Photon-separation to enhance the spatial resolution in pulsed STED microscopy

Abstract

Stimulated emission depletion microscopy (STED) is one of the pivotal super-resolution techniques. It overcomes the spatial resolution limit imposed by the diffraction by using an additional laser beam, the STED beam, whose intensity is directly related to the achievable resolution. Despite achieving nanometer resolution, much effort in recent years has been devoted to reduce the STED beam intensity because it may lead to photo-damaging effects. Exploring the temporal dynamics of the detected fluorescence photons and accessing the encoded spatial information has proven to be a powerful strategy, and has contributed to the separation by lifetime tuning (SPLIT) technique. The SPLIT technique uses the phasor analysis to efficiently distinguish photons emitted from the center and the periphery of the excitation spot. It thus improves the resolution without increasing the STED beam intensity. This method was proposed for architectures based on STED beam running in continuous wave (CW-STED microscopy). Here, we extend it to architectures based on pulsed STED beam (pSTED microscopy). We show, through simulated and experimental data, that the SPLIT-pSTED method reduces the detection volume of the pSTED microscope without significantly reducing the signal-to-noise ratio of the final image, thus effectively improving the resolution without increasing the STED beam intensity.

A phasor‐based approach to improve optical sectioning in any confocal microscope with a tunable pinhole

“…Notably, this work is the first demonstration that the SPLIT concept can have application also outside the superresolution field. Phasor‐based super‐resolution was originally developed in lifetime‐resolved STED microscopy (Coto Hernandez et al, 2019; Lanzano et al, 2015; Tortarolo et al, 2019; Wang et al, 2018) and later extended to other (non‐lifetime) STED configurations (Pelicci et al, 2020; Sarmento et al, 2018). Recently, we demonstrated that SPLIT can be applied also to structured illumination microscopy (SIM), using as the additional channel the illumination pattern translation step (Cainero et al, 2021).…”

“…In other words, the same phasor algorithm can be applied to microscopy images containing an additional channel with encoded spatial information. In STED microscopy, this additional channel can be represented by the fluorescence lifetime variations induced by a STED beam (Coto Hernandez et al, 2019; Lanzano et al, 2015; Lanzano et al, 2017; Tortarolo et al, 2019; Wang et al, 2018) or a tunable depletion power (Pelicci et al, 2020; Sarmento et al, 2018). In STED microscopy, the application of SPLIT produces images that have higher resolution and better contrast, compared to their counterpart STED images (Cerutti et al, 2021).…”

Focus-ISM for Sharp and Gentle Super-Resolved Microscopy

“…In this scenario, our STED-ISM implementation based on a SPAD array detector can become the gold standard: the proposed implementation (i) requires only minimal changes in a conventional STED microscopy architecture; (ii) preserves all functions of STED microscopy, such as multi-colour, threedimensional, and fast imaging; (iii) is fully compatible with all current approaches for photo-damage reduction and signal-to-background ratio improvement. In terms of this last point, the single-photon timing nature of the SPAD array detector allows the combination of STED-ISM microscopy and time-resolved STED microscopy [43,11,13] to further improve the resolution for a given STED beam intensity. Such a time-resolved STED implementation based on a SPAD array detector will provide benefits not only for imaging but also for fluorescence fluctuation spectroscopy (FFS) [44,45]: we have recently shown how the SPAD array detector improves the information context of a FFS experiment [46].…”

“…In other words, increasing the optical resolution comes at the cost of photo-damaging the sample and reducing the signal-to-background ratio (SBR), hence hindering the feasibility for long-term STED imaging in living cells and STED imaging in thick samples, respectively. Several STED microscopy implementations have been proposed to mitigate the photo-damage and the SBR reduction [7]: Time-resolved [8,9,10,11,12,13] and subtraction methods [14,15] remove the incomplete depletion background, aiming to reduce the STED beam intensity necessary to achieve a target resolution. Tomographic STED microscopy [16] obtains comparable intensity reductions by fusing multiple STED images collected with efficient two-dimensional STED beam intensity distributions.…”