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R. Roizenblatt, J. Weiland, S. Carcieri, M. Humayun, R.H. Chow; Nanobiolistic Delivery of Functional Indicators to the Living Mouse Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5383.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To describe a novel and broadly applicable methodology that allows delivery of fluorescent indicators to the retina using nanosized silver particles Methods: In recent years, a ballistic technique for indicator delivery has been proposed, in addition to other classic loading techniques, such as acetoxymethyl (AM) ester loading. We demonstrate a novel technique that uses a modified gene gun to propel 80 nanometer silver particles (NanodynamicsTM) coated with indicators into living mouse retinal neurons. The gun assembly consists of a solenoid valve triggered by a time–delay relay. Because many intracellular processes are calcium dependent, we used calcium indicator dyes (Oregon Green Bapta1®, Rhod dextran®) that are commonly used in biophysical imaging experiments. Results: Fluorescence–based indicators have been of particular importance for investigating ionic concentrations, protein localization and other physiological measurements. The nanobiolistic technique succeeded in widely distributing these indicators to different retinal cell types, verified by multiphoton microscopy imaging of fluorescent photoreceptors and ganglion cells in the wholemount mouse retina. In the nerve fiber layer, we identified capillaries and bundles of axons from distant ganglion cells, marked by a specific antibody (CD90.2) coupled to a green fluorescent probe; the red fluorescent Rhod dextran coating nanobullets penetrate the ganglion cell somas and are visible within the layer. Photoreceptors are visible with the red calcium indicators inside, which penetrated the external segments by means of nanobiolistics. Pictures are also shown of difficulties with the AM ester neuronal loading technique Conclusions: We show that this minimally invasive technique is useful for faithful physiological measurements in fragile tissues, such as the isolated retina.
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