Functional alterations are first signs of a starting pathological process. A device that measures parameter for the characterization of the metabolism at the human eye-ground would be a helpful tool for early diagnostics in stages when alterations are yet reversible. Measurements of blood flow and of oxygen saturation are necessary but not sufficient. The new technique of auto-fluorescence lifetime measurement (FLIM) opens in combination with selected excitation and emission ranges the possibility for metabolic mapping. FLIM not only adds an additional discrimination parameter to distinguish different fluorophores but also resolves different quenching states of the same fluorophore. Because of its high sensitivity and high temporal resolution, its capability to resolve multi-exponential decay functions, and its easy combination with laser scanner ophthalmoscopy, multi-dimensional time-correlated single photon counting was used for fundus imaging. An optimized set up for in vivo lifetime measurements at the human eye-ground will be explained. In this, the fundus fluorescence is excited at 446 or 468 nm and the time-resolved autofluorescence is detected in two spectral ranges between 510 and 560 nm as well as between 560 and 700 nm simultaneously. Exciting the fundus at 446 nm, several fluorescence maxima of lifetime t1 were detected between 100 and 220 ps in lifetime histograms of 40 degrees fundus images. In contrast, excitation at 468 nm results in a single maximum of lifetime t1 = 190 +/- 16 ps. Several fundus layers contribute to the fluorescence intensity in the short-wave emission range 510-560 nm. In contrast, the fluorescence intensity in the long-wave emission range between 560 and 700 nm is dominated by the fluorescence of lipofuscin in the retinal pigment epithelium. Comparing the lateral distribution of parameters of a tri-exponential model function in lifetime images of the fundus with the layered anatomical fundus structure, the shortest component (t1 = 190 ps) originates from the retinal pigment epithelium and the second lifetime (t2 = 1,000 ps) from the neural retina. The lifetime t3 approximately 5.5 ns might be influenced by the long decay of the fluorescence in the crystalline lens. In vitro analysis of the spectral properties of expected fluorophores under the condition of the living eye lightens the interpretation of in vivo measurements. Taking into account the transmission of the ocular media, the excitation of NADH is unlikely at the fundus.
Abstract. 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 τ i , amplitudes α i , and relative contributions Q i were statistically compared between corresponding groups in two spectral channels (490 < ch1 < 560 nm, 560 < ch2 < 700 nm). The change in single fluorophores was estimated by applying the Holm-Bonferroni method and by calculating differences in the sum histograms of lifetimes. Median and mean of the histograms of τ 2 , τ 3 , and α 3 in ch1 show the greatest differences between phakic diabetic patients and age-matched controls (p < 0.000004). The lack of pixels with a τ 2 of ∼360 ps, the increased number of pixels with τ 2 > 450 ps, and the shift of τ 3 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.
The supplementation of L, Z, O-3-LCPUFAs and antioxidants resulted in considerable increase in MPOD. There was no difference in accumulation of MPOD between both dosages. Thus, we believe that the used supplementation with L and Z seems to reach a saturation level in retinal cell structure. Additionally, the constant supplementation of L, Z, O-3-LCPUFAs and antioxidants in AMD patients seems to be useful, because MPOD reduces without supplementation. We conclude that the supplementation caused an increase of MPOD, which results in an improvement and stabilization in BCVA in AMD patients. Thus, a protective effect on the macula in AMD patients is assumed.
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.
ABSTRACT.Purpose: To determine alterations in the retina of patients with Alzheimer's disease (AD) by the newly developed technique of fluorescence lifetime imaging ophthalmoscopy (FLIO) in a pilot study. Methods: FLIO set-up uses a scanning laser ophthalmoscope (HRA2, Heidelberg Engineering, Germany), which was modified by the use of an excitation pulse laser BLD440 (Becker&Hickl, Berlin, Germany) and detection of fluorescence lifetime by time-correlated single photon counting (TCSPC; Becker&Hickl) in two spectral channels (channel 1: 490-560 nm, channel 2: 560-700 nm). Least square fit of three exponential functions was used for fluorescence decay analysis. That resulted in three fluorescent components with lifetimes s i , amplitudes a i and relative contributions Q i . 16 patients with AD (mean age 77.2 AE 7.0 years) were investigated. After regular ophthalmic investigation, FLIO examination and OCT examination were performed. Alzheimer-specific clinical data were collected (MMSE, cerebrospinal fluid (CSF) concentration of amyloid-b (1-42), total-tau and phosphorylated tau181 (p-tau181) protein). Results: The FLIO parameters of the second fluorescent component a 2 and Q 2 (channel 2) correlated significantly with MMSE score (Q 2 , R = À0.757, p = 0.007; a 2 , R = À0.618, p = 0.043) as well as p-tau181-protein concentration in CSF (Q 2 , R = 0.919, p = 0.009; a 2 , R = 0.881, p = 0.020) in patients with AD. OCT measurements of retinal nerve fibre layer thickness, optic disc excavation and macular thickness neither correlated with Alzheimer-specific CSF data nor MMSE score. Conclusions: Unlike conventional techniques, such as OCT, the new technique of FLIO revealed changes in the retina of patients with AD in relation to Alzheimer-specific markers in this pilot study.
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