Axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy

Investigating the dynamics and interactions of biomolecules within or attached to membranes of living cells is crucial for understanding biology at the molecular level. In this pursuit, classical, diffraction-limited optical fluorescence microscopy is widely used, but it faces limitations due to (1) the heterogeneity of biomembranes on the nanoscale and (2) the intrinsic motion of membranes with respect to the focus. Here we introduce a new confocal microscopy-based fluctuation spectroscopy technique aimed at alleviating these two problems, called axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy (axial ls-STED-FCS). Axial line scanning by means of a tunable acoustic gradient index of refraction lens provides a time resolution of a few microseconds, which is more than two orders of magnitude greater than that of conventional, lateral line-scanning fluorescence correlation spectroscopy (typically around 1 ms). Using STED excitation, the observation area on the membrane can be reduced 10-100 fold, resulting in sub-diffraction spatial resolution and the ability to study samples with densely labeled membranes. Due to these attractive properties, we expect that the axial ls-STED-FCS will find wide application, especially in the biomolecular sciences.