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Adi Schejter, Nairouz Farah, Limor Tsur, Shy Shoham; Single- and multi-photon fluorescence retinal imaging in the intact rodent eye: a comparison. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4880.
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Non-invasive fluorescence fundus imaging could prove to be an important tool for in-vivo small animal retinal imaging in a wide array of translational vision applications, including the tracking of fluorescently tagged cells and the expression of gene-therapy and optogenetic vectors. Recently, we demonstrated the ability to achieve cellular resolved images by means of 1P fluorescence micro-endoscopy in a retina transduced with fluorescent proteins and the GCaMP-family of optogenetic calcium indicators (Schejter et al,. 2012, TVST). Here we demonstrate two-photon imaging in the same animal models, and compare the two systems' retinal imaging capability.
A custom endoscope-based fundus system and a two-photon microscope were used to acquire fluorescence images from head-fixed animals expressing optogenetic probes in-vivo. These were compared to in-vitro images of the same structures and were analyzed.
Two-photon imaging with a 10X water-immersion objective yielded well-resolved fluorescence images of retinal fine structure which were axially sectioned to individual retinal layers. Interestingly, both two-photon imaging (without adaptive optics) and the endoscope-based images appeared to be robust to PSF distortions that completely smeared out cellular details in a conventional 1P fluorescence microscopic image of the same retina. The two methods have major differences in their sectioning ability, field of view, resolution, and the practicality of extending them to functional imaging.
This study demonstrates the ability to acquire fluorescent fundus images in-vivo by implementing two different imaging modalities. We will discuss the advantages and disadvantages of both methods and possible methods for improvement.
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