IsoSTED nanoscopy with intrinsic beam alignment.

Franziska Curdt,Tobias Lutz,Roman Schmidt,Simon J Herr,Steffen J Sahl,Stefan W Hell,Johann Engelhardt

doi:10.1364/oe.23.030891

Abstract

Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.

Highlights

Far-field super-resolved fluorescence microscopy or, in short, nanoscopy has raised fluorescence imaging to a new level
Since the axial and lateral resolution enhancements are brought about by different polarization components of the STimulated Emission Depletion (STED) focus [Fig. 1(c)], the shape of the effective point spread functions (PSFs) is influenced by the orientation distribution and mobility of the fluorophore dipoles
We have described an isoSTED setup with significantly reduced complexity and a high degree of mechanical robustness

Summary

Introduction

Far-field super-resolved fluorescence microscopy or, in short, nanoscopy has raised fluorescence imaging to a new level. While the spatial resolutions of methods including electron microscopy (EM), or scanning-probe microscopy, still surpass those of the optical far-field approaches, EM and scanning-probe techniques fail to provide imaging inside the volume of intact or living cells in three dimensions. Super-resolved far-field methods have the potential to realize non-destructive, three-dimensional imaging, with spatial resolution that is conceptually limited only by the size of the emitting fluorophores. Ongoing developments to fully deliver on this promise have opened the door to addressing research problems in structure and dynamics which could not be tackled with conventional diffraction-limited optical approaches. For the imaging of biological samples, the invasiveness of electron microscopy sample preparation procedures is a severe problem.

Methods

Results

Conclusion