Changes in drusen-associated autofluorescence over time observed by fluorescence lifetime imaging ophthalmoscopy in age-related macular degeneration.

PURPOSE: To observe fundus autofluorescence (FAF) lifetimes and peak emission wavelength (PEW) of drusen with respect to the pathology of the overlying RPE in the follow-up of AMD-patients. METHODS: Forty eyes of 38 patients (age: 75.1 ± 7.1 years) with intermediate AMD were included. FAF lifetimes and PEW were recorded by fluorescence lifetime imaging ophthalmoscopy (FLIO). Twenty-six eyes had a follow-up investigation between months 12 and 36, and 10 at months 37-72. AMD progression was retrieved from color fundus photography (CFP) and OCT. Drusen were classified with respect to changes in the overlying RPE into groups no, questionable or faint, and apparent hyperpigmentation based on CFP. RESULTS: Among the 210 hyperautofluorescent drusen found at baseline, those with hyperpigmentation had longer lifetimes and shorter PEW than those without. Drusen without hyperpigmentation had shorter lifetimes and PEW than neighboring RPE (all p < 0.001) at baseline, but drusen lifetimes increased, and PEW shortened further over follow-up. Eyes, showing AMD progression, had significantly longer FAF lifetimes at baseline than non-progressing eyes: 282 ± 102 ps versus 245 ± 98 ps, p < 0.001 and 365 ± 44 ps vs. 336 ± 48 ps, p = 0.025 for short and long wavelength FLIO channel, respectively. CONCLUSIONS: Depending on hyperpigmentation properties, drusen show lifetimes and PEW different from that of adjacent RPE which change over the natural history of AMD. This difference and change, however, might reflect progressive dysmorphia of the RPE rather than representing fluorescence of drusen material itself. Nevertheless, the observed FAF changes could make FLIO a useful tool for the early detection of AMD progression risk.

Fluorescence Lifetime and Spectral Characteristics of Subretinal Drusenoid Deposits and Their Predictive Value for Progression of Age-Related Macular Degeneration.

Purpose: To measure fundus autofluorescence (FAF) lifetimes and peak emission wavelengths (PEW) of subretinal drusenoid deposits (SDD) in age-related macular degeneration (AMD) and their development over time. Methods: Fluorescence lifetime imaging ophthalmoscopy (FLIO) was performed in 30 eyes with optical coherence tomography (OCT)-confirmed early or intermediate AMD and SDD. Contrasts of mean lifetimes in short- (SSC) and long-wavelength channels (LSC), PEW, and relative fluorescence intensity were determined as differences of the respective measures at individual SDD and their environment. Measurements were made at baseline and at follow-up intervals 1 (13-36 months) and 2 (37-72 months), respectively. Results: Of 423 SDD found at baseline, 259, 47, and 117 were hypoautofluorescent, isoautofluorescent, and hyperautofluorescent, respectively. FAF lifetimes of SDD were significantly longer than those of their environment by 14.5 ps (SSC, 95% confidence interval [CI], 13.3-15.7 ps) and 3.9 ps (LSC, 3.1-4.7 ps). PEW was shorter by 1.53 nm (1.07-1.98 nm, all contrasts P < 0.001) with higher contrasts for hyperfluorescent SDD. Over follow-up, SDD tended to hyperautofluorescence (relative intensities increased by 3.4% [95% CI, 2.9%-4.1%; P < 0.001] in follow-up 2). Hyperautofluorescence was associated with disruption of the ellipsoid zone on OCT. Disease progression to late-stage AMD was associated with higher lifetime contrast in SSC (15.9ps [14.2-17.6 ps] vs. 11.7 ps [9.9-13.5 ps], P < 0.001) at baseline. Conclusions: SDD show longer FAF lifetimes and shorter PEW than their environments. A high lifetime contrast of SDD in SSC might predict disease progression to late-stage AMD.

Optic disc blood perfusion and oxygenation in glaucoma.

