Fluorescence Lifetime Imaging Ophthalmoscopy Research Articles

Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy.

To demonstrate the first near-infrared adaptive optics fluorescence lifetime imaging ophthalmoscopy (NIR-AOFLIO) measurements in vivo of the human retinal pigment epithelial (RPE) cellular mosaic and to visualize lifetime changes at different retinal eccentricities. NIR reflectance and autofluorescence were captured using a custom adaptive optics scanning light ophthalmoscope in 10 healthy subjects (23-64 years old) at seven eccentricities and in two eyes with retinal abnormalities. Repeatability was assessed across two visits up to 8weeks apart. Endogenous retinal fluorophores and hydrophobic whole retinal extracts of Abca4-/- pigmented and albino mice were imaged to probe the fluorescence origin of NIR-AOFLIO. The RPE mosaic was resolved at all locations in five of seven younger subjects (<35 years old). The mean lifetime across near-peripheral regions (8° and 12°) was longer compared to near-foveal regions (0° and 2°). Repeatability across two visits showed moderate to excellent correlation (intraclass correlation: 0.88 [τm], 0.75 [τ1], 0.65 [τ2], 0.98 [a1]). The mean lifetime across drusen-containing eyes was longer than in age-matched healthy eyes. Fluorescence was observed in only the extracts from pigmented Abca4-/- mouse. NIR-AOFLIO was repeatable and allowed visualization of the RPE cellular mosaic. An observed signal in only the pigmented mouse extract infers the fluorescence signal originates predominantly from melanin. Variations observed across the retina with intermediate age-related macular degeneration suggest NIR-AOFLIO may act as a functional measure of a biomarker for in vivo monitoring of early alterations in retinal health.

Detection sensitivity of fluorescence lifetime imaging ophthalmoscopy for laser-induced selective damage of retinal pigment epithelium

PurposeTo investigate the sensitivity of fluorescence lifetime imaging ophthalmoscopy (FLIO) to detect retinal laser spots by comparative analysis with other imaging modalities.MethodsA diode laser with a wavelength of 514 nm was applied with pulse durations of 5.2, 12, 20, and 50 µs. The laser pulse energy was increased so that the visibility of the laser spot by slit-lamp fundus examination (SL) under the irradiator’s observation covers from the subvisible to visible range immediately after irradiation. The irradiated areas were then examined by fundus color photography (FC), optical coherence tomography (OCT), fundus autofluorescence (AF), FLIO, and fluorescein angiography (FA). The visibility of a total of over 2200 laser spots was evaluated by two independent researchers, and effective dose (ED) 50 laser pulse energy values were calculated for each imaging modality and compared.ResultsAmong examined modalities, FA showed the lowest mean of ED50 energy value and SL the highest, that is, they had the highest and lowest sensitivity to detect retinal pigment epithalium (RPE)-selective laser spots, respectively. FLIO also detected spots significantly more sensitively than SL at most laser pulse durations and was not significantly inferior to FA. AF was also often more sensitive than SL, but the difference was slightly less significant than FLIO.ConclusionConsidering its high sensitivity in detecting laser spots and previously reported potential of indicating local wound healing and metabolic changes around laser spots, FLIO may be useful as a non-invasive monitoring tool during and after minimally invasive retinal laser treatment.

Artificial Intelligence in Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) Data Analysis-Toward Retinal Metabolic Diagnostics.

The purpose of this study was to investigate the possibility of implementing an artificial intelligence (AI) approach for the analysis of fluorescence lifetime imaging ophthalmoscopy (FLIO) data even with small data. FLIO data, including the fluorescence intensity and mean fluorescence lifetime (τm) of two spectral channels, as well as OCT-A data from 26 non-smokers and 28 smokers without systemic and ocular diseases were used. The analysis was performed with support vector machines (SVMs), a well-known AI method for small datasets, and compared with the results of convolutional neural networks (CNNs) and autoencoder networks. The SVM was the only tested AI method, which was able to distinguish τm between non-smokers and heavy smokers. The accuracy was about 80%. OCT-A data did not show significant differences. The feasibility and usefulness of the AI in analyzing FLIO and OCT-A data without any apparent retinal diseases were demonstrated. Although further studies with larger datasets are necessary to validate the results, the results greatly suggest that AI could be useful in analyzing FLIO-data even from healthy subjects without retinal disease and even with small datasets. AI-assisted FLIO is expected to greatly advance early retinal diagnosis.

