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 utilizing a high-power depletion laser (increasing the risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here, we demonstrate a custom-built STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating tissue. We introduce and investigate a state of the art image denoising method by block-matching and collaborative 3D filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise and further enhance the image quality. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well established Fourier ring correlation metric. Results achieved through the combination of aberration correction and tailored image processing are experimentally validated through 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, respectively.
Bibliographical noteFunding Information:
This work was funded by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. 608133. The microscope development was supported by the Wolfson Foundation and the Scottish Universities Physics Alliance (SUPA). P.Z. and M.T. would like to acknowledge the support of the National Science Center, Poland, under the project OPUS 13 (UMO2017/25/B/ST7/02049), National Agency for Academic Exchange, and statutory funds of the Faculty of Mechatronics, Warsaw University of Technology. We thank Iain Porter, Michael Porter, and Lindsay Davidson for the preparation of the differentiated hiPSC derived neurons.
Copyright © 2019 American Chemical Society.
- aberration correction
- adaptive optics
- deep imaging
- fluorescence microscopy
- STED microscopy
- super-resolution microscopy