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L. Chen, N. Tian; Quantitative Analysis of in vivo Imaging of Retinal Ganglion Cells in Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1012.
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The goal of this study is to determine the reliability and precision of in vivo imaging of RGCs dendritic structure and to determine the possible laser damage to the imaged RGCs.
A confocal scanning laser microscope was used to image the yellow fluorescent protein (YFP) expressing RGCs in vivo. The in vitro images of the same cells were obtained immediately after in vivo imaging. To determine how precise could in vivo images illustrate the dendritic structure of RGCs, the dendritic branches from corresponding in vivo and in vitro images were manually traced and analyzed. To test the possible laser damage to RGCs, RGCs were time-lapse imaged with different amount laser and the cell number were counted before and several weeks after laser exposure.
We determined the reliability and precision of in vivo images by comparing the number and length of dendritic branches between in vivo and in vitro images. Our results showed that in vivo images have the same number of dendritic branches and a slight decrease of total dendritic length (less than 5% on average), suggesting that in vivo images can precisely illustrate the branch pattern of RGCs. The decrease of total dendritic length was most likely due the natural concave of retina in vivo. We examined the possible laser damage to RGCs by imaging 3 groups of RGCs with different laser exposure time and same laser intensity. The results showed that all the imaged cells can survive after exposure to laser for 3 ~ 4 min, 10 % of the imaged cells would disappear after exposure to laser for 6 ~ 10 min, and 20 % of the imaged cells would disappear when increase exposure time to 15 min.
Our results showed that in vivo image could reliably and precisely illustrate the dendritic structure of RGCs and laser could cause damage on RGCs during prolonged laser exposure.
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