Optical characterization of fully programmable MEMS diffraction gratings.

We have fabricated and characterized fully programmable diffraction gratings consisting of 64 silicon micro-mirrors. The mirrors are 700µm long and 50µm wide with a fill factor of 90%. They are actuated electrostatically and move down by 1.25µm while showing negligible cross-talk and bowing as small as 0.14µm over 700µm. Extinction ratio up to 100 has been achieved by adjusting only 3 adjacent micro-mirrors. The gratings could operate either as light modulators up to 5µm or spectra generators up to 2.5µm.

Near-field optical microscopy based on microfabricated probes.

We demonstrate high resolution imaging with microfabricated, cantilevered probes, consisting of solid quartz tips on silicon levers. The tips are covered by a 60-nm thick layer of aluminium, which appears to be closed at the apex when investigated by transmission electron microscopy. An instrument specifically built for cantilever probes was used to record images of latex bead projection patterns in transmission as well as single molecule fluorescence. All images were recorded in constant height mode and show optical resolutions down to 32 nm.

Fabrication and characterization of a silicon cantilever probe with an integrated quartz-glass (fused-silica) tip for scanning near-field optical microscopy.

A cantilever-based probe is introduced for use in scanning near-field optical microscopy (SNOM) combined with scanning atomic-force microscopy (AFM). The probes consist of silicon cantilevers with integrated 25-mum-high fused-silica tips. The probes are batch fabricated by microfabrication technology. Transmission electron microscopy reveals that the transparent quartz tips are completely covered with an opaque aluminum layer before the SNOM measurement. Static and dynamic AFM imaging was performed. SNOM imaging in transmission mode of single fluorescent molecules shows an optical resolution better than 32 nm.

Microfabrication of a combined AFM-SNOM sensor

The objective of this work is to fabricate a scanning probe sensor that combines the well-established method for atomic force microscopy, employing a micro-machined Si cantilever and integrated tip, with a probe for the optical near field. A photosensitive pn-junction is integrated into the tip for that purpose and an Al coating is applied to the tip. It comprises an aperture of 50-70 nm in diameter at the apex of the tip in order to spatially limit the interaction of the tip to the optical near field of the sample. Characterization of the tip and first results of simultaneously recorded force and photon images are presented.

Towards better scanning near-field optical microscopy probes--progress and new developments.

Several approaches are described with the aim of producing near-field optical probes with improved properties. Focused ion beam milling allows the fabrication of small apertures in a controlled fashion, resulting in probes with excellent polarization properties and increased transmission. Microfabrication processes are described that allow the production of apertures of 30-50 nm, facilitating the mass-fabrication of apertured tip structures that can be used in a combined force/near-field optical microscope. Finally, possible future developments are outlined.

Hyperthermia accelerates retinal light damage in rats.

PURPOSE: To study the time course of visual cell damage resulting from hyperthermic light exposure and the possible involvement of rod outer segment (ROS) lipids in the process. METHODS: Rats were acclimated in darkness for 2 hours in a hyperthermic chamber to elevate core body temperature and then exposed to intense green light for up to 4 hours during hyperthermia. After light exposure, the animals were either sacrificed immediately for biochemical or morphologic analysis of retinal light damage or returned to darkness for up to 2 weeks at ambient temperature before analysis. Rod outer segment lipid profiles were characterized, and visual cell loss was determined by rhodopsin and visual cell DNA measurements. Morphology was performed at the light and electron microscopic level. RESULTS: Retinal damage resulting from hyperthermic light exposure was found to be temperature, time, and light intensity dependent. At an elevated environmental temperature of 34.5 degrees, 50% visual cell loss was found after 1.5 hours of 1100 lux light exposure; the same degree of visual cell loss occurred after only 1 hour when rats were maintained at 37 degrees C. At ambient temperatures, 4 hours of light exposure had no effect on visual cell loss. Irrespective of environmental temperature, when rats were maintained in darkness no visual cell loss occurred. Whereas docosahexaenoic acid (22:6) was unchanged in the purest fraction of ROS isolated immediately after light treatment, a 5 mol% loss of the polyunsaturated fatty acid was found in ROS isolated 2 or 24 hours after light exposure. Rod outer segment lipid composition was largely unaffected by hyperthermic light exposure, but the density of some ROS increased. Morphologically, the ROS appeared to be nearly normal immediately after hyperthermic light exposure and structurally more abnormal 2 and 24 hours later. The retinal pigment epithelium exhibited damage immediately after exposure, which also increased 2 and 24 hours later. CONCLUSIONS: Hyperthermia in rats dramatically accelerates retinal light damage compared with light exposure under euthermic conditions. Over loss of ROS 22:6 does not occur during hyperthermic light exposure, but it is apparent during the 24-hour period after light treatment. This suggests that the disappearance of 22:6 from ROS occurs in tandem with the process of visual cell death resulting from retinal light damage.

