RESUMO
Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.
RESUMO
The minimum detection threshold of the infrared sensitive beetle, Melanophila acuminata, was measured with a helium-neon laser that emitted light at a wavelength of 3.39 microm. Extracellular recordings were taken both at the pit organ responsible for detection and at the interganglionic connectives in the thorax of the beetle. At the pit organ, generator and action potentials from single neurons were measured with a sharpened tungsten electrode. At the connectives that linked the fused second meso-/metathoracic and prothoracic ganglia, compound action potentials were measured with a tungsten hook electrode that encircled the connective. The latter recordings confirmed conveyance of infrared information through specific pathways to rostrally-situated sites in the nervous system of the beetle. The 50% probability irradiance threshold at which action potentials were elicited from the receptor and connectives occurred at 17.3 and 14.6 mW/cm(2), respectively. In addition to sensitivity threshold, several other characteristics of the response were quantified including dependence of generator potential latency, generator potential duration, spike frequency, and spike latency on irradiance, dependence of response strength (spike count) on exposure time, and flicker fusion frequency. The ability to detect infrared radiation is rare in nature, and these results provide valuable information necessary to understand this unique sensitivity.