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Neurophotonics. 2018 Apr;5(2):025001. doi: 10.1117/1.NPh.5.2.025001. Epub 2018 Feb 19.

Designing a large field-of-view two-photon microscope using optical invariant analysis.

Author information

1
Washington University in Saint Louis, Department of Biomedical Engineering, St. Louis, Missouri, United States.
2
Washington University School of Medicine, Department of Radiology, St. Louis, Missouri, United States.
3
Washington University in Saint Louis, Department of Biology, St. Louis, Missouri, United States.
4
Washington University School of Medicine, Department of Neurology, St. Louis, Missouri, United States.
5
Washington University in Saint Louis, Department of Physics, St. Louis, Missouri, United States.
6
Université Laval, Génie Physique et Optique, Département de Physique, Ville de Québec, Quebec, Canada.

Abstract

Conventional two-photon microscopy (TPM) is capable of imaging neural dynamics with subcellular resolution, but it is limited to a field-of-view (FOV) diameter [Formula: see text]. Although there has been recent progress in extending the FOV in TPM, a principled design approach for developing large FOV TPM (LF-TPM) with off-the-shelf components has yet to be established. Therefore, we present a design strategy that depends on analyzing the optical invariant of commercially available objectives, relay lenses, mirror scanners, and emission collection systems in isolation. Components are then selected to maximize the space-bandwidth product of the integrated microscope. In comparison with other LF-TPM systems, our strategy simplifies the sequence of design decisions and is applicable to extending the FOV in any microscope with an optical relay. The microscope we constructed with this design approach can image [Formula: see text] lateral and [Formula: see text] axial resolution over a 7-mm diameter FOV, which is a 100-fold increase in FOV compared with conventional TPM. As a demonstration of the potential that LF-TPM has on understanding the microarchitecture of the mouse brain across interhemispheric regions, we performed in vivo imaging of both the cerebral vasculature and microglia cell bodies over the mouse cortex.

KEYWORDS:

etendue; neurophysiology; optical design; optical invariant; scanning microscopy; two-photon microscopy

PMID:
29487876
PMCID:
PMC5818100
[Available on 2019-02-19]
DOI:
10.1117/1.NPh.5.2.025001

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