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

1. Neurophotonics. 2023 Oct;10(4):044402. doi: 10.1117/1.NPh.10.4.044402. Epub
2023 May 17.

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

Bancelin S(1), Mercier L(1), Roos J(1), Belkadi M(1), Pfeiffer T(1), Kim SK(2),
Nägerl UV(1).

Author information:
(1)University of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience,
IINS, UMR 5297, Bordeaux, France.
(2)Kyung Hee University, Graduate School, Department of Science in Korean
Medicine, Seoul, Republic of Korea.

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.

© 2023 The Authors.

DOI: 10.1117/1.NPh.10.4.044402
PMCID: PMC10197143
PMID: 37215638

Auteurs Bordeaux Neurocampus