Numerically Enhanced Stimulated Emission Depletion Microscopy with Adaptive Optics for Deep-Tissue Super-Resolved Imaging

Abstract

In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilising both a high power depletion laser (increasing risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here we demonstrate a custom-built 3D STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating, tissue. We introduce and investigate image denoising by block-matching and collaborative filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well-established Fourier ring correlation metric. Experimental validation of the proposed method is achieved through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under a 80µm thick layer of tissue with lateral and axial resolution of 256nm and 300nm, respectively..

Stimulated emission depletion microscopy for biological imaging in four dimensions: A review

“…The donut STED PSF is very sensitive to the refractive index mismatch, and degrades when image deeply. Adaptive optics by using SLM is used to reduce optical aberrations, such as Dual‐SLM STED (Zdańkowski et al, 2019), Easy 3D STED (Arizono et al, 2020), and 3D AO STED (Zdankowski et al, 2019).…”

“…(c) Confocal and 3D dynamic intensity minimum (DyMIN) STED recordings of nuclear pore complexes (NPCs) (shown in green) and lamina (red) (Heine et al, 2017). (d) An axon that grew above the layer of 80 μm of tissue, in the XZ view (Reprinted (adapted) with permission from Zdańkowski, Trusiak, McGloin, and Swedlow (2019). (e) STED images of dendrites belonging to hippocampal neurons recorded at various depths below the tissue surface (Urban, Willig, Hell, & Nägerl, 2011)…”

“…Coherent optical adaptive technique improves the spatial resolution of STED microscopy in thick samples (Coat‐based STED, depth at 36 μm) (Yan et al, 2017). 3D AO STED collaborating 3D filtering (BM3D) achieves super‐resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under an 80 μm thick layer of tissue with lateral and axial resolution of 204 and 310 nm as shown in Figure 2d (Zdańkowski et al, 2019). Using aberration‐reducing optics, it is possible to achieve STED super‐resolution imaging deep inside scattering biological tissue.…”

“…Line average reduces temporal resolution but improves SNR. 3D AO STED takes images of size 720 × 744 × 120 voxels with voxel size of 27 nm × 27 nm × 50 nm and line averaged 465 times, resulting in a single frame acquisition in 21.6 s (Zdańkowski et al, 2019).…”

3D super-resolution deep-tissue imaging in living mice

“…Previous implementations of AO in 3D-STED microscopy have determined the corrective phase modulation using a metric-based approach [ 12 , 36 – 38 ]. The applied correction is systematically adjusted to optimize the quality of the final image.…”