Comparing strategies for deep astigmatism-based single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) enables fluorescent microscopy with nanometric resolution. While localizing molecules close to the coverslip is relatively straightforward using high numerical aperture (NA) oil immersion (01) objectives, optical aberrations impede SMLM deeper in watery samples. Adaptive optics (AO) with a deformable mirror (DM) can be used to correct such aberrations and to induce precise levels of astigmatism to encode the z-position of molecules. Alternatively, the use of water immersion (WI) objectives might be sufficient to limit the most dominant aberrations. Here we compare SMLM at various depths using either WI or CH with or without AO. In addition, we compare the performance of a cylindrical lens and a DM for astigmatism-based z-encoding. We find that CH combined with adaptive optics improves localization precision beyond the performance of WI-based imaging and enables deep (> 10 mu m) 3D localization. (C) 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement