Direct supercritical angle localization microscopy for nanometer 3D superresolution

Anindita Dasgupta,Jonas Ries,Ralf Jungmann,Jan Becker,Rainer Heintzmann,Uwe Hübner,Ulf Matti,Joran Deschamps,Sebastian Strauss

doi:10.1038/s41467-021-21333-x

Abstract

3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

Highlights

1234567890():,; For many biological questions the 3D organization of proteins is of high interest, SMLM1–3 has been extended early on to go beyond 2D projections and to measure the 3D coordinates of single fluorescent emitters
We demonstrate on 3D DNA origami structures that dSALM can fulfill its potential in terms of 3D resolution and show on biological samples that a combination with astigmatism leads to a remarkable z resolution over an extended axial range
Since SAF depends on the orientation of the transition dipole moment of the fluorophore, dSALM should only be used with standard labeling approaches using fluorescent proteins, antibodies or self-labeling enzymes that allow for free rotation of the fluorophores

Summary

Introduction

1234567890():,; For many biological questions the 3D organization of proteins is of high interest, SMLM1–3 has been extended early on to go beyond 2D projections and to measure the 3D coordinates of single fluorescent emitters. Direct splitting of SAF and UAF had not been realized in SMLM because blocking the UAF in the SAF channel results in strong diffraction patterns dominating the SAF PSF23 (Supplementary Fig. 2), leading to an increased PSF size and preventing a reliable measurement of single-molecule intensities We overcome these challenges and realize the full potential of SALM by combining (a) direct measurement of SAF with greatly increased signal to noise ratio by splitting it from UAF with a custom elliptical mirror, (b) use of an ultrahigh NA objective to increase the SAF signal and to decrease the effect of diffraction on the SAF PSF, and (c) data analysis approaches that allow for precise determination of UAF and SAF intensities even in presence of a complex PSF. We demonstrate on 3D DNA origami structures that dSALM can fulfill its potential in terms of 3D resolution and show on biological samples that a combination with astigmatism leads to a remarkable z resolution over an extended axial range

Methods

Results

Conclusion