Three-dimensional adaptive optical nanoscopy for thick specimen imaging at sub-50-nm resolution

Abstract

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved isoSTED nanoscope, which utilizes an advanced adaptive optics strategy to achieve sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

All-optical visualization of specific molecules in the ultrastructural context of brain tissue

“…2 ll-qq,r ), consistent with previous work (Xiao et al, 1998). The expansion-corrected distributions of Bassoon, Homer1, and PSD-95 within the axial DP-PSD distances are all in agreement with previously published studies (Dani et al, 2010; Hao et al, 2021). For instance, the distance between Bassoon and the PSD is 103.7 ± 24.4 nm (mean ± s.d.…”

“…; N = 4 experiments; n = 44 synaptic profiles; Fig. 2n), which is consistent with the range of pre-and post-synaptic density distance measurements determined previously by super-resolution microscopy and electron tomography (Valtschanoff et al, 2001;Dani et al, 2010;Acuna et al, 2016;Hao et al, 2021;Yang and Annaert, 2021). Similarly, dividing the distance between neighboring DPs by the nuclear expansion factor, we obtained a value of 67.2 ± 15.4 nm (mean ± s.d.…”

Color centers in wide-bandgap semiconductors for subdiffraction imaging: a review

“…115 Other superresolution methods could also be explored in the future using CCs, such as the recently developed iso-STED, where adaptive optics is used to achieve sub-50-nm 3D resolution of structures in tissue. 171 While the bulk diamond NV has provided the highest localization and resolution, its resolution in NDs is limited by the size of the NDs, and, as such, the applicability to biological samples is limited. Nevertheless, using SMLM, NV − in NDs could be used for nanoscale imaging and sensing of a magnetic field in a cellular environment.…”

Active Aberration Correction with Adaptive Coefficient SPGD Algorithm for Laser Scanning Confocal Microscope

“…Nondestructive techniques such as the use of a laser scanning confocal microscope (LSCM) [ 1 , 2 , 3 ] provide the feasibility of investigating the characterization of biological cells. Scanning can be performed in the lateral as well as in the axial directions, and three-dimensional images of the sample can be generated in this way [ 2 ].…”

“…Scanning can be performed in the lateral as well as in the axial directions, and three-dimensional images of the sample can be generated in this way [ 2 ]. However, optical aberrations limit LSCMs to thin samples and have prevented their application to thick specimens [ 3 ]. In this paper, an image-based adaptive optics (AO) system with a fast convergence speed is designed for LSCMs to reduce the adverse effects of aberrations and improve imaging quality.…”