Fluorescent speckle microscopy in cultured cells

Abstract

After slightly more than a decade since it was first established, fluorescent speckle microscopy (FSM) has been intensively used to investigate macromolecular dynamics, such as microtubule flux in mitosis and meiosis, microtubule translocation in neurons, microtubule-binding proteins, and focal adhesion proteins, as well as the assembly of actin filaments. This state-of-the-art technique is based on nonuniform distribution of fluorescently labeled subunits diluted in the endogenous, unlabeled ones, resulting in microscopy-detectable speckled patterns. In order to enable sufficient contrast between neighboring diffraction-limited image regions, a low ratio between labeled and endogenous molecules is required, which can be achieved either by microinjection or by expression of limited amounts of fluorescently labeled subunits in cells. Over the years, the initial settings for FSM have been significantly improved by introduction of more sensitive cameras and spinning-disk confocal units, as well as by the development of specialized algorithms for image analysis. In this chapter, we describe our current FSM setup and detail on the necessary experimental approaches for its use in cultured cells, while discussing the present and future challenges of this powerful technique.