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SUBRATA BATABYAL, Kamal Dhakal, Gregory Cervenka, Weldon Wright, David G Birch, Young-tae Kim, Samrendra Mohanty; Optical delivery and broadband activation of polychromatic opsin lowers intensity threshold for photostimulation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):996.
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Here, we report ultrafast near-infrared laser-based spatially-targeted transfection of mono- and poly-chromatic opsins. The development will significantly lower intensity stimulation required for activating poly-chromatic sensitized cells may facilitate ambient white light-based restoration of vision for patients with widespread photoreceptor degeneration
We have devoloped a polychromatic ospin having higher photosensitivity. The newly devoloped opsin was confirmed by running DNA gel electrophpresis. We found significant higher photocurrent in case of polychormatic opsin compared to its single counterpart.
We have sucessfully optoportaed the HEK cell using femotsecond laser for the trageted delivery of the devoloped polychromatic opsin. To evaluate the efficacy of white light as compared to spectrally-filtered light for optogenetic stimulation, polychromatic opsin transfected HEK cells were subjected to different photo stimulation. Further, wavelength-dependent patch-clamp recordings were carried out to measure response of these opsins to individual narrow band (FWHM=10 nm) as well as wide-spectral (400-650 nm) activation. We observed higher inward current in case of polychromatic opsin sensitized in HEK cell stimulated by white light as compared to different spectrally-filtered components (total white light intensity: 0.12 mW/mm2). While all narrow-band spectrally-filtered components evoked similar inward current (15 pA), the white light was found to evoke order of magnitude higher inward current. Such a large enhancement in bandwidth sensitivity could not be achieved by using the exsisting opsins.
High fidelity of peak-photocurrent of polychromatic opsins in response to repetitive white light stimulation of varying pulse width was observed. Laser-assisted targeted delivery of polychromatic opsins encoding genes to peripheral retina/macula is ideal for sensitizing degenerated-retinal areas, thus paving the way for restoring lost-vision of RP/AMD patients. The significantly lower intensity stimulation required for activating white-opsin sensitized cells may facilitate ambient white light-based restoration of vision for patients with widespread photoreceptor degeneration.
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