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Proceedings Paper

Development of large field-of-view two photon microscopy for imaging mouse cortex (Conference Presentation)

Paper Abstract

Spontaneous neuronal activity has been measured at cellular resolution in mice, zebrafish, and C. elegans using optical sectioning microscopy techniques, such as light sheet microscopy (LSM) and two photon microscopy (TPM). Recent improvements in these modalities and genetically encoded calcium indicators (GECI’s) have enabled whole brain imaging of calcium dynamics in zebrafish and C. elegans. However, these whole brain microscopy studies have not been extended to mice due to the limited field of view (FOV) of TPM and the cumbersome geometry of LSM. Conventional TPM is restricted to diffraction limited imaging over this small FOV (around 500 x 500 microns) due to the use of high magnification objectives (e.g. 1.0 NA; 20X) and the aberrations introduced by relay optics used in scanning the beam across the sample. To overcome these limitations, we have redesigned the entire optical path of the two photon microscope (scanning optics and objective lens) to support a field of view of Ø7 mm with relatively high spatial resolution (<10 microns). Using optical engineering software Zemax, we designed our system with commercially available optics that minimize astigmatism, field curvature, chromatic focal shift, and vignetting. Performance of the system was also tested experimentally with fluorescent beads in agarose, fixed samples, and in vivo structural imaging. Our large-FOV TPM provides a modality capable of studying distributed brain networks in mice at cellular resolution.

Paper Details

Date Published: 24 April 2017
PDF: 1 pages
Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100690F (24 April 2017); doi: 10.1117/12.2251433
Show Author Affiliations
Jonathan Bumstead, Washington Univ. in St. Louis (United States)
Daniel C. Côté, Ctr. de Recherche de l'Univ. Laval Robert-Giffard (Canada)
Joseph P. Culver, Washington Univ. in St. Louis (United States)

Published in SPIE Proceedings Vol. 10069:
Multiphoton Microscopy in the Biomedical Sciences XVII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)

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