ns-time resolution for multispecies STED-FLIM and artifact free STED-FCS

Abstract

Stimulated Emission Depletion (STED) Microscopy has evolved into a well established method offering optical superresolution below 50 nm. Running both excitation and depletion lasers in picosecond pulsed modes allows for highest optical resolution as well as fully exploiting the photon arrival time information using time-resolved single photon counting (TCSPC). Non-superresolved contributions can be easily dismissed through time-gated detection (gated STED) or a more detailed fluorescence decay analysis (FLIM-STED), both leading to an even further improved imaging resolution. Furthermore, these methods allow for accurate separation of different fluorescent species, especially if subtle differences in the excitation and emission spectra as well as the fluorescence decay are taken into account in parallel. STED can also be used to shrink the observation volume while studying the dynamics of diffusing species in Fluorescence Correlation Spectroscopy (FCS) to overcome averaging issues along long transit paths. A further unique advantage of STED-FCS is that the observation spot diameter can be tuned in a gradual manner enabling, for example, determining the type of hindered diffusion in lipid membrane studies. Our completely pulsed illumination scheme allows realizing an improved STED-FCS data acquisition using pulsed interleaved excitation (PIE). PIE-STED-FCS allows for a straightforward online check whether the STED laser has an influence on the investigated diffusion dynamics.

Lipid Composition but Not Curvature Is the Determinant Factor for the Low Molecular Mobility Observed on the Membrane of Virus-Like Vesicles

“…Lipid mobility data was acquired for individual fluorescent vesicles of diameters of 100–200 nm (extruded) or 0.5–1 µm (non-extruded) using point STED-FCS in the time-correlated single photon-counting mode (TCSPC). TCSPC mode allowed us to remove non-depleted confocal contribution and residual laser scattering by fluorescence lifetime-based filtering, thus increasing the spatial resolution and signal-to-noise ratio of the acquired signal [ 26 , 27 ]. In addition, photobleaching correction was also applied using a local-averaging method ( Figures S2 and S3 ) [ 20 , 21 ].…”

Nanoscale Spatiotemporal Diffusion Modes Measured by Simultaneous Confocal and Stimulated Emission Depletion Nanoscopy Imaging

“…The only way to overcome this is the simultaneous recording of confocal and STED–FCS data, as done before in single-point FCS experiments (which lacks the spatial information). 20 , 21 …”

“…Unfortunately, it has not been managed to accurately characterize diffusion modes in these transient sites using sSTED–FCS so far because values of τ D could only be determined for one observation spot diameter d at a time. The only way to overcome this is the simultaneous recording of confocal and STED–FCS data, as done before in single-point FCS experiments (which lacks the spatial information). , …”

Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

“…Lipid mobility data was acquired for individual 200 nm or ~1 µm fluorescent vesicles using point STED-FCS in time-correlated single photon-counting mode (TCSPM). TCSPM mode was used in order to provide additional fluorescence lifetime-based filtering that allows for removal of residual laser scattering thus increasing signal-to-noise ratio of the acquired signal [19,20]. In addition, photobleaching correction was also applied using a local-averaging method [16].…”