2024/01/06 by Siqing Dai, Andrei Yu Kobitski, Amirhossein Barati Sedeh +3 · 1 voice
Biochemistry, Genetics and Molecular Biology · Physics and Astronomy · #Advanced Fluorescence Microscopy Techniques #Digital Holography and Microscopy #Advanced Electron Microscopy Techniques and Applications
paper · doi:10.1021/acsphotonics.3c01558
Stimulated emission depletion (STED) microscopy is a powerful super-resolution imaging technique for investigating the subcellular structure of biological samples in all three spatial dimensions. Its application to thick specimens is challenging, however, as sample-induced optical aberrations distort the intricate phase patterns of the STED beams and, consequently, their focal intensity distribution. Notably, the central intensity minimum, which is essential for resolution enhancement, is lifted at larger sample depths. Here we present a technique for correction of STED beam aberrations featuring minimal light exposure of the samples to avoid photobleaching and phototoxicity. We quantify STED beam aberrations by using modal wavefront sensing with spatial light modulators (SLMs) as adaptive optics (AO) elements based on reflection imaging of gold beads immobilized at the top and bottom surfaces enclosing the thick (≥100 μm) biological specimen. Based on this information, wavefront aberrations can be successfully compensated for all depths by linear interpolation of the correction parameters. We demonstrate the excellent imaging performance of AO 3D stimulated emission double depletion (STEDD) microscopy on cell nuclei of zebrafish embryos.