Impact of a tilted coverslip on two-photon and STED microscopy

The advent of super-resolution microscopy has opened up new avenues to unveil brain structures with unprecedented spatial resolution in the living state. Yet, its application to live animals remains a genuine challenge. Getting optical access to the brain in vivo requires the use of a ‘cranial windo...

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Bibliographic Details
Published in:Biomedical optics express Vol. 15; no. 2; pp. 743 - 752
Main Authors: Le Bourdellès, Guillaume, Mercier, Luc, Roos, Johannes, Bancelin, Stéphane, Nägerl, U. Valentin
Format: Journal Article
Language:English
Published: United States Optical Society of America - OSA Publishing 01.02.2024
Subjects:
Bioengineering
Engineering Sciences
Instrumentation and Detectors
Life Sciences
Optics
Photonic
Physics
cranial window
in vivo imaging
Microscopy
super-resolution
ISSN:2156-7085, 2156-7085
Online Access:Get full text
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Summary:The advent of super-resolution microscopy has opened up new avenues to unveil brain structures with unprecedented spatial resolution in the living state. Yet, its application to live animals remains a genuine challenge. Getting optical access to the brain in vivo requires the use of a ‘cranial window’, whose mounting greatly influences image quality. Indeed, the coverslip used for the cranial window should lie as orthogonal as possible to the optical axis of the objective, or else significant optical aberrations occur. In this work, we assess the effect of the tilt angle of the coverslip on STED and two-photon microscopy, in particular, image brightness and spatial resolution. We then propose an approach to measure and reduce the tilt using a simple device added to the microscope, which can ensure orthogonality with a precision of 0.07°.
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ISSN:2156-7085
2156-7085
DOI:10.1364/BOE.510512