Fluorescence Lifetime Imaging Ophthalmoscopy of Mouse Models of Age-related Macular Degeneration.

To investigate fluorescence lifetime of mouse models of age-related macular degeneration (AMD) by fluorescence lifetime imaging ophthalmoscopy (FLIO). Two AMD mouse models, apolipoprotein E knockout (ApoE-/-) mice and NF-E2-related factor-2 knockout (Nrf2-/-) mice, and their wild-type mice underwent monthly ophthalmic examinations including FLIO from 3months of age. After euthanasia at the age of 6 or 11months, blood plasma was collected to determine total antioxidant capacity and eyes were enucleated for Oil red O (ORO) lipid staining of chorioretinal tissue. In FLIO, the mean fluorescence lifetime (τm) of wild type shortened with age in both spectral channels. In short spectral channel, τm shortening was observed in both AMD models as well, but its rate was more pronounced in ApoE-/- mice and significantly different from the other strains as months of age progressed. In contrast, in long spectral channel, both model strains showed completely opposite trends, with τm becoming shorter in ApoE-/- and longer in Nrf2-/- mice than the others. Oil red O staining at Bruch's membrane was significantly stronger in ApoE-/- mice at 11months than the other strains. Plasma total antioxidant capacity was highest in ApoE-/- mice at both 6 and 11months. The two AMD mouse models exhibited largely different fundus fluorescence lifetime, which might be related to the different systemic metabolic state. FLIO might be able to indicate different metabolic states of eyes at risk for AMD. This animal study may provide new insights into the relationship between early AMD-associated metabolic changes and FLIO findings.

Characterization of a new fluorescence lifetime imaging ophthalmoscope

Aims/Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) allows in vivo measurement of autofluorescence intensity decays of endogenous fluorophores in the ocular fundus. So far, only devices from Heidelberg Engineering based on the Spectralis system have been used in FLIO research. Here, we present and characterize a new FLIO device based on the RETImap system from Roland Consult.Methods: The device is based on a confocal scanning laser ophthalmoscope (35° field, 512 × 512 px). A ps diode laser (BDL‐SMN 473 nm, Becker &amp; Hickl GmbH, Berlin, Germany) excites autofluorescence. The fluorescence photons are split into a short (498–560 nm, SSC) and a long (560–720 nm, LSC) spectral channel (one HPM‐100‐40 detector [Becker &amp; Hickl GmbH] each) and are detected by time‐correlated single photon counting (SPC‐160, Becker &amp; Hickl GmbH). We determined the maximum laser power (ILT2400, International Light Technologies, Inc. Peabody, MA, USA) and analysed the instrument's behaviour at three different laser power levels (150 μW, 200 μW and max.) in terms of laser spectrum (CAS140CT, Instrument Systems GmbH, Munich, Germany) and instrument response function (IRF). The IRF was determined using a 25 μM Eosin Y solution, mixed with a 5 M solution of potassium iodide, placed in a flat cuvette (110‐OS, Hellma GmbH &amp; Co. KG, Müllheim, Germany) in front of the objective lens of the FLIO device. Fluorescence measurements of approximately 1‐min duration were performed three times for all three laser powers. The IRF and the full width at half maximum (FWHM) were calculated using FLIMX software (www.flimx.de).Results: The max. laser power was 280 μW. The peak wavelengths of the laser spectra were 467.6 (150 μW), 467.9 (200 μW) and 468.0 nm (280 μW). IRF FWHM in SSC were 298.6 ± 1.1 ps (150 μW), 341.0 ± 2.5 ps (200 μW) and 347.5 ± 6.0 ps (280 μW). In LSC, the IRF FWHM were 290.4 ± 3.8 ps (150 μW), 344.0 ± 3.4 ps (200 μW) and 358.8 ± 1.3 ps (280 μW). Results are mean ± standard deviation.Conclusions: A new fluorescence lifetime imaging ophthalmoscope has been characterized. The device offers a high laser power for fluorescence excitation, a large field of view, a high spatial resolution, and a sufficiently high time resolution. Thus, it is suitable for fluorescence lifetime studies.