PURPOSE: To investigate the haemoglobin concentration and oxygenation in the optic disc in glaucoma patients vs. controls. METHODS: Thirty-one eyes of primary open angle glaucoma patients (mean age: 64.9 ± 2.1 years) and 31 eyes of 31 healthy controls (65.5 ± 2.0 years) were included. Perimetry, optical coherence tomography (OCT), and OCT angiography were performed. Multispectral imaging was used to record the optic disc reflectance at wavelengths 522 nm, 548 nm, 555 nm, 586 nm, and 610 nm, and haemoglobin concentration and oxygenation (SO2) were calculated from these measures. This was done in the rest and under stimulation of neuronal activity by flicker light. RESULTS: The haemoglobin concentration was significantly lower (p < 0.001) in the rim (40.0 ± 6.3) and the excavation (35.7 ± 8.0) of the glaucoma patients' discs than in controls (45.7 ± 7.5). SO2 was not different in general, but lower in a subgroup of 18 glaucoma patients with ischaemic disc rims than in non-ischaemic ones (median 26.8%, interquartile range (IQR): 29.5% vs. 51.9%, IQR 32.0%, p = 0.02) as well as in controls (41.0%, IQR 30.6%, p = 0.01). Flicker light stimulation significantly increased the haemoglobin concentration in the controls (+ 1.3 ± 3.6, p = 0.048) as well as in the rim of glaucoma discs (+ 2.6 ± 5.0, p = 0.006) and SO2 in the controls only (+ 15.4 ± 23.6%, p = 0.001). The haemoglobin concentration was significantly correlated with the perimetric mean defect, retinal nerve fibre layer (RNFL) thickness and para-papillary perfusion density. CONCLUSIONS: The optic disc haemoglobin concentration and oxygenation are quantifiable from multispectral imaging and reduced in glaucoma. The correlation of haemoglobin concentration with perfusion density, RNFL thickness and visual field loss indicates its implication in glaucoma pathology.

Fundus Autofluorescence Lifetimes and Spectral Features of Soft Drusen and Hyperpigmentation in Age-Related Macular Degeneration.

Purpose: To investigate the autofluorescence lifetimes as well as spectral characteristics of soft drusen and retinal hyperpigmentation in age-related macular degeneration (AMD). Methods: Forty-three eyes with nonexudative AMD were included in this study. Fluorescence lifetime imaging ophthalmoscopy (FLIO), which detects autofluorescence decay over time in the short (SSC) and long (LSC) wavelength channel, was performed. The mean autofluorescence lifetime (τm) and the spectral ratio (sr) of autofluorescence emission in the SSC and LSC were recorded and analyzed. In total, 2760 soft drusen and 265 hyperpigmented areas were identified from color fundus photographs and spectral domain optical coherence tomography (SD-OCT) images and superimposed onto their respective AF images. τm and sr of these lesions were compared with fundus areas without drusen. For clearly hyperfluorescent drusen, the local differences compared to fundus areas without drusen were determined for lifetimes and sr. Results: Hyperpigmentation showed significantly longer τm (SSC: 341 ± 81 vs. 289 ± 70 ps, P < 0.001; LSC: 406 ± 42 vs. 343 ± 42 ps, P < 0.001) and higher sr (0.621 ± 0.077 vs. 0.539 ± 0.083, P < 0.001) compared to fundus areas without hyperpigmentation or drusen. No significant difference in τm was found between soft drusen and fundus areas without drusen. However, the sr was significantly higher in soft drusen (0.555 ± 0.077 vs. 0.539 ± 0.081, P < 0.0005). Hyperfluorescent drusen showed longer τm than surrounding fundus areas without drusen (SSC: 18 ± 42 ps, P = 0.074; LSC: 16 ± 29 ps, P = 0.020). Conclusions: FLIO can quantitatively characterize the autofluorescence of the fundus, drusen, and hyperpigmentation in AMD. Translational Relevance: The experimental FLIO technique was applied in a clinical investigation. As FLIO yields information on molecular changes in AMD, it might support future diagnostics.

Simplified approach to least-square fitting of fluorescence lifetime ophthalmoscopy (FLIO) data by fixating lifetimes.

