Background reduction in STED-FCS using coherent-hybrid STED

Abstract

Fluorescence correlation spectroscopy (FCS) is a valuable tool to study the molecular dynamics of living cells. When used together with a super-resolution stimulated emission depletion (STED) microscope, STED-FCS can measure diffusion processes at the nanoscale in living cells. In twodimensional (2D) systems like the cellular plasma membrane, a ring-shaped depletion focus is most commonly used to increase the lateral resolution, leading to more than 25-fold decrease in the observation volumee, reaching the relevant scale of supramolecular arrangements. However, STED-FCS faces severe limitations when measuring diffusion in three dimensions (3D), largely due to the spurious background contributions from undepleted areas of the excitation focus that reduce the signal quality and ultimately limit the resolution. In this paper, we investigate how different STED confinement modes can mitigate this issue. By simulations as well as experiments with fluorescent probes in solution and in cells, we demonstrate that the coherent-hybrid (CH) depletion pattern reduces background most efficiently and thus provides superior signal quality under comparable reduction of the observation volume. Featuring also the highest robustness to common optical aberrations, CH-STED can be considered the method of choice for reliable STED-FCS based investigations of 3D diffusion on the sub-diffraction scale.

Background Reduction in STED-FCS Using a Bivortex Phase Mask

“…Finally, the impact of tilt, which in this situation corresponds to a misalignment between excitation and depletion foci, is useful to be assessed to estimate the impact of chromatic aberrations 25 or thermal and mechanical drift. 19 We found that 2D-STED was the most sensitive to tilt, followed by z-STED and CH-STED, which can be linked to the respective sizes of their central areas, with a smaller central area leading to a higher vulnerability to misalignment (Figure 6f).…”

“…To assess the robustness of STED-FCS measurements with different confinement modes, we used the wavefront shaping capabilities of the SLM to quantify the effects of optical aberrations introduced as loworder Zernike modes, which had previously been reported to be most often present while imaging common biological specimens. 22 We introduced either 0, 0.5, or 1 rad rms of each mode using the SLM, corresponding respectively to no aberrations, to the maximum amplitude of aberrations we experienced in our microscope across this study (see Supporting Information, Figure S5) and to highly aberrating situations that can be encountered, for instance, when focusing deep inside a medium with a refractive index mismatch 19 or when focusing deep through an optically inhomogeneous specimen. For each aberration value, we acquired a set of STED-FCS measurements of freely diffusing Abberior Star Red dye in a water/glycerol solution.…”

“…Attempting to overcome these issues, sophisticated experimental approaches, relying on the introduction of additional laser pulses, 5 have been developed, requiring the acquisition of time-tagged photon streams, or by advanced dynamic aberration corrections. 19 On the contrary, we here aimed at improving STED-FCS in 3D environments by a technically simpler approach, adopting the alternative depletion pattern designed to reduce the background fluorescence in imaging via bivortex, CH-STED. 20 This implementation is straightforwardly introduced in setups incorporating an SLM (available also in certain commercial implementations of STED), as it only requires the modulation of the STED laser with a bivortex phase mask.…”

“…However, this configuration is more sensitive to optical aberrations, 17,18 which can only be mitigated with advanced implementation of adaptive optics. 19 Even aberration-free z-STED-FCS, though, exhibits spurious contributions from undepleted areas that damp the amplitude of the autocorrelation function, bias measurements of the numbers of molecules, and reduce the signal-to-noise ratio. 3,10 A combination of both 2D and z-STED depletion patterns (3D-STED) has also been used in STED-FCS, 5,10 but did not significantly reduce background contributions.…”

Multi-scale sensorless adaptive optics: application to stimulated emission depletion microscopy

“…We followed the procedure in [ 29 ] to align the SLM in the depletion branch. Note that, since the beam is reflected twice over the SLM at and , it accumulates static aberration due to both of these distinct locations over the SLM window.…”