Imaging fluorescence fluctuation spectroscopy: new tools for quantitative bioimaging

Fluorescence fluctuation spectroscopy (FFS) techniques provide information at the single-molecule level with excellent time resolution. Usually applied at a single spot in a sample, they have been recently extended into imaging formats, referred to as imaging FFS. They provide spatial information at the optical diffraction limit and temporal information in the microsecond to millisecond range. This review provides an overview of the different modalities in which imaging FFS techniques have been implemented and discusses present imaging FFS capabilities and limitations. A combination of imaging FFS and nanoscopy would allow one to record information with the detailed spatial information of nanoscopy, which is ∼20 nm and limited only by fluorophore size and labeling density, and the time resolution of imaging FFS, limited by the fluorescence lifetime. This combination would provide new insights into biological events by providing spatiotemporal resolution at unprecedented levels.