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Ryuji Hisamura, Shigeru Nakamura, Imada Toshihiro, Yusuke Izuta, Kazuo Tsubota; Two-photon imaging of nerve projection in optically cleared mouse lacrimal gland. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5656.
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© ARVO (1962-2015); The Authors (2016-present)
Two-photon laser-scanning microscopy enables us to perform deep imaging of the biotissue. Tissue optical clearing makes it possible for laser to reach the deep tissue. In this study we carried out Two-photon laser-scanning microscopy imaging in optically cleared lacrimal gland (LG) by 2, 2’-Thiodiethanol (TDE) solution to identify the nerve projection of mouse LG.
For imaging the peripheral nerve projection into LG, we used transgenic mice which expressed yellow fluorescent protein (YFP) in the Schwann cells (PLP-tTA::tetO-ChR2-EYFP double transgenic, PLP-YFP). The exorbital LG in the male and female 10-week-old PLP-YFP mouse was immersed in the 60% TDE solution for 0.5 to 2 hours without removing. After clearing, the nerve projection images were obtained using a Two-photon laser-scanning microscopy (FV1200, Olympus) with a 4x objective lens (XL Fluor 4x/340, NA 0.28, Olympus).
After immersion in TDE for 2 hours the LG became transparent enough to deep imaging without YFP quenching. On the lateral side of LG YFP-positive nerve bundle was localized along the rostral lacrimal duct from dorsal to ventral LG with trifurcation at the dorsal end. On the medial side of LG two YFP-positive nerve bundles were observed. One nerve bundle was localized along the rostral lacrimal duct and the other was localized along the caudal blood vessels from dorsal to ventral LG with ramification.
Combination of Two-photon laser-scanning microscopy with tissue optical clearing by TDE is useful imaging method for identification of the nerve projection of LG.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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