Spectroscopic measurement of light that is reflected from biological tissue in vivo is being investigated for various clinical applications. One special object of investigation using optical methods is the human ocular fundus. A fundus reflectometer that enables the simultaneous acquisition of up to 192 spectra arranged in a horizontal line across the fundus is described. The underlying optical principle of the device is the confocal imaging of an illuminated narrow, slitlike field at the fundus to the entrance slit of a spectrograph. This is imaged by the grating of the spectrograph onto a two-dimensional CCD chip that records the local distribution of ocular fundus reflectance spectra within a wavelength range of 400 up to 710 nm with a resolution better than 2 nm and a local resolution of 23 mm in a field dimension of 1.5 mm. The performance of the device was investigated, the effects of confocal and nonconfocal imaging are discussed, and some representative measurements are presented.
Multispectral images can provide useful information for objective diagnosis, control of the effect of therapy and for a patient-specific optimization of therapy regime in ophthalmology. Laser scanner systems have the advantage of a high radiation power also in case of small spectral bandwidth. Additionally, the flying spot principle reduces the irradiation of the patient. Commercial laser scanner ophthalmoscopes (LSO) are developed till now only for qualitative, visual interpretation. Maximal four fixed wavelengths are available with a stabilized radiation power. Using the spectral properties of fundus pigments like xanthophyll, rhodopsin or of pathological alterations, e.g. hard exudates, its optical density or local distribution can be determined in this way before and after therapy. As also three wavelengths can be chosen which are best suited for determination of oxygen saturation (OS) in the blood, the validity of the 3-(lambda) -method for 2D calculation of OS can be tested. These investigations are first steps in functional diagnosis of the metabolism in the human ocular fundus.
An imaging spectrometer, developed at the University of Jena, enables locally resolved spectral measurements at the fundus of the human eye. From the measured profile of a blood vessel the value of vessel diameter may be detected and the weakening of intensity is a result of wavelength- dependent absorption and scattering in blood. Although extinction spectra of hemolyzed oxygenated and reduced hemoglobin are known very exactly, in vivo measurements of oxygen saturation at streaming whole blood in single vessels are difficult. The extension of Lambert-Beer's law by a scattering part is used as an attempt for the calculation of the oxygen saturation from the locally resolved reflectance spectra of the fundus. Measurements of vessel diameter according to the principle of optical centroid are insensitive to variations of real illumination. This principle results in exact values if there are at least three observations in each edge of vessel profile. Practical measurements of oxygen saturation over vessel cross section and of vessel diameter are given.
Ocular fundus reflectometry is known as a method for the determination of the optical density of pigments at the eye ground. This has been described for diagnostic investigations at single locations. The new technique of imaging spectroscopy enables the recording of one dimensional local distribution of spectra from the fundus which is illuminated confocal to the entrance slit of a spectrograph. A fundus reflectometer consisting of a Zeiss fundus camera, an imaging spectrograph, and an intensified CCD-camera are presented. The local resolved spectra gained by this apparatus are approximated by a mathematical model on the basis of the anatomy of the fundus as a structure of layers with different optical properties. Each spectrum is assumed to be described by a function of the absorption spectra of the pigments found in the retinal and choroidal tissue. Assuming the existence of parameters which are independent from the fundus location we have to approximate the measured local distribution of spectra by a system of coupled non-linear equations. By a least square fit the local distribution of the extinction of melanin, xantophyll and hemoglobin may be obtained as well as the extension of pathologic alterations at the fundus. The benefits of the method for clinical diagnostics are discussed at first measurements from physiological and pathological examples.
The detection of alterations in the microcirculation requires both the measurement of the blood-flow and the measurement of the oxygen saturation in whole blood. The basis for the non-invasive estimation of the oxygen saturation is the difference between extinction-spectra of hemoglobin and of oxyhemoglobin. Further in whole blood the scattering at the erythrocytes has to be taken into account. In principle spectral measurements at three neighboring wavelengths are sufficient for the calculation of the oxygen saturation, the concentration- thickness-geometry product and the scattering intensity. Caused by the maximal permissible exposure, the signal/noise ratio is very low in fundus reflectometry. But if the wavelength- range from 520 nm to 620 nm is evaluated, the requirement at the signal/noise ratio is reduced. This reduction corresponds to the square root of the number of discrete wavelengths at which the ocular fundus reflectance is measured. So the oxygen saturation can be calculated with an error lower than +/- 4%. For this purpose the extinction spectrum of whole blood is approximated by a model, including besides the above mentioned unknowns the spectral dependency of the scattering. The experimental arrangement for the measurement of the oxygen saturation is an imaging ophthalmo-spectrometer which allows reflectance measurements with a good spectral (< 3 nm) and local (> 3 micrometers ) resolution simultaneously at a vessel and in its neighborhood. The extinction of blood is calculated as the logarithm of the ratio of the reflectance of the neighborhood and of the vessel. In this calculation the influences of the ocular media, of the background and of eye movements are eliminated. The sensitivity of the detector system has to be very high in order to detect the light which is reflected at a dark background and travels through the blood. The new method was tested by a comparison the oxygen saturation of the blood in an arteriole and a venule in the brain of a piglet measured by reflectometry with the oxygen saturation of the left ventricular and the venous blood measured by a laboratory hemoximeter. First results of the measurement of the oxygen saturation in human retinal vessels are demonstrated.
A double beam spectrometer, which works in single photon counting technique, was developed for measuring the reflectance spectra at selected fundus sites in a wavelength range from 430 to 700 nm. By forming logarithmic difference spectra between the reflectance of the normal fundus and the fundus reflectance in different stages of glaucoma only substance or structure specific glaucomatous alterations appear. The approximation of logarithmic difference spectra is demonstrated by a linear model. The primary information in the modelling is only the relative spectral course of important fundus substances like oxyhemoglobin, melanin and xanthophyll. The influence of scattering is formulated as a wavelength-independent intensity, multiplied by a term, containing a power of the wavelength. Found by the spectra-deconvolution, a lack of oxyhemoglobin in the papillo-macular bundle is the first sign of a damaged microcirculation in case of relative losses of the visual field function. The reduced scattering intensity and the altered scattering exponent point to a thinning of the nerve fiber layer.