Electrical responses of the rat's retinal pigment epithelium to azide and thiocyanate.

In the rat under urethane anesthesia, a fast intravenous injection of a bolus of sodium azide elicited a transient cornea-positive change in transocular potential (azide response). A bolus injection of sodium thiocyanate (NaSCN) produced a cornea-negative response (SCN- response) with a similar rising phase as the azide response, but with a faster return from the peak. The peak amplitude depended on bolus volume, concentration, animal strain, and age. For more than 24 h, the azide and SCN- responses could be recorded repeatedly from a single rat with little variation in peak amplitudes. Following an administration of iodate, known to degenerate the retinal pigment epithelium (RPE), the transocular d.c. potential decreased; the azide response became smaller and then was inverted in polarity, whereas the SCN- response became larger. Azide and SCN- are assumed to depolarize and hyperpolarize the basal membrane of RPE, respectively. The equilibrium potential of ions passing through the putative azide-sensitive channels is assumed less negative than resting potential of RPE cells. The SCN- response probably represents a diffusion potential of SCN- permeating through anionic channels at a higher rate than Cl-. Results demonstrate the feasibility of in vivo electrophysiological measurement of the functional state and the structural integrity of RPE under pathological conditions.

In vivo response of the rat's retinal pigment epithelium to azide: changes induced by light damage.

Functional changes in retinal pigment epithelium (RPE) associated with light-induced retinal damage were studied by measuring transocular potential changes evoked by injections of azide and thiocyanate (SCN-). The retinal damage by light in the rat is classified into two types: Type 1, rod cell death associated with RPE deterioration; Type 2, the loss of rod cells without RPE deterioration. To study the type 1 damage, littermate pairs of long-term dark-adapted adult albino rats were tested at 1 h and 10 d after the exposure to green light of 1,200 lx for 1/2 to 24 h. Time course of the damage progress was also followed for 12 h. We found that 1) RPE was affected rapidly by the damaging light, 2) the exposure length determined the ultimate degree of RPE damage, 3) damaging effects on RPE proceeded slower and weaker after exposure than during continuous light, 4) progress of the damage in RPE was two-phasic; during the first phase, the SCN- response was enhanced and the azide response was reduced; both responses were decreased rapidly in the second phase. The first phase was assumed to indicate a depolarization of the basolateral membrane of RPE, and the second phase to manifest the structural deterioration of RPE. The type 2 damage was studied in young rats with exposure to weak light for 28 d. At 30 d after the exposure, a-wave of the ERG and number of rod cells were substantially reduced but azide and SCN- responses were affected slightly.

In vivo response of the rat's retinal pigment epithelium to azide at advanced stages of hereditary retinal dystrophy.

Electrophysiological properties of the retinal pigment epithelium (RPE) were studied in the rat with hereditary retinal dystrophy (rdy). Transocular potential changes evoked by intravenous bolus injections of azide and thiocyanate (SCN-) are the only available indication of RPE state when degeneration of rods is in progress. Also determined were age-dependent decrease in retinal DNA content and in counts of cones that survive after degeneration of rods. The azide response in the pigmented and albino rdy rat was already reduced at the earliest age tested (60 d) and continued to decrease till the age of 2 years. The SCN-response was similarly affected but seemed to decline faster than the azide response. The azide/SCN- response ratio was significantly increased in albino mutants, especially around the age of 400 d. At the age of 10 months and later, the azide and SCN- responses became slower than those of normals. A prolonged exposure of 1,200 1x light to dystrophic rats older than 110 did not affect the azide and SCN- responses whereas the same exposure abolishes the responses of normal rats and of the dystrophic rats at early stages. In rdy rats, the electrophysiological changes were considered to correlate with structural changes of the junctional RPE complex and with abnormal membrane enzyme distribution discovered by others. These RPE changes may contribute to the decreasing cone cell number after rod cell disappearance.

Rod outer segment lipids in vitamin A-adequate and -deficient rats.

