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Robin Sharma, Ying Geng, Lu Yin, William H. Merigan, David R. Williams, Jennifer J. Hunter; In Vivo Two-Photon Imaging Of Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5600.
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Our goal is to develop two-photon fluorescence imaging of retinal cells in the living mouse eye. Important advantages of two-photon imaging in the eye include imaging molecules in structures that are otherwise transparent and measuring cellular function at wavelengths that minimally excite the visual response. The mouse is an excellent model for developing two-photon retinal imaging because of (1) the substantially higher spatial resolution and light collecting efficiency of its eye compared to that of humans, (2) the availability of disease models and, (3) the ability to fluorescently label cells for both structural and functional imaging.
A two-photon adaptive optics scanning laser ophthalmoscope (2PAOSLO) designed for the mouse eye was equipped with a dispersion-compensated wavelength tunable Ti:Sapphire laser source. Viral vectors were used to express green fluorescent protein (GFP) or the calcium indicator G-CaMP3 in ganglion or Müller cells. Fluorescence was excited using <70 femtosecond pulses at 920 nm and a repetition rate of 80 MHz. Emission was collected between 400 and 680 nm. Eye motion during imaging was computed from high signal-to-noise ratio (SNR) near-infrared reflectance images acquired with a second imaging channel, allowing for the registration of large numbers of low SNR two-photon images.
In the living mouse eye, we successfully obtained two-photon fluorescence images of individual cells. To confirm the presence of two-photon fluorescence, we noted that the magnitude of the signal varied with dispersion. We identified strong fluorescence from somas of ganglion cells and processes of Müller cells that were expressing GFP or G-CaMP3. We were also able to resolve the dendrites and axons of ganglion cells.
We have demonstrated the utility of the 2PAOSLO for imaging of cells in the living mouse eye. This method will eventually be useful for structural imaging of cells that are transparent in reflected light and may also be useful for monitoring and controlling the functional activity of neurons throughout the retina.
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