posted on 2024-01-06, 18:29authored bySiqing Dai, Andrei Kobitski, Amirhossein Barati Sedeh, Süheyla Eroğlu-Kayıkçı, Lennart Hilbert, G. Ulrich Nienhaus
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.