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
Hybridization of nucleic acids (NAs) is a fundamental molecular mechanism that drives many cellular processes and enables new biotechnologies as well as therapeutics. However, existing methods that measure hybridization kinetics of nucleic acids are either performed at the ensemble level or constrained to non-native physiological conditions. Recent advances in 3D single-molecule tracking techniques break these limitations by allowing multiple annealing and melting events to be observed on a single oligonucleotide freely diffusing inside a live mammalian cell. This review provides an overview of diverse approaches to measuring NA hybridization kinetics at the single-molecule level and in live cells, and concludes with a synopsis of unresolved challenges and opportunities in the live-cell hybridization kinetics measurements. Important discoveries made by NA kinetics measurements and biotechnologies that can be improved with a deeper understanding of hybridization kinetics are also described.