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

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.

Multifocal imaging for precise, label-free tracking of fast biological processes in 3D

“…Alternative methods to derive depth information from a single-plane image use chromatic and spherical aberrations or astigmatism [50][51][52][53][54][55][56] . Like our method, these methods cannot resolve the z-position of objects that are stacked above each other in z or that are very close.…”

Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique

“…Even thicker samples can be imaged through the use of Selective Plane Illumination Microscopy (SPIM) [64]. For monolayer samples, 3D SMLM is also achievable in wide-field setups, for example through engineering of the point spread function (PSF) to produce astigmatism whereby a cylindrical lens breaks the symmetry of the PSF above and J o u r n a l P r e -p r o o f below the focal plane [65] or by using two focal planes as in biplane imaging [58].…”

Single-particle tracking photoactivated localization microscopy of membrane proteins in living plant tissues

“…depth in z), which is determined in our experimental set-up by the depth of the evanescence wave 14 . Note that the z-position of fluorescent emitters can be resolved with higher accuracy by using additional methods (not discussed hereafter) based on astigmatic or engineered point spread functions (PSF) [27][28][29][30][31] . From single molecule tracks, it is then possible to obtain a range of quantitative parameters, including a spatial map of the trajectories, their diffusion coefficient, their molecular displacement, their diffusion type (active, Brownian or constrained), and their transition state (between the different diffusion types) 23 .…”

“…Calculation of the mean square displacement and instantaneous diffusion). [29][30][31][32][33][34]. Third, we describe how to detect and localize the position of fluorescent emitters and track them through time using published tracking software (i.e.…”