Retinal imaging biomarkers of Alzheimer’s disease: clinical evidence and perspectives

AbstractA growing body of research is devoted to retinal imaging biomarkers of Alzheimer’s disease (AD). Imaging methods such as optical coherence tomography (OCT) provide qualitative and quantitative measures of retinal and optic nerve neurodegeneration, whereas OCT‐angiography yields depth‐resolved maps of the retinal microvasculature in exquisite detail. Similarly, conventional and widefield retinal photography enable measurements of the retinal vascular geometry. Other emerging technologies, including hyperspectral imaging, retinal curcumin‐fluorescence imaging and fluorescence lifetime imaging ophthalmoscopy, provide insights into retinal changes at the molecular level, such as the accumulation of amyloid beta.Navigating the evidence in this field is challenging. Of the many studies that have examined associations between retinal imaging findings and AD, most have been based on clinical case ascertainment in the absence of biomarker findings. In this presentation, we will provide an accessible overview of this rapidly evolving field, with an emphasis on studies of retinal imaging biomarkers of the disease that align with the NIA‐AA Research Framework. Underpinning this are the findings of a systematic review and meta‐analysis that we have recently conducted (in press). Drawing on the anatomical and pathological insights provided by fellow speakers in the symposium, we highlight opportunities for further clinicopathological correlation of key imaging biomarkers. Finally, as a prelude to the last talk in the symposium, we highlight gaps in the existing clinical literature and point to efforts to harmonise study methods to generate the evidence needed for adoption of retinal imaging biomarkers of AD in practice.

Retina Oculomics in Neurodegenerative Disease.

Ophthalmic biomarkers have long played a critical role in diagnosing and managing ocular diseases. Oculomics has emerged as a field that utilizes ocular imaging biomarkers to provide insights into systemic diseases. Advances in diagnostic and imaging technologies including electroretinography, optical coherence tomography (OCT), confocal scanning laser ophthalmoscopy, fluorescence lifetime imaging ophthalmoscopy, and OCT angiography have revolutionized the ability to understand systemic diseases and even detect them earlier than clinical manifestations for earlier intervention. With the advent of increasingly large ophthalmic imaging datasets, machine learning models can be integrated into these ocular imaging biomarkers to provide further insights and prognostic predictions of neurodegenerative disease. In this manuscript, we review the use of ophthalmic imaging to provide insights into neurodegenerative diseases including Alzheimer Disease, Parkinson Disease, Amyotrophic Lateral Sclerosis, and Huntington Disease. We discuss recent advances in ophthalmic technology including eye-tracking technology and integration of artificial intelligence techniques to further provide insights into these neurodegenerative diseases. Ultimately, oculomics opens the opportunity to detect and monitor systemic diseases at a higher acuity. Thus, earlier detection of systemic diseases may allow for timely intervention for improving the quality of life in patients with neurodegenerative disease.

