Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.

Abstract

Fluorescent speckle microscopy (FSM) is a technology used to analyze the dynamics of macromolecular assemblies in vivo and in vitro. Speckle formation by random association of fluorophores with a macromolecular structure was originally discovered for microtubules. Since then FSM has been expanded to study other cytoskeleton and cytoskeleton-binding proteins. Specialized software has been developed to convert the stochastic speckle image signal into spatiotemporal maps of polymer transport and turnover in living cells. These maps serve as a unique quantitative readout of the dynamic steady state of the cytoskeleton and its responses to molecular and genetic interventions, allowing a systematic study of the mechanisms of cytoskeleton regulation and its effect on cell function. Here, we explain the principles of FSM imaging and signal analysis, outline the biological questions and corresponding methodological advances that have led to the current state of FSM, and give a glimpse of new FSM modalities under development.