Biophysical Imaging

Fluorescence correlation spectroscopy (FCS) is a powerful tool to study molecular diffusion. However, the spatial resolution of FCS experiments is restrained by the diffraction limit and many diffusion processes happening at the nanoscale cannot be resolved by conventional FCS. To overcome this limitation, FCS can be used together with stimulated emission depletion (STED) microscopy, and this STED-FCS approach has already found multiple applications, particularly when studying the cellular plasma membrane. STEDFCS has however rarely been used to study 3-dimensional diffusion, mainly because with the most widely used STED depletion pattern, a ring-shaped focus (“doughnut”, 2D STED), the non-depleted out-of-focus volumes contribute to high background noise and deteriorate the effective spatial resolution. A solution to this problem consists in using a bottle-shaped pattern as the depletion beam (3D STED); but this pattern suffers from an exacerbated sensitivity to optical aberrations. We present here an adaptive optics method that corrects aberrations affecting 3D STED-FCS experiments. Using a spatial light modulator (SLM) as a wavefront shaping device in the depletion path of a STED microscope, we corrected aberrations affecting STED-FCS measurements in the cytoplasm of living cells, leading to increased resolution, improved signal quality, and larger accessible depth range for measurements.