Photoreceptor Material Research Articles

Ultrastructural Localization and Expression of TRPM1 in the Human Retina

Transient receptor potential subfamily melastatin (TRPM)1 cation channels of retinal ON-bipolar cells are modulated via a mGluR6 (GMR6) signaling cascade. While light-microscopy shows these channels are located on the tips of ON-bipolar cells dendrites, near rod and cone synaptic ribbons, TRPM1 localization at the electron-microscope level is currently not described. The authors report here the ultrastructural localization of TRPM1 in the human retina. TRPM1 was localized in postmortem human retinas by immunohistochemistry at both the light and electron microscope levels. Additionally, TRPM1 expression was studied using in situ hybridization, laser dissection microscopy, and PCR techniques. TRPM1-immunoreactivity was located on the dendrites and soma of ON-bipolar cells at the light microscope level. At the electron microscope level TRPM1-immunoreactivity was located on the tips of ON-bipolar cell dendrites that were invaginating cone pedicles and rod spherules. In addition, TRPM1-immunoreactivity was occasionally found on the rod spherules ribbons, suggesting that at least a proportion of rods may also express TRPM1. In situ hybridization showed TRPM1 encoding RNA in inner nuclear layer somata and in some photoreceptors. The presence of TRPM1-RNA in photoreceptors was confirmed by PCR in pure photoreceptor material obtained with a laser dissection microscope. In the human retina TRPM1 is expressed on ON-bipolar cell dendrites that invaginate photoreceptor terminals. TRPM1 is also expressed on the synaptic ribbons of a subclass of rods, suggesting a dual function for TRPM1 in the ON-pathway.

IPS cells ‐ an alternative source of RPE?

AbstractPurpose We have previously shown that human embryonic stem cells spontaneously differentiate into RPE, and, after transplantation into the dystrophic RCS rat, maintain visual acuity. Recent advances in the generation of stem cells from somatic cells have led us to investigate the use of induced pluripotent stem cells (iPS) as a potential source of therapeutic RPE.Methods iPS cells spontaneously differentiate after removal of bFGF from the culture medium. Isolated pigmented colonies were dissociated for expansion. The enriched population was then characterised morphologically and at the gene and protein expression level. Functionality of iPS‐derived RPE cells was assessed by RPE phagocytosis of retinal debris in vitro and in vivo. The therapeutic effect of iPS‐derived RPE was examined by injection of cells into the subretinal space of the dystrophic RCS rat.Results In culture iPS‐RPE were morphologically similar to, and expressed many markers of developing and mature RPE. These cells also phagocytose photoreceptor material, in vitro and in vivo following transplantation into the RCS dystrophic rat. Preservation of the neural retina layers and maintenance of visual acuity was observed 13 weeks following transplantation, however at this stage very few human cells remained and a number of large pigmented macrophages were observed in the subretinal space.Conclusion iPS cells can readily differentiate into functional RPE which facilitate the short‐term maintenance of photoreceptors. However, long‐term maintenance of the dystrophic RCS rat neural retina is likely to be due to a protective host response initiated against the xenografted cells. The use of iPS‐RPE as a cell therapy requires further investigation, however these cells provide a new model system in which to study RPE specific diseases in vitro.

Why are retinal pigment epithelial cells so resistant to oxidative stress?

Abstract Purpose The functional integrity of postmitotic retinal pigment epithelial (RPE) cells is crucial for photoreceptor survival, and RPE malfunction is considered an important factor behind age‐related macular degeneration (AMD). Although RPE cells daily phagocytose large amounts of fatty acid‐rich distal photoreceptor material, their lysosomal accumulation of lipofuscin (LF) is nevertheless slow. Only late in life do humans have LF‐loaded RPE cells, which then not only have a reduced capacity to endocytose photoreceptor material but also are less capable of keeping up with normal autophagy. The latter activity is important for normal turnover of the cell’s own components. Methods ARPE‐19 cells in culture were evaluated with respect to lysosomal stability at oxidative stress. Redox‐active iron, autophagic activity (evaluation of LC3‐I and LC3‐II protein), and anti‐oxidative proteins were assayed using cytochemistry (Sulphide‐Silver Method), electron spin resonance (ESR) and western blotting for a variety of stress proteins. Results The lysosomal compartment of the ARPE‐19 cells was found remarkably resistant to oxidative stress. The SSM and ESR methods suggested low lysosomal amounts of redox‐active iron and no enhanced autophagy following oxidative stress. The normal autophagy rate was higher than in other professional scavengers. Conclusion The found low amount of lysosomal redox‐active iron explains the slow formation of lipofuscin over time and the remarkable stability that ARPE‐19 cells and their lysosomes show even under severe oxidative stress. The molecular mechanisms behind this phenomenon may be an unusual capacity to quickly export low mass iron from lysosomes to the cytosol and/or a high normal degree of autophagy of iron‐binding stress proteins.