Impact of cigarette smoking on fluorescence lifetime of ocular fundus

Cigarette smoking is known to adversely affect cellular metabolism and is a risk factor for various retinal diseases. Fluorescence lifetime imaging ophthalmoscopy (FLIO) has the potential to detect metabolic changes in the ocular fundus. Aim of this study was to analyze the influence of cigarette smoking on fluorescence lifetime (FLT) of healthy eyes using FLIO. Twenty-six non-smokers and 28 smokers aged between 20 and 37 years without systemic and ocular diseases were investigated by FLIO (excitation: 473 nm, emission: short spectral channel (SSC) 498–560 nm, long spectral channel (LSC) 560–720 nm). The FLT at the ETDRS grid regions were analyzed and compared. In SSC, the mean FLT (τm) of smokers was significantly longer in the ETDRS inner ring region, whereas the τm in LSC was significantly shorter in the outer ring. For the long component (τ2), smokers with pack year < 7.11 showed significantly shorter τ2 in SSC than non-smokers and the smokers with pack year ≥ 7.11. There were no significant differences in retinal thickness. The lack of obvious structural differences implies that the observed FLT changes are likely related to smoking-induced metabolic changes. These results suggest that FLIO may be useful in assessing retinal conditions related to lifestyle and systemic metabolic status.

The value of pre-symptomatic genetic risk assessment for age-related macular degeneration: the Moran AMD Genetic Testing Assessment (MAGENTA) study—a study protocol for a randomized controlled trial

BackgroundAge-related macular degeneration (AMD) is an irreversible blinding eye condition with complex genetic and environmental etiologies. Genetic testing for AMD for previously identified multiple-risk single nucleotide polymorphisms can help determine an individual’s future susceptibility. However, such testing has been discouraged until evidence shows that providing such information to symptomatic or pre-symptomatic individuals will alter their disease course. Therefore, we designed this study to investigate whether knowledge of AMD risk could stimulate the adoption of a healthier lifestyle that could lower the incidence of AMD later in life. We hypothesize that pre-symptomatic individuals informed of a high genetic risk of AMD are more likely to make quantifiable, positive lifestyle changes relative to participants informed of lower genetic risk or randomized to deferred disclosure of genetic testing results.MethodsThe Moran AMD Genetic Testing Assessment (MAGENTA) study is a phase 2, single-center, prospective, double-masked, randomized controlled trial conducted at the John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. Participants are randomized by a 3:1 allocation ratio to immediate and deferred disclosure groups and followed for 12 months. Skin, ocular, and serum carotenoid status, as well as nutritional and social surveys, are assessed at study visits. Skin carotenoid assessment is by resonance Raman spectroscopy and reflectance spectroscopy, ocular carotenoids are measured with Heidelberg Spectralis autofluorescence imaging and fluorescence lifetime imaging ophthalmoscopy (FLIO), and serum carotenoids are quantified using high-performance liquid chromatography. The primary outcome evaluates changes in skin carotenoid status in response to genetic risk disclosure. The secondary outcomes examine changes in ocular and serum carotenoid status in response to genetic risk disclosure. Also, we will correlate AMD genetic risk with baseline ocular and systemic carotenoid status and FLIO.DiscussionMAGENTA will provide much-needed evidence on whether pre-symptomatic testing for AMD risk can lead to quantifiable long-term changes in behavior and lifestyle associated with a lower incidence of AMD later in life. Findings from the MAGENTA trial will facilitate the design of a future larger, longer-term, multicenter phase 3 trial that could feature subgroup analysis, expanded measures of lifestyle modification, and potential active nutritional interventions.Trial registrationClinicalTrials.gov NCT05265624. Registered on March 3, 2022.

FLUORESCENCE LIFETIME IMAGING OPHTHALMOSCOPY IN PATIENTS WITH MACULAR TELANGIECTASIA TYPE 2 WITH AND WITHOUT DIABETES.

