Axial Line-Scanning STED-FCS

Abstract

We present axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy (axial ls-STED-FCS), a super-resolution method to investigate the dynamics and interactions of biomolecules associated with the plasma membrane of live cells. Intrinsic membrane fluctuations are detrimental for FCS, and line scanning of the confocal spot through the membrane is an effective tool to cope with this problem. In contrast to earlier lateral (within the focal area) line scanning approaches, we employ axial (along the optical axis) line scanning at ultra-high speed by using a tunable acoustic gradient index of refraction (TAG) lens. This device allows us to sweep the observation volume through the membrane within a few microseconds and thus about two orders of magnitude faster than with lateral line scanning using galvanometric scanners. A key advantage of axial lsFCS is that its observation area on the membrane is circular whereas the one of lateral lsFCS is elliptical and larger due to the lower axial resolution of the confocal microscope. As a result, the correlation decay is significantly faster with axial lsFCS, allowing shorter overall measurement times to reach a comparable precision, and its simpler model function makes fitting more robust. Moreover, it is easier to find suitable spots in live imaging of cultured cells. With STED excitation, the observation area can be markedly reduced, as was shown with giant unilamellar vesicles (GUVs) labeled with Atto 647N. Axial ls-STED-FCS enables measurements on smaller spatial scales and with greater biomolecule densities and, therefore, is a valuable extension of standard axial lsFCS, which we have recently introduced for precise measurements of ligand-membrane receptor binding affinities in the (sub)nanomolar range.