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Proceedings Paper

In vivo three-photon imaging of deep cerebellum
Author(s): Mengran Wang; Tianyu Wang; Chunyan Wu; Bo Li; Dimitre G. Ouzounov; David Sinefeld; Akash Guru; Hyung-Song Nam; Mario R. Capecchi; Melissa R. Warden; Chris Xu
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Paper Abstract

We demonstrate three-photon microscopy (3PM) of mouse cerebellum at 1 mm depth by imaging both blood vessels and neurons. We compared 3PM and 2PM in the mouse cerebellum for imaging green (using excitation sources at 1300 nm and 920 nm, respectively) and red fluorescence (using excitation sources at 1680 nm and 1064 nm, respectively). 3PM enabled deeper imaging than 2PM because the use of longer excitation wavelength reduces the scattering in biological tissue and the higher order nonlinear excitation provides better 3D localization. To illustrate these two advantages quantitatively, we measured the signal decay as well as the signal-to-background ratio (SBR) as a function of depth. We performed 2-photon imaging from the brain surface all the way down to the area where the SBR reaches ~ 1, while at the same depth, 3PM still has SBR above 30. The segmented decay curve shows that the mouse cerebellum has different effective attenuation lengths at different depths, indicating heterogeneous tissue property for this brain region. We compared the third harmonic generation (THG) signal, which is used to visualize myelinated fibers, with the decay curve. We found that the regions with shorter effective attenuation lengths correspond to the regions with more fibers. Our results indicate that the widespread, non-uniformly distributed myelinated fibers adds heterogeneity to mouse cerebellum, which poses additional challenges in deep imaging of this brain region.

Paper Details

Date Published: 23 February 2018
PDF: 6 pages
Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 1049816 (23 February 2018); doi: 10.1117/12.2285406
Show Author Affiliations
Mengran Wang, Cornell Univ. (United States)
Tianyu Wang, Cornell Univ. (United States)
Chunyan Wu, Cornell Univ. (United States)
Bo Li, Cornell Univ. (United States)
Dimitre G. Ouzounov, Cornell Univ. (United States)
David Sinefeld, Cornell Univ. (United States)
Akash Guru, Cornell Univ. (United States)
Hyung-Song Nam, The Univ. of Utah (United States)
Mario R. Capecchi, The Univ. of Utah (United States)
Melissa R. Warden, Cornell Univ. (United States)
Chris Xu, Cornell Univ. (United States)


Published in SPIE Proceedings Vol. 10498:
Multiphoton Microscopy in the Biomedical Sciences XVIII
Ammasi Periasamy; Peter T. C. So; Karsten König; Xiaoliang S. Xie, Editor(s)

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