Macular telangiectasia type 2 (MacTel) is a vision-altering retinal disease with a high prevalence of diabetes. Differences between patients with MacTel with and without diabetes were investigated using fluorescence lifetime imaging ophthalmoscopy (FLIO). Eighty-six patients with MacTel (59 ± 12 years) were included. 40 patients (46%) did not have diabetes, 16 patients (19%) were prediabetic, and 30 patients (35%) were diabetic. Of these, seven had diabetic retinopathy. 18 diabetic patients without MacTel and 42 age-matched healthy controls were included. FLIO lifetimes (FLTs) were obtained in short (SSC, 498-560 nm) and long (LSC, 560-720 nm) spectral channels from different areas of interest using a Heidelberg Engineering FLIO. Fundus autofluorescece lifetimes did not show significant differences when comparing diabetic with nondiabetic MacTel eyes (MacTel zone, SSC, diabetic: 243 ± 65 ps; nondiabetic: 232 ± 51 ps; P = 1.0; LSC, diabetic: 327 ± 66 ps; nondiabetic: 309 ± 54 ps; P = 0.582). Longitudinal changes were similarly unrelated to diabetes status. A nonsignificant trend of increased FLT progression with higher body mass index was found. Fundus autofluorescece lifetimes in diabetic patients without MacTel were significantly shorter within the MacTel zone and longer in the periphery compared with diabetic patients with MacTel. Although MacTel has a high prevalence of diabetes, FLTs from the MacTel zone are unrelated to diabetes. Fluorescence lifetime imaging ophthalmoscopy retains diagnostic abilities in patients with MacTel even in the presence of prediabetes, diabetes, and advanced diabetic retinopathy. The lack of diabetic FLT changes in the periphery of diabetic patients with MacTel is an interesting finding that needs further investigation.

Review of Retinal Imaging Modalities for Hydroxychloroquine Retinopathy.

This review provides an overview of conventional and novel retinal imaging modalities for hydroxychloroquine (HCQ) retinopathy. HCQ retinopathy is a form of toxic retinopathy resulting from HCQ use for a variety of autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Each imaging modality detects a different aspect of HCQ retinopathy and shows a unique complement of structural changes. Conventionally, spectral-domain optical coherence tomography (SD-OCT), which shows loss or attenuation of the outer retina and/or retinal pigment epithelium-Bruch's membrane complex, and fundus autofluorescence (FAF), which shows parafoveal or pericentral abnormalities, are used to assess HCQ retinopathy. Additionally, several variations of OCT (retinal and choroidal thickness measurements, choroidal vascularity index, widefield OCT, en face imaging, minimum intensity analysis, and artificial intelligence techniques) and FAF techniques (quantitative FAF, near-infrared FAF, fluorescence lifetime imaging ophthalmoscopy, and widefield FAF) have been applied to assess HCQ retinopathy. Other novel retinal imaging techniques that are being studied for early detection of HCQ retinopathy include OCT angiography, multicolour imaging, adaptive optics, and retromode imaging, although further testing is required for validation.

Fluorescence lifetime imaging ophthalmoscopy and the influence of oral lutein/zeaxanthin supplementation on macular pigment (FLOS) – A pilot study

Oral lutein (L) and zeaxanthin (Z) supplementation enhances macular pigment optical density (MPOD) and plays a protective role in the development of age-related macular degeneration (AMD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a novel invivo retinal imaging method that has been shown to correlate to classical MPOD measurements and might contribute to a metabolic mapping of the retina in the future. Our aim was to show that oral supplementation of L and Z affects the FLIO signal in a positive way in patients with AMD. This was a prospective, single center, open label cohort study. Patients with early and intermediate AMD received oral L and Z supplementation during three months, and were observed for another three months after therapy termination. All visits included measurements of clinical parameters, serum L and Z concentration, MPOD measurements using heterochromatic flicker photometry, dual wavelength autofluorescence imaging, and FLIO. Correlation analysis between FLIO and MPOD were performed. Twenty-one patients completed the follow up period. Serum L and Z concentrations significantly increased during supplementation (mean difference 244.8ng/ml; 95% CI: 81.26-419.9, and 77.1ng/ml; 95% CI: 5.3-52.0, respectively). Mean MPOD units significantly increased (mean difference 0.06; 95% CI: 0.02-0.09; at 0.5°, 202; 95% CI: 58-345; at 2°, 1033; 95% CI: 288-1668; at 9° of eccentricity, respectively) after three months of supplementation with macular xanthophylls, which included L and Z. Median FLIO lifetimes in the foveal center significantly decreased from 277.3ps (interquartile range 230.2-339.1) to 261.0ps (interquartile range 231.4-334.4, p=0.027). All parameters returned to near-normal values after termination of the nutritional supplementation. A significant negative correlation was found between FLIO and MPOD (r2=0.57, p<0.0001). FLIO is able to detect subtle changes in MPOD after L and Z supplementation in patients with early and intermediate AMD. Our findings confirm the previous described negative correlation between FLIO and MPOD. Macular xanthophylls seem to contribute to short foveal lifetimes. This study is registered at ClinicalTrials.gov (identifier number NCT04761341).

