Quantitative fluorescent speckle microscopy

Fluorescent speckle microscopy (FSM) is a fluorescence imaging modality uniquely suited for the analysis of macromolecular assemblies in living cells. Highly substoichiometric fluorescent labeling of subunits in the assembly produces by diffraction-limited imaging a random fluctuation pattern of fluorescence on a dim background, whose temporally stable local maxima are referred to as 'speckles.' Speckles are fiduciary marks of the motion and turnover of subunits within the assembly and/or of the movement and deformation of the entire assembly within the cell. Because of the stochastic nature of the speckle fluorescent signal, the dynamics of a large number of speckles have to be integrated in computational models of the possible behaviors of the assembly. This article reviews the requirements in terms of specimen preparation, imaging, and image analysis for quantitative FSM (qFSM) experiments. Applications of qFSM have enabled critical discoveries of the architectural dynamics of the actin filament and microtubule cytoskeletons and their regulation during cell migration and cell division. The article also reviews the development of the qFSM methodology in the context of single-molecule imaging approaches that make use of the stochastic sampling paradigm to reconstruct the architecture of macromolecular assemblies with super-resolution.