Robust adaptive optics for localization microscopy deep in complex tissue

Marijn E Siemons,Lukas C Kapitein,Maarten H P Kole,Naomi A K Hanemaaijer

doi:10.1038/s41467-021-23647-2

Abstract

Single-Molecule Localization Microscopy (SMLM) provides the ability to determine molecular organizations in cells at nanoscale resolution, but in complex biological tissues, where sample-induced aberrations hamper detection and localization, its application remains a challenge. Various adaptive optics approaches have been proposed to overcome these issues, but the exact performance of these methods has not been consistently established. Here we systematically compare the performance of existing methods using both simulations and experiments with standardized samples and find that they often provide limited correction or even introduce additional errors. Careful analysis of the reasons that underlie this limited success enabled us to develop an improved method, termed REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of up to 1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. After its quantitative validation, we demonstrate that REALM enables to resolve the periodic organization of cytoskeletal spectrin of the axon initial segment even at 50 μm depth in brain tissue.

Highlights

Single-Molecule Localization Microscopy (SMLM) provides the ability to determine molecular organizations in cells at nanoscale resolution, but in complex biological tissues, where sample-induced aberrations hamper detection and localization, its application remains a challenge
Because SMLM acquisitions are comprised of pseudo-random point sources, these methods effectively probe and optimize the magnitude transfer function (MTF)
REALM estimated the aberrations to be ~1 rad RMS, whereas the method of Mlodzianoski et al resulted in a 3-fold lower estimate, leaving an average residual aberration of ~0.7 rad RMS. These results demonstrate that REALM achieves a 3-fold improvement in aberration correction over previous methods and enables robust single-molecule imaging at 60 μm depth through brain tissue with improved resolution

Summary

Introduction

Single-Molecule Localization Microscopy (SMLM) provides the ability to determine molecular organizations in cells at nanoscale resolution, but in complex biological tissues, where sample-induced aberrations hamper detection and localization, its application remains a challenge. Careful analysis of the reasons that underlie this limited success enabled us to develop an improved method, termed REALM (Robust and Effective Adaptive Optics in Localization Microscopy), which corrects aberrations of up to 1 rad RMS using 297 frames of blinking molecules to improve single-molecule localization. Careful analysis of the reasons that underlie this limited success enabled us to develop an improved method, termed Robust and Effective Adaptive Optics in Localization Microscopy (REALM), which corrects aberrations of up to 1 rad RMS using 297 frames of blinking molecules, thereby enabling robust SMLM at 50 μm depth and even up to 80 μm depth when pre-correcting spherical aberration

Objectives

Methods

Results

Conclusion