Time of flight transients analysis in photo-excited molecular doped polymers

Charge transport in molecular doped polymers (MDP) is analyzed by a new formalism taking into account spatial dispersion effects also. Charge packets are injected in thin films of photoreceptor materials by pulsed UV photo-excitation to perform time of flight (TOF) experiments. The transient currents are related in this work to thermal and electric field excitation mechanisms using new dispersion parameters. The parameters used for describing the charge spatial dispersion are the velocity distribution within the moving charge packet and the trapping time. The formalism uses the classical electrodynamic equations with proper limiting conditions. This work analyzes the experimental transients using the equation of current displacement, and a simple deep trapping model. The results are different from those obtained for semiconductors.

Growth of phthalocyanine doped anthracene crystal using a three-zone furnace by vacuum sublimation method

Anthracene is an organic semiconductor extensively investigated over many decades. It finds applications as photoreceptor material, scintillation counter etc. Anthracene is optically active in the UV region of the electromagnetic spectrum whereas H2Pc is active from visible through IR regions. Hence phthalocyanine (H2Pc) doped anthracene crystal formed is expected to be active in the entire region UV–VIS–IR, and the compound material could be a viable alternative for a possible solar cell material. In the present study doped crystal was grown by two methods namely co-crystallization and vacuum sublimation using a special type three-zone furnace. The co-crystals of anthracene and H2Pc were formed using dimethyl sulphoxide as the common solvent, and were subjected to various analysis including electroreflectance method. The electroreflectance method gives the band gap accurately and the calculations show that there is a decrease in the band gap as the concentration of H2Pc increases. Also the doped samples were found to be photo active and hence H2Pc could be used as a suitable dopant to increase the conductivity of anthracene. A special type three-zone furnace is designed to grow H2Pc doped anthracene single crystal. The melting point of anthracene is 217°C while H2Pc decomposes above 550°C, but sublimates around 460°C in vacuum. Anthracene and H2Pc were placed in two side arms of a main stem and the tube is sealed in vacuum. The sealed tube is heated for few days to enhance the growth of a single crystal. The compound crystal is formed along with the main stem at a convenient temperature gradient.

Study on the optoelectronic properties of amorphous selenium-based photoreceptors

Due to its superior photosensitivity in the visible wavelength region, amorphous selenium-based material is widely used as the basic photoreceptor material in xerography. The optoelectronic properties of the samples were measured and calculated with photoinduced discharge curve (PIDC) by using Electrostatic Paper Analyzer, and the initial surface potential (V so), dark decay time (t d), photosensitivity (E 1/2) and residual potential (V r) could be obtained. It is found that the contrast voltage which affects directly the resolution of image can be improved by the blocking layer between the bulk (such as a-Se, a-As2Se3) and substrate (Al). Furthermore, the relations between thickness and optoelectronic properties of photoreceptor could be identified, and compared with different blocking layers and isothermal annealing effect for the photoreceptors.

Morphology and excited state dynamics in thin organic molecular films probed by femtosecond time-resolved photoemission spectroscopy

We have studied the lifetimes of the excited electron states of thin N,N′bis(phenethyl)perylene-3,4:9,10-bis(dicarboximide) (DPEP) films, prepared in situ, using femtosecond time-resolved photoemission spectroscopy. DPEP is an organic compound similar to photoreceptor materials widely used in many imaging applications. By controlling the evaporation conditions, we have been able to grow films of different morphologies, and found that the relaxation dynamics depends on the morphology. We have investigated two distinct films characterized by very different absorption spectra. We have found that for the film with absorption maximum at 500 nm, a typical lifetime is 55 fs at 2.1 eV above the highest occupied molecular orbital level. For the other film with absorption maximum at 630 nm, the relaxation rate is almost twice as fast, resulting in a lifetime of 30 fs at the same energy. We attribute the extremely short lifetimes to a rapid internal conversion process from S2 and S1 levels to the vibrational manifolds of S1 and S0 states and the vibrational relaxation of the latter. The dependence of the lifetimes on the morphology can be explained by the formation of extended states in the better-ordered DPEP film and by fast transitions within these states.

Phthalocyanine-C 60 composites as improved photoreceptor materials?

-Pc, in contact with C60 is studied using photoelectron spectroscopy with ultraviolet and X-ray radiation (UPS and XPS, respectively), X-ray absorption near edge spectroscopy (XANES), and optical transmission spectroscopy. A possible improvement of the charge-carrier generation efficiency, which is essential for the performance as photoreceptor material, is thereby found for these materials upon doping with C60. No ground-state charge transfer is detected for the Pcs in contact with C60. The effect of an enhanced photoconductivity is demonstrated for τ-H2-Pc when it is doped by 5% C60.

