The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included in the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τi, amplitudes αi, and relative contributions Qi were statistically compared between corresponding groups in two spectral channels (490450 ps, and the shift of τ3 from ∼3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine dinucleotide at the fundus. AGE also accumulated in the crystalline lens.
Measurements of time-resolved autofluorescence (FLIM) at the human ocular fundus of diabetic patients permit the
detection of early pathologic alterations before signs of diabetic retinopathy are visible. The measurements were
performed by the Jena Fluorescence Lifetime Laser Scanner Ophthalmoscope applying time-correlated single photon
counting (TCSPC) in two spectral channels (K1: 490-560 nm, K2:560-700ps). The fluorescence was excited by 70 ps
pulses (FWHM) at 448 nm. The decay of fluorescence intensity was triple-exponentially approximated. The frequency
of amplitudes, lifetimes, and relative contributions was compared in fields of the same size and position in healthy
subjects and in diabetic patients. The most sensitive parameter was the lifetime T2 in the short-wavelength channel,
which corresponds to the neuronal retina. The changes in lifetime point to a loss of free NADH and an increased
contribution of protein-bound NADH in the pre-stage of diabetic retinopathy.
A new simple method for two-dimensional determination of optical density of macular pigment xanthophyll (ODx) in clinical routine is based on a single blue-reflection fundus image. Individual different vignetting is corrected by a shading function. For its construction, nodes are automatically found in structureless image regions. The influence of stray light in elderly crystalline lenses is compensated by a correction function that depends on age. The reproducibility of parameters in a one-wavelength reflection method determined for three subjects (47, 61, and 78 years old) was: maxODx = 6.3%, meanODx = 4.6%, volume = 6%, and area = 6% already before stray-light correction. ODx was comparable in pseudophakic and in an eye with a crystalline lens of the same 11 subjects after stray-light correction. Significant correlation in ODx was found between the one-wavelength reflection method and the two-wavelength autofluorescence method for pseudophakic and cataract eyes of 19 patients suffering from dry age-related macular degeneration (AMD) (R2 = 0.855). In pseudophakic eyes, maxODx was significantly lower for dry AMD (n = 45) (ODx = 0.491±0.102 ODU) than in eyes with healthy fundus (n = 22) (ODx = 0.615±0.103 ODU) (p = 0.000033). Also in eyes with crystalline lens, maxODx was lower in AMD (n = 125) (ODx = 0.610±0.093 ODU) than in healthy subjects (n = 45) (ODx = 0.674±0.098 ODU) (p = 0.00019). No dependence on age was found in the pseudophakic eyes both of healthy subjects and AMD patients.
During clinical application of the fluorescence lifetime laser scanner ophthalmoscope, a stepped slope of ocular
autofluorescence was found. Fitting of fluorescence results in wrong lifetimes if the left border of the fitting interval is
set at the time channel of first appearance of fluorescence. A better fit was reached at least for the fluorescence decay if
the left border of the fitting interval is set near the maximum of detected fluorescence. Analysing the appearance of the
stepped slope, its origination was found by the different appearance time of fluorescence from the crystalline lens and
from the fundus fluorescence. The extension of the exponential model function by a parameter tci results in an optimal fit
of both the slope and of the fluorescence decay. This new parameter describes differences in the appearance time of
fluorescence originating from different layers. Taking into account the refractive index between layers, the geometrical
distance between them can be determined. In this way, functional information (lifetimes) and geometrical information
(distances) can be determined by the same measurement. To reach geometrical resolution comparable with OCT, pulses
and time resolution are required in the order of 30 fs.
A laser scanner ophthalmoscope was developed for in vivo fluorescence lifetime measurements at the human retina.
Measurements were performed in 30 degree fundus images. The fundus was excited by pulses of 75 ps (FWHM). The
dynamic fluorescence was detected in two spectral channels K1(490-560nm), K2(560-700 nm) by time-correlated single
photon counting. The decay of fluorescence was three-exponentially. Local and global alterations in lifetimes were
found between healthy subjects and patients suffering from age-related macular degeneration, diabetic retinopathy, and
vessel occlusion. The lifetimes T1, T2, and T3 in both channels are changed to longer values in AMD and diabetic
retinopathy in comparison with healthy subjects. The lifetime T2 in K1 is most sensitive to metabolic alterations in
branch arterial vessel occlusion.
First pathological alterations occur at cellular level, most in metabolism. An indirect estimation of metabolic
activity in cells is measurement of microcirculation. Measurements of tissue autofluorescence are potentially
suited for direct investigation of cellular metabolism. Besides redox pairs of co-enzymes (NADH-NAD,
FADH2-FAD) several other fluorophores are excited in tissue. In addition, a number of anatomical structures are
simultaneously excited, when investigating the eye-ground. In this study, spectral and time resolved comparison
was performed between purified substances, single ocular structures and in vivo measurements of the time-resolved
autofluorescence at the human eye. In human eyes, the ageing pigment lipofuscin covers other
fluorophores at the fundus in long - wave visible range. Applying lifetime measurements, weakly emitting
fluorophores can be detected, when the lifetimes are different from the strongly emitting fluorophore. For this,
the autofluorescence was excited at 468 nm and detected in two spectral ranges (500 nm-560 nm, 560 nm-700
nm). In tri-exponential fitting, the short lifetime corresponds to retinal pigment epithelium, the mean lifetime
corresponds probably to neural retina and the long lifetime is caused by fluorescence of connective tissue.
Measurements of endogeous fluorophores open the possibility for evaluation of metabolic state at the eye. For
interpretation of 2-dimensional measurements of time-resolved auto fluorescence in 2 separate spectral ranges at the
human eye, comparing measurements were performed on porcine eyes. Determining excitation and emission spectra,
attention was drawn of proof of coenzymes NADH and FAD in isolated anatomical structures cornea, aqueous
humor, lens, vitreous, neuronal retina, retinal pigment epithelium (RPE), choroid, and sclera. All these structures
exhibit auto fluorescence, highest in lens. Excitation at 350 nm results in local fluorescence maxima at 460 nm,
corresponding to NADH, in all structures. This short-wave excitation allows metabolic studies only at the anterior
eye, because of the limited transmission of the ocular media. During excitation at 446 nm the existence of FAD is
expressed by local fluorescence maxima at 530 nm. The composition fluorescence spectra allow no discrimination
between single ocular structures. Approximating the dynamic fluorescence by a double exponential function, the
shortest lifetimes were detected in RPE and neuronal retina. The histograms of mean lifetime tM cover each other on
lens with cornea and also on sclera with choroid. Despite the lifetimes are close between RPE and neuronal retina,
the relative contributions Q1 are wide different. The gradient of trend lines in cluster diagrams of amplitudes α2 vs.
α1 allows a discrimination of ocular structures.
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