Abstract
Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.
Highlights
The plasma membrane is a dynamic environment that plays host to a variety of interacting molecular species
The functional behaviour of the constituent species is mediated through a variety of protein–protein or protein–lipid interactions, some of which are supported by membrane-associated structures such as the cortical cytoskeleton, and all of which restrict the free lateral movement of the constituent species [1,2,3,4]
supported lipid bilayers (SLBs) were made from a lipid stock solution with 1 mg/mL DOPC (1,2-dio leoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids) and the fluorescent lipid analogue of interest dissolved in Chloroform: MeOH (2:1 v:v) (Sigma Aldrich Co Ltd, UK)
Summary
The plasma membrane is a dynamic environment that plays host to a variety of interacting molecular species. The functional behaviour of the constituent species is mediated through a variety of protein–protein or protein–lipid interactions, some of which are supported by membrane-associated structures such as the cortical cytoskeleton, and all of which restrict the free lateral movement of the constituent species [1,2,3,4]. These interactions are often short-lived, and in addition, many of the involved molecules are very mobile. The direct imaging of such molecular interactions using optical microscopy is challenging. The acquisition time of scanning confocal microscopy is usually too slow to follow dynamics such as the formation and disassembly of transient molecular clusters.
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