Abstract
Abstract: :
Purpose: To understand the retinoid metabolism, particularly the retinyl ester storage, in the mouse RPE cells. Methods: Two–photon excitation microscopy was performed using a Zeiss LSM 510 MP–NLO confocal microscope (Carl Zeiss) equipped with mode–locked Ti: Sapphire laser (Mira–900, Coherent). Mouse eyecups or intact eyes were cultured on glass bottomed 35mm dish and perfused by oxygenized Ames buffer at 37oC. Short pulse laser (730 nm) was focused on RPE cells in culture, and emission signals were directed to a photomultiplier tube to obtain temporally and spatially resolved images of retinol/retinyl ester distribution in RPE cells. Retinoids were analyzed by normal phase HPLC. Results: After flash stimulation of eyes in culture, changes in fluorescence intensity and the amount of fluorescent retinoid were measured. The change in fluorescence was high in filamentous subcellular compartments in RPE cells. These structures are perpendicularly aligned to the RPE cell layer and often located proximal to lateral plasma membrane. The kinetics of this process paralleled accumulation of the esters as determined by the HPLC analysis of retinoids. To further characterize the storage of all–trans–retinyl esters, mouse eye cups were prepared on microscope stage. Addition of an excess amount of all–trans retinol to RPE cell culture resulted in the formation of all–trans–retinyl ester, which corresponded to the increase in fluorescent intensity in the novel subcellular structure, termed retinosomes. Conclusions: Two–photon microscopy is a useful method for real–time imaging of retinol and retinyl esters in the eye. In mouse RPE cells, retinyl esters accumulate in highly organized subcellular structures (retinosomes). In conjunction with HPLC, this imaging technique will allow us to analyze in more detail the retinoid flow in the vertebrate retina in native and genetically altered animal models.
Keywords: retinal pigment epithelium • microscopy: confocal/tunneling • retinoids/retinoid binding proteins