Single-molecule fluorescence imaging in living cells

The transition of single-molecule fluorescence detection and imaging from in vitro to living cells has greatly enriched our knowledge on the behavior of single biomolecules in their native environments and their roles in cellular processes. Here we review recent advances of single-molecule biophysical approaches to live-cell studies based on fluorescence imaging. We start by discussing the practical considerations in designing single-molecule fluorescence imaging in cells, including the choice of fluorescent probes, labeling methods, instrumentation, and imaging techniques. We then describe representative examples in detail to illustrate the physicochemical parameters that can be obtained by imaging individually labeled biomolecules in cells and what can be learned from such characterizations.