Single-Molecule Imaging of Endogenous Proteins

Single-molecule imaging is a technique of choice to investigate the dynamic nanoscale organization of proteins of interest, revealing a wealth of new information on how proteins perform their biological function. However, overexpression, which is often used in this context, can alter biological functions. It is therefore useful to implement tools to visualize and track endogenous proteins in living cells and observe their dynamic clustering and motion within their native environment. Herein, we describe two approaches: Fluorescent intrabody Localization Microscopy (FiLM) and gene-editing (CRISPR/Cas9) and provide a workflow including the design of plasmid backbones used to track endogenous proteins by either i) introducing nanobodies that target endogenous proteins to perform single-particle tracking or ii) gene-editing cells in culture to express tagged endogenous proteins at biologically relevant expression levels.