Weanling albino rats were fed a vitamin-A-adequate diet or vitamin-A-deficient diet and maintained in a cyclic light or dark environment for up to 14 weeks. One half of the rats were supplemented with additional dietary linolenic acid in the form of linseed oil. The lipid composition and rhodopsin-opsin contents of isolated rod outer segments were determined after 6-7 weeks or 12-14 weeks on diet. This study shows that feeding rats a standard vitamin A-adequate or -deficient diet results in an age-dependent loss of omega three docosahexaenoic acid and a concomitant increase in omega six docosapentanoic acid in the rod outer segments. The loss of docosahexaenoate appears to be caused by insufficient dietary omega three fatty acids. The increase in omega six docosapentanoic acid appears to arise from the high concentration of linoleic acid in standard diets containing either cottonseed, or peanut oil or supplemental corn oil. Feeding rats diets supplemented with linseed oil, however, results in a rod outer-segment lipid profile which is the same as for chow-fed animals. The same effects were seen in the fatty-acid profile of lipids from liver, although the content of polyunsaturates was much lower than in rod outer segments. Vitamin A deficiency, itself, does not lead to changes in the fatty-acid composition of either the rod outer segments or liver. After 6-7 weeks on A+ or A- diet, rhodopsin levels were, as expected, higher in dark-reared rats than in cyclic-light animals. Although the rhodopsin levels in dark-reared vitamin A-adequate rats were significantly higher than in vitamin A-deficient animals, measurements of the lipid to opsin ratio of rod outer segments indicate that the rods of vitamin A-deficient rats are not markedly different than those of vitamin A-adequate rats. It is concluded that these diets may be useful in providing a means for evaluating the role of docosahexaenoic acid in visual cell death from damaging light.

Eccentricity perception in the periphery of normal observers and those with retinitis pigmentosa.

The perception of the eccentricity of a suprathreshold light flash (III/4e) presented in a Goldmann perimeter was measured in four ophthalmologically normal observers and three young observers with retinitis pigmentosa (RP). The task was to indicate the perceived distance from central fixation of each light flash by making a mark on a 180-mm line drawn on a sheet of paper. In the center of the line was a hash mark. Observers were instructed that the hash mark should correspond to fixation and the ends of the line to the limits of the visual field. The vertical, horizontal, and both 45 degree oblique meridians were tested. The results showed that: 1. The extent of the field along the tested meridian determined perceived eccentricity, i.e., eccentricity of 45 degrees in the temporal field of normals was perceived as equally eccentric as 30 degrees in the superior field. 2. The central 20 degrees were taken to represent more of the perceptual field than any other 20 degrees. 3. Along a meridian, the eccentricity which extends from fixation to 16% of the way to the field limit along the meridian is perceived as extending to 25% of the field by normal observers. 4. On the other hand, observers with RP reported that along a meridian the eccentricity extending from fixation to 8% of the field limit is perceived as extending to 25% of the field. Kinetic primary with the II/4 target showed no evidence of field constriction in observers with RP. However, losses in sensitivity are evident starting at about 10 degrees. Full field single flash electroretinograms were unrecordable in the observers with RP.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral visual field is radially organized.

The visibility of vertical, horizontal, and 45 degrees oblique square wave gratings was measured at 10 to 45 degrees of eccentricity along the vertical, horizontal, and the 45 degrees oblique meridians. A forced-choice orientation identification response to the stimulus grating and a subjective rating of the confidence in response accuracy were the response measurements. The best seen grating orientation was always the one parallel to the meridian upon which it was presented. Along the vertical and horizontal meridians the most poorly seen orientation was the one perpendicular to the meridian. Along the 45 degrees oblique meridians the horizontal grating was as poorly seen as the meridian perpendicular to the grating orientation, except in the infranasal quadrant of the field. Pinhole experiments show these results unlikely to be due to off-axis optical aberrations. It is hypothesized that the radial organization of the peripheral visual field is due to underlying neural factors ontogenically determined, not due to environmental adaptations.

Ultrastructure of remnant photoreceptors in advanced hereditary retinal degeneration.

The outer layers of the retinas of pigmented rats affected with hereditary retinal degeneration (rdy) were studied at an advanced stage in the degenerative process, ie, in 200 day old animals. At this age, most of the photoreceptors that survived the degenerative process were cones. The chromatin pattern of their nuclei clearly differentiated them from rods, displaced pigment cells and/or presumptive macrophages that also were found in the outer nuclear layer. None of the cones encountered had outer segments although structures resembling outer segment discs were found with a single cilium. Cones that had inner segments were found in regions of the retina that contained large accumulations of cellular debris. Cones that had lost both inner or outer segments, on the other hand, were found in regions that contained less debris. In such cells, the perikaryon of the cone was rich in mitochondria and other organelles; and the cilium arose directly from the cell body. The morphology of the cones and the fact that they were found in regions of the retina that contained different amounts of cellular debris suggested that cones with inner segments were in an early stage of degeneration while those that had lost inner segments were in a later stage of degeneration. All the cones encountered contained a variety of organelles including: free ribosomes, rough endoplasmic reticulum, and the Golgi apparatus. The cones that survived retinal degeneration therefore appeared to retain the cellular organelles needed for the production of photosensitive pigments. As a consequence, they may be capable of photoreceptor functions.