isoSTED nanoscopy with intrinsic beam alignment

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.

Super-Resolution Imaging of Tight and Adherens Junctions: Challenges and Open Questions

“…Pulsed excitation and time-gated detection have enabled resolutions below 50 nm [55]. Using a two-objective configuration or 3D STED depletion patterns a lateral and axially similar resolution of 50-70 nm has been demonstrated [56]. The overall STED resolution is mostly limited by depletion laser power, alignment quality and, most importantly, by labelling density and the availability of bright and photostable dyes with an optimal 5%-10% normalized emission intensity at the depletion laser wavelength.…”

Aberrations in 4Pi Microscopy

“…For example, the high NA causes mixing of one polarization direction into the other two [19] which has visible impact on the symmetry of 4Pi focal intensities for some contra-variant 4Pi modes. In some 4Pi systems, polarization manipulation has even been applied as an effective strategy for PSF engineering [20]. For these reasons, we further investigated the combined effects of aberrations and polarizations.…”

High-Resolution 3D Light Microscopy with STED and RESOLFT

“…From [57], reproduced with permission at millisecond imaging times for ultrafast dynamics in small fields of view [56], the demonstration of a RESOLFT strategy to neutralize the diffraction limit of light-sheet fluorescence microscopy [57] (compare Fig. 1.18), efficient STED nanoscopy with quantum dots as fluorescent labels [58]; highest levels of 3D isotropic resolution (<30 nm in x,y,z simultaneously) with a new, stable design of 4Pi-based isoSTED [59] (compare Fig. 1.17 for more details on the isoSTED approach), and the several-thousand fold "massive" parallelization of RESOLFT and even STED without resolution compromises for faster imaging of large fields [60,61] as well as extended multicolor capabilities [62] and nanoscopy in living animals ( Fig.…”