Dietrich Schweitzer

Optical properties of ocular fundus tissues-an in vitro study using the double-integrating-sphere technique and inverse Monte Carlo simulation

Various models have been published calculating the light transport at the ocular fundus either for interpretation of in vivo reflectance measurements or for the prediction of photocoagulation effects. All these models took the absorption spectra of the pigments located at the ocular fundus, melanin, haemoglobin, xanthophyll, and the photoreceptor pigments, into account. However, light scattering inside the single fundus layers has not been investigated in detail and was, therefore, neglected in the calculations or only considered by very rough approximations. This paper presents measurements on specimens of retina, retinal pigment epithelium, choroid, and sclera using the double-integrating-sphere technique. Absorption coefficients, scattering coefficients, and anisotropy of scattering were calculated by an inverse Monte Carlo simulation from the measured collimated and diffuse transmittance and diffuse reflectance. Conclusions are drawn for the interpretation of fundus reflectance measurements, which are a useful tool in diagnostics and photocoagulation dosimetry.

Single scattering by red blood cells

A highly diluted suspension of red blood cells (hematocrit 0.01) was illuminated with an Ar or a dye laser in the wavelength range of 458-660 nm. The extinction and the angle-resolved intensity of scattered light were measured and compared with the predictions of Mie theory, the Rayleigh-Gans approximation, and the anomalous diffraction approximation. Furthermore, empirical phase functions were fitted to the measurements. The measurements were in satisfactory agreement with the predictions of Mie theory. However, better agreement was found with the anomalous diffraction model. In the Rayleigh-Gans approximation, only small-angle scattering is described appropriately. The scattering phase function of erythrocytes may be represented by the Gegenbauer kernel phase function.

In vivo measurement of the oxygen saturation of retinal vessels in healthy volunteers

A new method for the spatially resolved measurement of the oxygen saturation of retinal vessels is described. Imaging spectrometry was used for both measurements of transmission and reflectance spectra of whole blood in cuvettes as well as for fundus reflectance spectra. A model was developed for the calculation of the oxygen saturation, valid in the wavelength range between 510 nm and 586 nm, in that the internal reflectance is constant and only the transmitted light depends on layer thickness and hematocrit. Altogether 265 measurements were performed in different number at 30 eyes. In each measurement, the oxygen saturation was simultaneously determined for 193 locations along a line of 1.5 mm at the fundus. The mean oxygen saturation in retinal arteries was (92.2/spl plusmn/4.1)% and (57.9/spl plusmn/9.9)% in retinal veins. The mean retinal arterio-venous difference of the oxygen saturation was (35.1/spl plusmn/9.5)%. The venous oxygen saturation depended on distance from the optic disc. The measured mean of the arterio-venous difference of the oxygen saturation corresponded well to the value of the brain (34%). The utilization of oxygen in the temporal quadrants (inferior: 39.4/spl plusmn/10.4%) is significantly (p=0.05) higher than in the nasal quadrants (inferior: 31.3/spl plusmn/6.7%).

Retinal vessel oximetry-calibration, compensation for vessel diameter and fundus pigmentation, and reproducibility

The purpose of this study was to measure the hemoglobin oxygenation in retinal vessels and to evaluate the sensitivity and reproducibility of the measurement. Using a fundus camera equipped with a special dual wavelength transmission filter and a color charge-coupled device camera, two monochromatic fundus images at 548 and 610 nm were recorded simultaneously. The optical densities of retinal vessels for both wavelengths and their ratio, which is known to be proportional to the oxygen saturation, were calculated. From 50-deg images, the used semiautomatic vessel recognition and tracking algorithm recognized and measured vessels of 100 microm or more in diameter. On average, arterial and venous oxygen saturations were measured at 98+/-10.1% and 65+/-11.7%, respectively. For measurements in the same vessel segments from the five images per subject, standard deviations of 2.52% and 3.25% oxygen saturation were found in arteries and veins, respectively. Respiration of 100% oxygen increased the mean arterial and venous oxygen saturation by 2% and 7% respectively. A simple system for noninvasive optical oximetry, consisting of a special filter in a fundus camera and software, was introduced. It is able to measure the oxygen saturation in retinal branch vessels with reproducibility and sensitivity suitable for clinical investigations.

