Development of an Alignment Method for Retarders in isoSTED Microscopy

초록

The use of stimulated emission depletion (STED) microscopy has significantly improved resolution beyond the limits imposed by diffraction; Furthermore, STED microscopy adopts a 4Pi-geometry to achieve an isotropic improvement in resolution. In isoSTED microscopy, a polarizing beam splitter and retarders are used in a 4Pi cavity to split beams of identical power, generating constructive and destructive interference for lateral and axial resolution improvements, respectively. The precise alignment of the retarders is crucial for optimizing the performance of isoSTED microscopy, because this orientation affects the quality of the depletion focus, necessitating zero intensity at the center. Incomplete destructive interference can lead to unwanted fluorescence inhibition, resulting in degraded resolution and contrast. However, measuring the intensity and polarization state in each optical path of the 4Pi cavity is complex and requires additional devices such as a power meter. Here, we propose a simple and accurate alignment method for the 4Pi cavity in isoSTED microscopy. Our approach demonstrates the equal allocation of power between upper and lower beam paths and achieves complete destructive interference using a polarizing beam displacer and a single CCD camera positioned outside the 4Pi cavity.

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