Charge decay in amorphous selenium layers with an intermediate aluminum oxide film

Amorphous selenium is often used as the basic photoreceptor material in xerography in the form of a layer on an aluminum substrate. It is found that the dark decay rate of charge on the selenium surface can be reduced by the presence of a thin aluminum oxide film between the selenium and the substrate metal. In this study, different methods were explored for forming this film, including direct thermal oxidation and various glow-discharge methods. It was found that with thermal oxidation and low voltage glow discharge, the film thicknesses were limited to about 200 Å (1 Å = 10−10 m). However, with dc reactive sputtering, aluminum oxide films with thicknesses of more than 2000 Å could be prepared. Evaluation of the films, with the selenium layer present, indicated that the low voltage glow-discharge and direct thermal oxidation methods gave the best results xerographically.

Kinematical transformations in amorphous selenium alloys used in xerography

Differential scanning calorimetric (DSC) studies were carried out on xerographic photoreceptor materials of amorphous selenium (a-Se), a-Se1&#x2203-xTex and a-Se:0.5 wt% As alloys with various amounts of chlorine doping. Glass transformation kinetics of bulk and vapour-deposited film samples were essentially identical indicating a similar amorphous structure for both forms. The glass transition temperature,Tg, in the a-Se1&#x2203-xTexsystem was found to increase monotonically with the tellurium content up to ∼13 wt% which was accompanied by a fall in the activation energy,Egl, for the structural relaxation. 0.5wt% As increasedTg, whereas chlorine doping in the amounts used (∼100 ppm) had no effect onTg. Crystallization exotherms were analysed using the Kissinger method which was shown to remain a valid analysis by integrating the fundamental Kolmogoroff equation describing crystallization transformations by nucleation and growth. The signifiance of the activation energy,EA, and the frequency factor,C0, in the Kissinger analysis is discussed and used in interpreting the different crystallization behaviour exhibited by the various alloys. The apparent activation energy,EA, of the crystallization kinetics was found to fall with the temperature range accessed in the DSC experiment. When other authors' previous crystallization studies by other methods were also considered, then 1/EA was found to fall nearly linearly with the mean reciprocal temperature, 〈1/T〉, used in the experiment. It is argued that this behaviour arises if the crystal growth rate scales with the inverse viscosity of the melt and the latter is described by a Vogel-Tammann-Fulcher type of behaviour. The additives tellurium and arsenic retard the crystallization essentially by increasing the viscosity. Chlorine doping was also found to have an inhibitory effect but due probably to a change in the nucleation kinetics, rather than a viscosity rise.

Light regulation of adenosine 3′, 5′ cyclic monophosphate levels in vertebrate photoreceptors

The regulation of adenylyl cyclase activity (measured as the production of labeled cyclic-AMP (cAMP) from labeled ATP) in frog rod outer segment photoreceptor membranes has been studied. The activity was found to be more than 20 times higher in fully dark-adapted than in bleached material. A number of experimental parameters must be carefully regulated to obtain this large ratio of dark to light activities. The disrupted rod outer segments must not be washed, since a soluble factor is essential for inhibition of activity by light. The photoreceptor membranes should be assayed for adenylyl cyclase immediately after preparation since the specific activity and light-dark differences are substantially reduced on storage at 4°C. Mechanical trauma (homogenization or sonication) causes some decrease in dark activity and a lessening of sensitivity to illumination. Preparation and assay of the dark material must be carried out using infrared light since there is a marked disproportionality between photic bleaching of rhodopsin and loss of cyclase activity. This disproportionality was further studied by mixing dark and totally bleached material in various proportions. With as little as 2% of the material bleached the cyclase activity is the same as in fully bleached material. The inhibitory action of the bleached material is destroyed by heating but is not affected by dialysis or treatment with tryspin. If the photoreceptor material is fractionated by sedimentation at 60 000 × g and 200 000 × g, the rhodopsin and adenylyl cyclase are primarily in the 60 000 × g pellet. The inhibitor is present in both pellets but not in the high speed supernatant. However, the high speed supernatant is essential for action of the inhibitor on unbleached material. Bleached (but not dark) photoreceptor material was found to inhibit cyclase in brain, liver and melanoma. Recent work carried out after presentation of the above findings has indicated that although the activity of cAMP phosphodiesterase is not affected by light under the usual assay conditions, in the presence of ATP light produces a marked stimulation of this activity. This stimulation of phosphodiesterase exhibits many of the characteristics previously attributed to the inhibition of cyclase and accounts completely or in large part for the apparent light dark differences in cyclase activity. At present the evidence points to phosphodiesterase as the main locus of light regulation of cAMP concentrations in photoreceptors, though a role for the light regulation of cyclase activity is not excluded.