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new imaging modality in ophthalmology. For clinical investigations, the amplitude-weighted mean of two or three lifetime components is usually analyzed. In this study, we investigated the effects of fixation of lifetime components. This resulted in slightly higher fit errors but mean lifetimes were highly correlated to those from fits with variable individual lifetimes. Furthermore, this approach resulted in a similarly good discrimination of diabetic retinopathy patients from controls, a reduction of the computational workload, a de-noising of the mean lifetime images and allows higher local resolution. Thus, fixation of lifetimes in the fit of FLIO data could be superior for clinical routine analysis of FLIO data.

Monitoring macular pigment changes in macular holes using fluorescence lifetime imaging ophthalmoscopy.

PURPOSE: To investigate the impact of macular pigment (MP) on fundus autofluorescence (FAF) lifetimes in vivo by characterizing full-thickness idiopathic macular holes (MH) and macular pseudo-holes (MPH). METHODS: A total of 37 patients with MH and 52 with MPH were included. Using the fluorescence lifetime imaging ophthalmoscope (FLIO), based on a Heidelberg Engineering Spectralis system, a 30° retinal field was investigated. FAF decays were detected in a short (498-560 nm; ch1) and long (560-720 nm; ch2) wavelength channel. τm , the mean fluorescence lifetime, was calculated from a three-exponential approximation of the FAF decays. Macular coherence tomography scans were recorded, and macular pigment's optical density (MPOD) was measured (one-wavelength reflectometry). Two MH subgroups were analysed according to the presence or absence of an operculum above the MH. A total of 17 healthy fellow eyes were included. A longitudinal FAF decay examination was conducted in nine patients, which were followed up after surgery and showed a closed MH. RESULTS: In MH without opercula, significant τm differences (p < 0.001) were found between the hole area (MHa) and surrounding areas (MHb) (ch1: MHa 238 ± 64 ps, MHb 181 ± 78 ps; ch2: MHa 275 ± 49 ps, MHb 223 ± 48 ps), as well as between MHa and healthy eyes or closed MH. Shorter τm , adjacent to the hole, can be assigned to areas with equivalently higher MPOD. Opercula containing MP also show short τm . In MPH, the intactness of the Hele fibre layer is associated with shortest τm . CONCLUSIONS: Shortest τm originates from MP-containing retinal layers, especially from the Henle fibre layer. Fluorescence lifetime imaging ophthalmoscope (FLIO) provides information on the MP distribution, the pathogenesis and topology of MH. Macular pigment (MP) fluorescence may provide a biomarker for monitoring pathological changes in retinal diseases.

Fundus autofluorescence lifetimes are increased in non-proliferative diabetic retinopathy.

PURPOSE: To discriminate non-proliferative diabetic retinopathy (NPDR) patients from healthy controls by fluorescence lifetime imaging ophthalmoscopy (FLIO). METHODS: A prototype FLIO (Heidelberg-Engineering, Heidelberg, Germany) was used to examine the retina of 33 patients and 28 controls. As increased fluorescence of the diabetic lens is known, the lenses of 34 patients and 24 controls were investigated as well. Time-resolved decay was detected in two spectral channels (ch1: 498-560 nm, ch2: 560-720 nm) and approximated by a series of three exponential functions yielding in lifetimes (τ1 , τ2 , τ3 ), amplitudes (α1 , α2 , α3 ) and their amplitude-weighted means (τm ). RESULTS: Significant differences between patients and controls were found for all fundus lifetime components (τm , τ1 -τ3 ) as for the amplitude α3 in both spectral channels. Channel 1 showed the largest differences: the average of mean fluorescence lifetime τm in the macula was 259 ± 137 ps in the patients versus 147 ± 69 ps in the controls. A logistic regression model allowed discrimination between study and control group with a sensitivity of 90.09% and a specificity of 71.4% (area under the curve: 0.865). Significantly shorter τm in the patients group than in the control group was detected in channel 2 in the crystalline lens (1587 ± 326 ps versus 1854 ± 384 ps, p = 0.006). CONCLUSIONS: Fundus Fluorescence lifetimes are significantly increased in NPDR while lens lifetimes are shorter in the patient group. Lifetime changes might be indicative for the accumulation of advanced glycation end products (AGEs) which enables detection of the disease with high sensitivity and specificity possibly bearing diagnostic merit.

