and high-sensitivity protein detection in tissue samples. Finally, I presented a method that enables highly multiplexed, high-sensitivity and super-resolution imaging in situ by conjoining Immuno-SABER and Expansion Microscopy (ExM). I concluded the dissertation with a summary of the evolution of described techniques and a discussion of current challenges in highly multiplexed in situ protein detection using DNA-barcoded antibodies.