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 (OI) 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 OI 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 OI combined with adaptive optics improves localization precision beyond the performance of WI-based imaging and enables deep ( > 10 µm) 3D localization. in bypass mode. The AO-module consists of 3 lenses and 4 broadband mirrors. The DM itself has a silver coating with a reflectance of 98% at 15 degree incidence. These results in a total transmission of 93% for the AO-module.

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