Dependence of diameters and oxygen saturation of retinal vessels on visual field damage and age in primary open-angle glaucoma.

PURPOSE: To investigate the interrelationship between the oxygen supply of the retina and its regulation with the severity of primary open-angle glaucoma (POAG). METHODS: Central retinal artery (CRAE) and vein (CRVE) diameters and oxygen saturation of peripapillary retinal vessels in 41 patients suffering from POAG (64.1 ± 12.9 years) and 40 healthy volunteers (63.6 ± 14.1 years) were measured using the retinal vessel analyzer. All measures were taken before and during flicker light stimulation. The mean retinal nerve fiber layer thickness (RNFLT) was determined by OCT and the visual field mean defect (MD) was identified using perimetry. RESULTS: In glaucoma patients, CRAE (r = -0.48 p = 0.002) and CRVE (r = -0.394 p = 0.014) at baseline were inversely related to MD, while arterial and venous oxygen saturation showed no significant dependence on the severity of the damage. However, the flicker light-induced change in arterio-venous difference in oxygen saturation was correlated with the MD (r = 0.358 p = 0.027). The diameters of arteries and veins at baseline decreased with reduction of the mean RNFLT (arteries: r = 0.718 p < 0.001; veins: r = 0.685 p < 0.001). CONCLUSION: Vessel diameters showed a strong correlation with RNFLT and MD. This, as well as the reduction of stimulation-induced change in arterio-venous oxygen saturation difference with visual field loss, may be explained by a reduction of the retinal metabolic demand with progressive loss of neuronal tissue in glaucoma.

Impact of Macular Pigment on Fundus Autofluorescence Lifetimes.

PURPOSE: To characterize the macular region and to investigate the influence of the macular pigment (MP) on fundus autofluorescence (FAF) lifetimes in vivo. METHODS: Forty-eight healthy subjects with a mean age of 24.1 ± 3.6 years (range, 20-37 years) were included. A 30° retinal field was investigated using the fluorescence lifetime imaging ophthalmoscope (FLIO), based on a Heidelberg Engineering Spectralis system, detecting FAF decays in a short (498-560 nm; ch1)- and a long (560-720 nm; ch2)-wavelength channel. The mean fluorescence lifetime τm was calculated from a 3-exponential approximation of the FAF decays. Macular pigment optical density (MPOD) was measured by one-wavelength reflectometry, and macular optical coherence tomogram (OCT) scans were recorded. Correlations between τm and MPOD were analyzed. RESULTS: The τm showed shortest values at the macular region with a mean of 82 ps (ch1) and 126 ps (ch2). We found a strong correlation of τm to the MPOD (ch1: r = -0.760; ch2: r = -0.663; P < 0.001), as well as a topologic agreement of shortest τm with highest MPOD. CONCLUSIONS: Macular pigment, which is known to have very short fluorescence decays, considerably contributes to the macular autofluorescence (AF). This study gives indirect evidence for a strong impact of MP on macular τm, although no direct measurement of MP autofluorescence lifetimes in vivo is possible at this point. Potentially, imaging the FAF lifetimes could lead to a novel methodology for the detection of macular pigment properties and pathology-induced changes in the living human retina.

Anterior optical coherence tomography: a non-contact technique for anterior chamber evaluation.

PURPOSE: To assess a new non-contact anterior optical coherence tomography (OCT) system for anterior chamber evaluation. METHODS: A new commercial 131-nm infrared light anterior OCT system was used for anterior chamber evaluation. Forty-four eyes of 35 subjects (18 normal subjects, 17 subjects with anterior chamber abnormalities) were enrolled in the study. RESULTS: Eyes were divided into those with narrowed, broadened and normal anterior chamber depth. Anterior chamber angle dynamics were assessed in three patients with angle-closure glaucoma and cataract extraction. Anterior OCT was also used for visualization of the anterior chamber (OCT goniometry) in a subject with multiple local anaesthetic allergies with phobia regarding conventional contact gonioscopy. CONCLUSIONS: Anterior OCT is a new, easy-to-handle, non-contact technique that allows exact evaluation of anterior chamber parameters such as anterior chamber depth, chamber angle dynamics, corneal curvature and corneal thickness.