In vivo measurement of time-resolved autofluorescence at the human fundus

An experimental setup for measurement of time-resolved autofluorescence of the human eye fundus is demonstrated. The method combines laser scanning technique and time-correlated single photon counting. The light source is a laser diode, delivering pulses of about 100 ps duration at a repetition rate of 40 MHz. The excitation wavelength is 446 nm and the cutoff wavelength of fluorescence detection is at 475 nm. The autofluorescence can be determined with a spatial resolution of 80 x 80 microm2 and 25 ps time resolution. The fluorescence decay is optimally approximated by a biexponential model. The dominating lifetime tau1 is shortest in the macula (320 to 380 ps) and reaches 1500 ps in the optic disk. The lifetime tau2 varies between 2 ns and 5 ns, but the spatial distribution is more homogeneous. Respiration of 100% oxygen for 6 min leads to changes in the fluorescence lifetime pointing to detection of coenzymes. Diagrams of lifetime tau2 versus tau1 are well suited for comparison of substances. Such lifetime clusters of a 20 deg macular field of a young healthy subject and of a patient suffering from dry age-related macular degeneration overlap only partially with tau2-tau1 clusters of lipofuscin.

A scattering phase function for blood with physiological haematocrit

Though the optics of red blood cells as well as whole blood has been studied extensively, an effective scattering phase function for whole blood is still needed. The interference of waves scattered by neighbouring cells cannot be neglected in highly concentrated suspensions such as whole blood. As a result, the phase function valid for single erythrocytes may fail to describe a single scattering process in whole blood with physiological haematocrit (Hct approximately 0.4). In this study we compared the results obtained in goniophotometric measurements of blood samples with the results of angle-resolved Monte Carlo simulations. The results show that a Henyey-Greenstein phase function with an anisotropy factor of 0.972 is an adequate approximation for the effective scattering phase function of whole blood with high haematocrit at a wavelength of 514 nm.

Light paths in retinal vessel oximetry

The oxygen utilization and, therefore, the metabolic state, of a distinctive area of the retina may be calculated from the diameter of the supplying artery and vein, the haemoglobin oxygenation, and the velocity of the blood. The first two parameters can be determined by imaging spectrometry at the patients ocular fundus. However the reflected light emerging from a vessel followed different pathways through the ocular fundus layers and the vessel embedded in the retina. The contribution of the single pathways to the vessel reflection profile is investigated by a Monte Carlo simulation. Considering retinal vessels with diameters of 25-200 /spl mu/m we found the reflection from a thin vessel to be determined by the single and double transmission of light at 560 nm. The backscattering from the blood column determines the reflectance in the case of a thick vessel. However, both components are in the same order of magnitude. This has to be considered in the calculation of the oxygen saturation of blood in retinal vessels from their reflection spectra.

Retinal venous oxygen saturation increases by flicker light stimulation.

PURPOSE Luminance flicker stimulation of the photoreceptors is known to increase retinal blood flow. Elevated blood velocity was determined using laser Doppler velocimetry, and increased vascular diameters during flicker were observed by measurements with a retinal vessel analyzer. Oxygen supply may be the target of the regulation of retinal blood flow. Thus, the oxygen saturation (SO(2)) in retinal arterioles and venules was investigated along with their diameters. METHODS Dual-wavelength (548 nm and 610 nm) fundus images were taken in 19 healthy volunteers (mean age, 26 ± 2.5 years) before (baseline) and during luminance flicker stimulation (12.5 Hz; modulation depth, 1:25). Retinal vessel SO(2) (dual-wavelength optical oximetry) and diameters (central retinal arterial and venous equivalents [CRAE and CRVE]) were determined. RESULTS CRAEs and CRVEs of 193 ± 20 μm and 228 ± 20 μm at baseline increased statistically significant to a maximum of 202 ± 19 μm (P < 0.0005) and 242 ± 17 μm (P < 0.0005), respectively, under flicker stimulation. Although the arterial SO(2) remained unchanged at 98%-99%, an increase of the venous saturation from 60% ± 5.7% to 64% ± 5.9% (P < 0.0005) was found. CONCLUSIONS In agreement with earlier investigations, the vessel dilation found here indicates an elevation of retinal blood flow by luminance flicker stimulation. This increase of the flow should meet the enhanced metabolic need of the neural retina under a physiological stimulus. The augmentation of venous oxygenation may indicate a higher capillary oxygen concentration, necessary to provide a sufficient diffusion rate of oxygen from the capillaries to the inner retinal tissue.

A new method for the measurement of oxygen saturation at the human ocular fundus.

The investigation of changes in the metabolism as a functional diagnosis is a promising way for the detection of early reversible pathological alterations before they get manifest. Such a functional parameter of the microcirculation is the oxygen saturation of blood. The oxygen saturation in an artery describes in the connection with the blood flow the supply of oxygen in a certain region of the fundus. The consumption of oxygen is proportional to the product of the arterio-venous difference in the oxygen saturation and the blood flow.

Time-resolved autofluorescence imaging of human donor retina tissue from donors with significant extramacular drusen.

PURPOSE Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen. METHODS Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted. RESULTS RPE can be discriminated clearly from Bruchs membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected. CONCLUSIONS The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.