Effects of aberrations on isoSTED microscopy

Abstract

Aberrations are inevitable in optical microscopes and affect the image quality. In STED microscopy, the optical resolution is especially dominated by the performance of depletion pattern. To quench fluorescence only in the periphery of the excitation focus, the depletion focus ideally has a zero-intensity point that is surrounded by a high-intensity crest. Otherwise, distortion of the depletion beam causes unwanted fluorescence inhibition. The influence of aberrations on isoSTED microscopy differs from that on STED microscopy because isoSTED microscopy generates a more complicated shape of the depletion focal spot with two opposing objectives which have different focusing properties. We present analysis that elucidates the effects on the shape, the central minimum intensity, and the peak intensity of the isoSTED pattern regarding primary aberrations such as astigmatism, coma, and spherical aberration when those aberrations are added in the shared beam path and each beam path in the 4Pi-cavity.

Investigation of the effects of polarisation and alignment method of retarders in isoSTED microscopy

“…The general implementation of isoSTED microscopy utilises two orthogonally polarised depletion beams with a polarising beam splitter (PBS) and retarders to provide the required relative phase differences [12,13,17]. Two halfwave plates (HWPs) were used before and after the PBS to equally split the power of the laser and rotate the beams to be in line with the beams in the other beam path of the 4Pi-cavity.…”