Abstract
Stimulated emission depletion (STED) microscopy is a powerful tool for observing subcellular structures beyond the diffraction-limited resolution. To achieve improved isotropic three-dimensional (3D) resolution in STED microscopy, 4Pi-geometry, which is equipped with two opposing objectives, is adopted; this approach is known as isoSTED microscopy. In isoSTED microscopy, constructive and destructive interference occur at the shared focal plane for lateral and axial depletion, respectively. The performance of isoSTED microscopy depends critically on the polarisation status and optical power differences of the two beams of the 4Pi cavity, which are adjusted using retarders. Misalignment of the retarders creates a nonzero core of the depletion focus owing to partial destructive interference. We analytically investigated the effects of retarder misalignments and proposed a simple and accurate alignment method for retarders. We also demonstrate that the proposed method can generate a completely destructive interference pattern and consequently achieve a sub-40-nm 3D resolution.
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