Confocal laser scanning microscopy of retinal rod outer segment intact disks: new labeling technique

Abstract

Abstract. Vertebrate retinal rod outer segment disks house the proteins involved in the phototransduction cascade that converts light into neuronal signal. We develop a technique for the immunofluorescent labeling of osmotically intact isolated rod outer segment disks for confocal laser scanning microscopy imaging. Osmotically intact Ficoll-flotation isolated bovine disks are directly labeled with antibodies in solution. For the first time, osmotically intact single disks can be visualized. Thus, imaging of purified disks, based on advanced optical techniques, may serve as a powerful complement to other methods in studies on phototransduction. In fact, even though much is known about the rod outer segment photoresponse, some unanswered questions remain, particularly about ATP supply, light adaptation, and morphogenesis. Oct. 3, 2007. Confocal laser scanning microscopy ͑CLSM͒ is a valuable tool for obtaining high-resolution images and 3-D reconstructions. 1 Vertebrate rod photoreceptors, the neurons responsible for visual phototransduction, mediate vision at low-light intensities using a modified cilium ͑called the outer segment͒ for the detection of light. The rod outer segment ͑rod OS͒ is packed with hundreds of membrane sacs, called disks, on the surface of which the initial biochemical reactions of visual transduction take place. 2,3 The disks, with dimensions of about 1 m diam, 4,5 are regularly stacked on top of each other with a repeat distance of 300 Å. 4 The visual pigment rhodopsin ͑Rh͒, an integral membrane protein, accounts for about 90% 6 of total disk protein content. Photoreceptor disks are continuously renewed through formation of new disks at the base of the outer segment, displaced distally along the length of the outer segment, and eventual detachment and phagocytosis occurs by adjacent pigment epithelial cells. 7 Several microscopy techniques have been used to analyze the morphological organization of rods and to identify the membrane proteins in rod OS. For example, measurements of the ellipsoid mitochondria density, sizes, and shapes of inner segments were conducted by electron microscopy and Nomads differential interference contrast ͑NDIC͒ imaging. 8 Studies on the stability of Rh in native membranes were done by single-molecule force spectroscopy. 9 Moreover, a recent progress in modeling the native conformation of Rh was based on topography of the disk membranes recorded by an atomic force microscope ͑AFM͒. 9 CLSM was only used on whole retina or rod OS to observe the protein distribution in the disk incisures. 10 Therefore, even though rod imaging has been achieved in many ways, to the best of our knowledge, until now no study has been reported regarding the application of immunofluorescence for confocal optical imaging of isolated intact disks. In fact, even though fluorescent optical imaging is among the most widely used approach for studying cells in vivo, it is not easily applicable for subcellular fractions. Apart from the classic inclusion methods, it is difficult to isolate an...

Hypothesis of an Energetic Function for Myelin

“…Biochemical assays, Western Blot analysis, Confocal Laser Scanning (CLSM) and Transmission Electron Microscopy (TEM) of purified Intact Myelin Vesicles (IMV) showed that IMV are able to produce ATP through the operation of a H ? gradient across their membranes [16,17]. Oximetric measurements showed that IMV consume O 2 at a rate comparable to that of mitochondria [15].…”

Proteomic Analysis of the Retinal Rod Outer Segment Disks

“…Panel B1 shows that the visual pigment is uniformly distributed on disks, as previously reported. 13 Panel B2 shows the transmission image of the same disk sample merged to the fluorescence of a Cy5-labeled secondary Ab bound to an Ab against R1 subunit of Na + /K + - ATPase. The low immunoreactivity suggests a little RIS contamination.…”

Live imaging of mammalian retina: rod outer segments are stained by conventional mitochondrial dyes

“…8,9 In past years we have investigated adenosine triphosphate ͑ATP͒ production in the ROS. 10 Recently, we reported a new protocol for the imaging of disks for immunofluorescence confocal laser scanning microscopy ͑CLSM͒, 11 and the first comprehensive proteomic analysis of purified rod disks, which were obtained by combining the results of 1-D or 2-D gel electrophoresis separation of disk proteins to matrix-assisted laser desorption/ionization-time-of-flight ͑MALDI-TOF͒ or liquid chromatography electrospray ionisation tandem mass spectrometry ͑nLC-ESI-MS/MS͒ mass spectrometry techniques. Proteins involved in vision as well as in aerobic metabolism were found, among which were the five complexes of oxidative phosphorylation.…”