Single-molecule Tracking and Kinetic Analysis in Living Cells and Multicellular Organisms

In a living organism, the interplay of stochastically interacting molecules brings forth structures and processes robustly organized in space and time. Single-molecule localization microscopy and tracking emerged as important techniques to visualize the super-resolved spatial distribution and real-time motion of individual fluorescently labeled molecules in a living cell or multicellular organism. Thereby, single-molecule tracking (SMT) enables quantifying kinetic mechanisms underlying vital organismal processes. This review covers the methodology of SMT in living cells and multicellular organisms, including labeling approaches and microscopy techniques, with a focus on recent developments in temporal excitation patterns facilitating extracting kinetic properties and the analysis of molecular kinetic parameters accessible by SMT. Emphasis lies on the application of this methodology to quantifying the kinetics of proteins such as transcription factors, and some recent examples are highlighted. The review concludes by envisioning future perspectives of SMT in living systems.