Fluorescence lifetime distribution in phakic and pseudophakic healthy eyes.

To investigate the influence of the lens status and to describe fundus autofluorescence lifetimes (FLT) in a large cohort of healthy eyes across a wide age range. FLT data were acquired from healthy phakic and pseudophakic eyes using fluorescence lifetime imaging ophthalmoscopy (FLIO). Retinal autofluorescence was excited with a 473 nm laser and emitted autofluorescence was detected in a short and a long spectral channel (SSC: 498-560 nm; LSC: 560-720 nm). 141 healthy eyes from 141 participants (56 ± 18 years) were included. The shortest mean FLTs were measured within the macular center, followed by the temporal inner and outer ETDRS (Early Treatment Diabetic Retinopathy Study) grid segments, and the remaining areas of the inner and the outer ETDRS ring. In phakic participants (81%), mean, short and long FLTs correlated with the age (SSC: r2 = 0.54; LSC: r2 = 0.7; both p<0.0001) with an increase of about 33 ps in the SSC resp. 28 ps in the LSC per decade. In pseudophakic subjects (19%), mean FLTs only correlated with age in the long spectral channel (r2 = 0.44; p = 0.0002) but not in the short spectral channel (r2 = 0.066; p = 0.2). Fundus autofluorescence lifetimes are age dependent. FLTs in the SSC are more susceptible to lens opacities but less dependent on age changes, whereas FLTs in the LSC are largely independent of the lens status but display a higher degree of age dependency. ClinicalTrials.gov NCT01981148.

Novel imaging techniques for hydroxychloroquine retinopathy.

Hydroxychloroquine retinopathy is an increasingly recognized cause of iatrogenic, irreversible visual impairment due to the expanding use of hydroxychloroquine in combination with improvements in disease detection following advances in retinal imaging techniques. The prevalence of disease is estimated to be greater than 5% amongst individuals who have used the drug for 5 years or more. In addition to conventional imaging modalities, such as spectral-domain optical coherence tomography (OCT) and fundus autofluorescence (FAF), novel retinal imaging techniques such as en face OCT, OCT angiography, fluorescence lifetime imaging ophthalmoscopy, quantitative autofluorescence, and retromode imaging are capable of detecting structural changes in the retina. These novel retinal imaging techniques have shown promise in detecting earlier disease than is possible with current mainstream imaging modalities. Moreover, these techniques may identify disease progression as well as enabling functional correlation. In the future, these novel imaging techniques may further reduce the risk of visual loss from hydroxychloroquine retinopathy through the earlier detection of pre-clinical disease.

Combining fluorescence lifetime with spectral information in fluorescence lifetime imaging ophthalmoscopy (FLIO).

Fluorescence lifetime imaging ophthalmoscopy (FLIO) provides information on fluorescence lifetimes in two spectral channels as well as the peak emission wavelength (PEW) of the fluorescence. Here, we combine these measures in an integral three-dimensional lifetime-PEW metric vector and determine a normal range for this vector from measurements in young healthy subjects. While for these control subjects 97 (±8) % (median (interquartile range)) of all para-macular pixels were covered by this normal vector range, it was 67 (±55) % for the elderly healthy, 38 (±43) % for age-related macular degeneration (AMD)-suspect subjects, and only 6 (±4) % for AMD patients. The vectors were significantly different for retinal pigment epithelium (RPE) lesions in AMD patients from that of non-affected tissue (p < 0.001). Lifetime- PEW plots allowed to identify possibly pathologic fundus areas by fluorescence parameters outside a 95% quantile per subject. In a patient follow-up, changes in fluorescence parameters could be traced in the lifetime-PEW metric, showing their change over disease progression.

