Imaging dendritic spines in the hippocampus of a living mouse by 3D-stimulated emission depletion microscopy

Stéphane Bancelin; Luc Mercier; Johannes Roos; Mohamed Belkadi; Thomas Pfeiffer; Sun Kwang Kim; U. Valentin Nägerl

doi:10.1117/1.NPh.10.4.044402

17 May 2023 Imaging dendritic spines in the hippocampus of a living mouse by 3D-stimulated emission depletion microscopy

Stéphane Bancelin, Luc Mercier, Johannes Roos, Mohamed Belkadi, Thomas Pfeiffer, Sun Kwang Kim, U. Valentin Nägerl

Author Affiliations +

Neurophotonics, Vol. 10, Issue 4, 044402 (May 2023). https://doi.org/10.1117/1.NPh.10.4.044402

Abstract

Significance

Stimulated emission depletion (STED) microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples, such as cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging.

Aim

In previous work, we established chronic STED imaging in the hippocampus in vivo but the gain in spatial resolution was restricted to the lateral plane. In our study, we report on extending the gain in STED resolution into the optical axis to visualize dendritic spines in the hippocampus in vivo.

Approach

Our approach is based on a spatial light modulator to shape the focal STED light intensity in all three dimensions and a conically shaped window that is compatible with an objective that has a long working distance and a high numerical aperture. We corrected distortions of the laser wavefront to optimize the shape of the bottle beam of the STED laser.

Results

We show how the new window design improves the STED point spread function and the spatial resolution using nanobeads. We then demonstrate the beneficial effects for 3D-STED microscopy of dendritic spines, visualized with an unprecedented level of detail in the hippocampus of a living mouse.

Conclusions

We present a methodology to improve the axial resolution for STED microscopy in the deeply embedded hippocampus in vivo, facilitating longitudinal studies of neuroanatomical plasticity at the nanoscale in a wide range of (patho-)physiological contexts.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Stéphane Bancelin, Luc Mercier, Johannes Roos, Mohamed Belkadi, Thomas Pfeiffer, Sun Kwang Kim, and U. Valentin Nägerl "Imaging dendritic spines in the hippocampus of a living mouse by 3D-stimulated emission depletion microscopy," Neurophotonics 10(4), 044402 (17 May 2023). https://doi.org/10.1117/1.NPh.10.4.044402

Received: 7 February 2023; Accepted: 14 April 2023; Published: 17 May 2023

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