Abstract
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.
Highlights
In Single-Molecule Localization Microscopy (SMLM) the diffraction limit is circumvented by analyzing the Point Spread-Function (PSF) of single fluorescent molecules to obtain precise information about the molecule’s position in x and y [1,2]
For 2D-SMLM we found that when using oil immersion (OI) at imaging depths above 5 μm, the use of a deformable mirror (DM) to correct aberrations improves localization precision, effectively doubling the amount of localizations with a precision below 15 nm at 24 μm depth
OI + Adaptive optics (AO) results in a 21% improvement in localization precision compared to water immersion (WI) for relevant photon levels
Summary
In Single-Molecule Localization Microscopy (SMLM) the diffraction limit is circumvented by analyzing the Point Spread-Function (PSF) of single fluorescent molecules to obtain precise information about the molecule’s position in x and y [1,2]. A common way to determine the z-position of fluorophores is by inducing astigmatism in the detection path, either by introducing a cylindrical lens (CL) [4] or an adaptive optical element [5]. The PSF of a fluorophore positioned in the AFP appears round, while the PSFs of fluorophores outside AFP appear elliptical. The orientation of the major and minor axis of an elliptical PSF indicates whether a fluorophore is positioned above or below the AFP and the distance to the AFP can be determined from the amount of ellipticity
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