Fluorescent speckle microscopy of microtubules: how low can you go?

Fluorescent speckle microscopy (FSM) is a new technique for visualizing the movement, assembly, and turnover of macromolecular assemblies like the cyt oskeleton in living cells. In this method, contrast is created by coassembl y of a small fraction of fluorescent subunits in a pool of unlabeled subuni ts, Random variation in association creates a nonuniform "fluorescent speck le" pattern. Fluorescent speckle movements in time-lapse recordings stand o ut to the eye and can be measured, Because fluorescent speckles represent f iduciary marks on the polymer lattice, FSM provides the opportunity for the first time to see the 2- land 3-dimensional trajectories of lattice moveme nts within large arrays of polymers as well as identifying sites of assembl y and disassembly of individual polymers, The technique works with either m icroinjection of fluorescently labeled subunits or expression of subunits l igated to green fluorescent protein (GFP). We have found for microtubules a ssembled in vitro that speckles containing one fluorophore can be detected and recorded using a conventional wide-field epi-fluorescence light microsc ope and digital imaging with a low noise cooled CCD camera. In living cells , optimal speckle contrast occurs at fractions of labeled tubulin of simila r to 0.1-0.5% where the fluorescence of each speckle corresponds to one to seven fluorophores per resolvable unit (similar to 0.27 mu m) in the micros cope. This small fraction of labeled subunits significantly reduces out-of- focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells.