Defocused imaging exploits supercritical-angle fluorescence emission for precise axial single molecule localization microscopy

Abstract

Single molecule localization microscopy (SMLM) is one of the key techniques that break the classical resolution limit in optical imaging. It is based on taking multiple recordings of a sample, each showing only a sparse arrangement of spatially well separated fluorescent molecules which can be localized at nanometer precision. While localizing along the lateral directions is usually straightforward, estimating axial positions at a comparable precision is known to be much harder, which is due to the relatively large depth of focus provided by the microscope optics. Whenever a molecule is sufficiently close to the coverslip, it becomes feasible to draw additional information from near field coupling effects: super-critical angle fluorescence (SAF) appears and can be exploited to boost the axial localization precision. Here we propose defocused imaging as a SMLM strategy that is capable of leveraging the information contained in SAF. We show that, regarding axial localization precision, our approach is superior to established SAF-based approaches. At the same time it is simple and can be conducted on any research-grade microscope where controlled defocusing on the order of a few hundred nanometers is possible.

Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

“…1e), and to assign experimental localization precisions to every single molecule. The CRLB can be calculated from the inverse of the Fisher Information matrix FI , 19,32 :…”

“…To this end, we start with eq. 3, use the definition ( ) = / and apply Gaussian error propagation 19 :…”

“…Interestingly, SAF has a strong impact on the shape of a defocused PSF. Thus, it can be exploited for precise 3D localization in a simple single-channel setup using an ultra-high NA objective 19 . However, this approach requires precise knowledge of the z-dependent PSF, which is not easy to calibrate in presence of aberrations.…”

Direct supercritical angle localization microscopy for nanometer 3D superresolution

“…Interestingly, SAF has a strong impact on the shape of a defocused PSF. Thus, it can be exploited for precise 3D localization in a simple single-channel setup using an ultrahigh NA objective 19 . However, this approach requires precise knowledge of the z-dependent PSF, which is not easy to calibrate in presence of aberrations.…”

25th Anniversary of STED Microscopy and the 20th Anniversary of SIM: feature introduction

“…In this collection there are 4 articles on SMLM. Philipp Zelger et al describe a new method for axial localization [56]. Sha An et al discuss a method for SMLM along axial cross-sections [57].…”