Suppression of natural lens fluorescence in fundus autofluorescence measurements: review of hardware solutions.

Fluorescence lifetime imaging ophthalmoscopy (FLIO), a technique for investigating metabolic changes in the eye ground, can reveal the first signs of diseases related to metabolism. The fluorescence of the natural lens overlies the fundus fluorescence. Although the influence of natural lens fluorescence can be somewhat decreased with mathematical models, excluding this influence during the measurement by using hardware enables more exact estimation of the fundus fluorescence. Here, we analyze four 1-photon excitation hardware solutions to suppress the influence of natural lens fluorescence: aperture stop separation, confocal scanning laser ophthalmoscopy, combined confocal scanning laser ophthalmoscopy and aperture stop separation, and dual point confocal scanning laser ophthalmoscopy. The effect of each principle is demonstrated in examples. The best suppression is provided by the dual point principle, realized with a confocal scanning laser ophthalmoscope. In this case, in addition to the fluorescence of the whole eye, the fluorescence of the anterior part of the eye is detected from a non-excited spot of the fundus. The intensity and time-resolved fluorescence spectral data of the fundus are derived through the subtraction of the simultaneously measured fluorescence of the excited and non-excited spots. Advantages of future 2-photon fluorescence excitation are also discussed. This study provides the first quantitative evaluation of hardware principles to suppress the fluorescence of the natural lens during measurements of fundus autofluorescence.

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

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. 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. 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. 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.

Insights into Metabolic Activity and Structure of the Retina through Multiphoton Fluorescence Lifetime Imaging Microscopy in Mice.

Fluorescence lifetime imaging microscopy (FLIM) evaluates the metabolic state of tissue based on reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging ophthalmoscopy (FLIO) can image the fundus of the eyes, but cannot detect NAD(P)H. We used multiphoton FLIM to study the metabolic state of the retina in fixed eyes of wild-type mice C57BL6/J. We sectioned the eye using a polyacrylamide gel-embedding technique and estimated the percentage of bound NAD(P)H. We found that oxidative phosphorylation was the predominant metabolic state, particularly in the inner retina, when a fixed retina was used. We also demonstrated the feasibility of FAD imaging of the retina. In addition, we demonstrated that autofluorescence and various FLIM channels, such as hemoglobin, melanin and collagen, can be used to evaluate the structure of the retina and other parts of the eye without any special staining.

Adaptive optics fluorescence lifetime imaging ophthalmoscopy of in vivo human retinal pigment epithelium.

The intrinsic fluorescence properties of lipofuscin - naturally occurring granules that accumulate in the retinal pigment epithelium - are a potential biomarker for the health of the eye. A new modality is described here which combines adaptive optics technology with fluorescence lifetime detection, allowing for the investigation of functional and compositional differences within the eye and between subjects. This new adaptive optics fluorescence lifetime imaging ophthalmoscope was demonstrated in 6 subjects. Repeated measurements between visits had a minimum intraclass correlation coefficient of 0.59 Although the light levels were well below maximum permissible exposures, the safety of the imaging paradigm was tested using clinical measures; no concerns were raised. This new technology allows for in vivo adaptive optics fluorescence lifetime imaging of the human RPE mosaic.

Adaptive optics two-photon excited fluorescence lifetime imaging ophthalmoscopy of photoreceptors and retinal pigment epithelium in the living non-human primate eye.

Fluorescence lifetime imaging has demonstrated promise as a quantitative measure of cell health. Adaptive optics two-photon excited fluorescence (TPEF) ophthalmoscopy enables excitation of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle, providing in vivo visualization of retinal structure and function at the cellular scale. Combining these technologies revealed that macaque cones had a significantly longer mean TPEF lifetime than rods at 730 nm excitation. At 900 nm excitation, macaque photoreceptors had a significantly longer mean TPEF lifetime than the retinal pigment epithelium layer. AOFLIO can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes.