Achim Kolb

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.

Basic investigations for 2-dimensional time-resolved fluorescence measurements at the fundus

Fluorescence measurements are considered as a tool in the discovering of the pathomechanism of ocular diseases especially in age-related macular degeneration. The presentation of fluorescence images is a kind of 2-dimensional functional diagnosis. For an interpretation of fluorescence images, it is necessary to know, which substances are detected at different fundus sites. For that reason, it was the goal of the investigation is to find out a method, which allows both the discrimination of different fluorophores and the 2-dimensional distribution of autofluorescent substances at the fundus. The main problem in fluorescence measurements at the fundus is the very weak detectable signal, which cannot be increased because of the limitation of the excitation by the maximal permissible exposure. As the eye is a moving object, an averaging of a number of fluorescence images to improve the signal to noise ratio is possible up to a certain degree, but it requires additional expenditures. A further limitation for fluorescence measurements is given by the spectral transmission of the ocular medida, which allows spectral investigations only in a wavelength-range between about 400 nm and 900 nm.

Zeitaufgelöste Messung der Autofluoreszenz

ZusammenfassungErkannte Veränderungen im Stoffwechsel eröffnen Möglichkeiten zur Intervention bei noch reversiblen pathologischen Veränderungen. Messungen der Sauerstoffsättigung sind auf das Gefäßsystem beschränkt. Messungen der Autofluoreszenz von Koenzymen gestatten es, Änderungen der Konzentration von Sauerstoff um 3 Größenordnungen empfindlicher als durch die Detektion der Sauerstoffsättigung nachzuweisen. Da infolge der begrenzten Transmission der Okularmedien keine spezifische Anregung innerer Fluorophore erreicht werden kann, wurde untersucht, ob eine Messung der substanzspezifischen Fluoreszenzlebensdauer nach Pulsanregung am lebenden Auge möglich ist. Unter Verwendung eines Laser-Scanner-Ophthalmoskopes und eines aktiv modengekoppelten Lasers gelang es, mittels des zeitkorrelierten Einzelphotonenzählens Bilder der Fluoreszenzlebensdauer vom lebenden Augenhintergrund zu erhalten. Bei monoexponentieller Approximation wird in der Papille eine mittlere Fluoreszenzlebensdauer von ca. 5 ns und im parapapillären Gebiet von ca. 1,5 ns gemessen. Aus dem Histogramm der Lebensdauern folgt, dass wahrscheinlich Lipofuszin, freies Flavin und Collagen am gesunden Auge nachweisbar sind. Es wurden Vergleichsmessungen am Funduspräparat und an gelöstem Flavinadenindinucleotid ausgeführt.AbstractThe detection of metabolic changes opens the possibility for intervention of reversible pathological alterations. Measurements of oxygen saturation are limited to the blood vessel system. Detection of alterations in oxygen concentrations are up to 3 orders of magnitude more sensitive by autofluorescence of coenzymes than by measurement of oxygen saturation. Because of limited transmission of the ocular media no specific excitation of endogenous fluorophores can be realised. For this reason it was investigated if the fluorescence lifetime after pulse excitation can be detected at the human fundus. Applying a laser scanner ophthalmoscope and mode-locked Ar+ laser as well as time-correlated single photon counting, lifetime images of the living fundus were obtained. In mono-exponential approximation, a mean lifetime of 5 ns was detected from the optic disc and large vessels whereas about 1.5 ns were detected in the parapapillary area. By evaluating the frequency of lifetimes, lipofuscin, free FAD, and collagen are probably detectable. Comparative measurements were performed in fundus specimens and on free FAD.

Optical properties of ocular fundus tissues determined by optical coherence tomography

Abstract Goal: To determine the extinction coefficient and the anisotropy of scattering as well as the refractive indices in the retina and in the choroid non-invasively in vivo. Method: The power of coherent reflected light versus fundus depth is recorded by optical coherence tomography (OCT). The ratio of the refractive indices is derived from the height of the reflection peaks. The extinction coefficient and anisotropy of scattering are calculated from the offset and the slope of the signal behind the reflection peaks. Results: Values similar to those known from in vitro measurements were found for the extinction coefficient and scattering anisotropy of the retina and the choroid. Conclusions: The OCT, usually employed for the measurement of intraocular distances and tomographic imaging, is capable to determine further interesting parameters of single ocular layers.

Autofluorescence lifetime measurements in images of the human ocular fundus

Measurements of the autofluorescence at the fundus prove to be an important tool in early diagnosis and in discovering the pathomechanism, e.g., in age-related macular degeneration. In addition to the action of lipofuscin in the aging process, flavines play an important role as prosthetic groups. As metabolic changes occur at cellular level, patient-specific optimized therapy should be possible according to endogenous fluorophores, before morphological alterations are manifest. As a first tool for the detection of dynamic autofluorescence, a laser scanner opthalmoscope will be presented permitting lifetime measurements at the living human eye-ground under extremely weak detectable light. Considering histograms of lifetimes after excitation at 457.8 nm and determined at the living human eye ground in parapapillary region, a lifetime (rho) approximately equal to 1.38nm was calculated most frequently in the long-wave emission range ((lambda)

Scattering properties of the retina and the choroids determined from OCT-A-scans

GTR550 nm). This points to the main contribution of lipfuscin. If the emission range is extended down to 515 nm, components with longer lifetimes are additionally detectable. Lifetime measurements at a human fundus specimen confirmed the lifetime of 1.38nm in lipfuscin-rich pigment epithelium, whereas the mean lifetime of an intact fundus was 2.04ns. A comparison of lifetimes before, during, and after breathing 100% oxygen results in a quenching of the mean lifetime of 0.15ns by oxygen.

Non-invasive measurement of the concentration of melanin, xanthophyll, and hemoglobin in single fundus layers in vivo by fundus reflectometry

Goal: To determine the coefficient and the anisotropy of scattering as well as the refractive indices in the retina and in the choroid noninvasively in vivo.Methods: The power of coherent reflected light versus fundus depth is recorded in OCT-A-scans. The ratio of refractive indices is derived from the height of the reflection peaks. Providedthat the absorption coefficient is known from fundus reflectometry, the scattering coefficient and anisotropy are calculated from the offset and the slope of the signal behind the reflection peaks on the basis of a single backscattering model.Results: We found scatteringcoefficients of 12/mm (retina) and 27.5/mm (choroid) as well as anisotropy values of 0.97 (retina) and 0.90 (choroid).Discussion: The OCT is usually employed for the measurement of intra-ocular distances. The new technique described here gives the unique opportunity to determine further interesting parameters of single ocular layers. The values given above are in good agreement with in vitro results.

Investigations on the measurement of oxygen saturation in retinal vessels using polarized light

A new model of the reflection of the human ocular fundus on the basis of the adding-doubling method, an approximate solution of the radiative transport equation, is described. This model enables the calculation of the concentration of xanthophyll in the retina, of melanin in theretinal pigment epithelium and in the choroid, and of hemoglobin in the choroid from fundus reflectance spectra. The concentration values found in 12 healthy subjects are in excellent agreement with literature data. In single cases of pathologic fundus alterations possiblebenefits to the ophthalmologic diagnostics is demonstrated.

Verfahren und Anordnung zur Bestimmung von Fluorophoren an Objekten, insbesondere am lebenden Augenhintergrund

SummaryThe accuracy of the spectrometric measurement of the oxygen saturation in retinal vessels is limited by its signal-to-noise ratio. The aim of this study was to investigate the possibility of enhancement of the reflection signal by the use of polarized light. Materials and method: The Jena ophthalmospectrometer was equipped with two polarizing filters: one in the illumination and the other in front of the detector. Reflection spectra of erythrocytes streaming through a cuvette in the focus of an artificial eye were recorded. The influence of the polarization on the reflection spectra was investigated by rotating the polarizer in front of the detector. Furthermore, the degree of polarization of the light reflected from retinal vessels in vivo was determined. Results: The degree of polarization of the light reflected from the erythrocytes was 0.6–0.8, whereas the polarization of light reflected by a standard white reflectance target was virtually zero. Conclusion: Polarized light can be used for the reduction of error in retinal vessel oximetry.ZusammenfassungHintergrund: Die Genauigkeit der spektrometrischen Messung der Sauerstoffsättigung in retinalen Gefäßen ist begrenzt durch das Signal-Rausch-Verhältnis. Eine Erhöhung der Reflexion durch die Verwendung polarisierten Lichtes könnte die Genauigkeit verbessern. Material und Methode: In die Beleuchtungs- und in die Beobachtungsoptik des Jenaer Ophthalmospektrometers wurde je ein Polarisationsfilter eingefügt. So wurde eine mit einer Erythrozytensuspension durchströmte Küvette im Fokus eines Modellauges mit linear polarisiertem Licht beleuchtet. Die Reflexionsspektren der Erythrozyten wurden zweimal aufgenommen. Dabei waren die Polarisationsrichtungen der beiden Filter bei der ersten Messung parallel zueinander ausgerichtet, bei der zweiten Messung senkrecht. Ferner wurde der Polarisationsgrad des von retinalen Gefäßen in vivo reflektierten Lichtes bestimmt. Ergebnisse: Das von Erythrozyten reflektierte Licht weist einen Polarisationsgrad von 0,6–0,8 auf. Die Polarisation des von einem als Referenz verwendeten Weißstandard reflektierten Lichtes ist dagegen auch bei linear polarisierter Beleuchtung isotrop. Schlußfolgerung: Die Verwendung polarisierten Lichtes kann zu einer sichereren Messung der Sauerstoffsättigung in retinalen Gefäßen führen.