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

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Serine and Lipid Metabolism in Macular Disease and Peripheral Neuropathy.

BACKGROUND: Identifying mechanisms of diseases with complex inheritance patterns, such as macular telangiectasia type 2, is challenging. A link between macular telangiectasia type 2 and altered serine metabolism has been established previously. METHODS: Through exome sequence analysis of a patient with macular telangiectasia type 2 and his family members, we identified a variant in SPTLC1 encoding a subunit of serine palmitoyltransferase (SPT). Because mutations affecting SPT are known to cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), we examined 10 additional persons with HSAN1 for ophthalmologic disease. We assayed serum amino acid and sphingoid base levels, including levels of deoxysphingolipids, in patients who had macular telangiectasia type 2 but did not have HSAN1 or pathogenic variants affecting SPT. We characterized mice with low serine levels and tested the effects of deoxysphingolipids on human retinal organoids. RESULTS: Two variants known to cause HSAN1 were identified as causal for macular telangiectasia type 2: of 11 patients with HSAN1, 9 also had macular telangiectasia type 2. Circulating deoxysphingolipid levels were 84.2% higher among 125 patients with macular telangiectasia type 2 who did not have pathogenic variants affecting SPT than among 94 unaffected controls. Deoxysphingolipid levels were negatively correlated with serine levels, which were 20.6% lower than among controls. Reduction of serine levels in mice led to increases in levels of retinal deoxysphingolipids and compromised visual function. Deoxysphingolipids caused photoreceptor-cell death in retinal organoids, but not in the presence of regulators of lipid metabolism. CONCLUSIONS: Elevated levels of atypical deoxysphingolipids, caused by variant SPTLC1 or SPTLC2 or by low serine levels, were risk factors for macular telangiectasia type 2, as well as for peripheral neuropathy. (Funded by the Lowy Medical Research Institute and others.).

Gut microbiome and plasma metabolome changes in rats after oral gavage of nanoparticles: sensitive indicators of possible adverse health effects.

BACKGROUND: The oral uptake of nanoparticles is an important route of human exposure and requires solid models for hazard assessment. While the systemic availability is generally low, ingestion may not only affect gastrointestinal tissues but also intestinal microbes. The gut microbiota contributes essentially to human health, whereas gut microbial dysbiosis is known to promote several intestinal and extra-intestinal diseases. Gut microbiota-derived metabolites, which are found in the blood stream, serve as key molecular mediators of host metabolism and immunity. RESULTS: Gut microbiota and the plasma metabolome were analyzed in male Wistar rats receiving either SiO2 (1000 mg/kg body weight/day) or Ag nanoparticles (100 mg/kg body weight/day) during a 28-day oral gavage study. Comprehensive clinical, histopathological and hematological examinations showed no signs of nanoparticle-induced toxicity. In contrast, the gut microbiota was affected by both nanoparticles, with significant alterations at all analyzed taxonomical levels. Treatments with each of the nanoparticles led to an increased abundance of Prevotellaceae, a family with gut species known to be correlated with intestinal inflammation. Only in Ag nanoparticle-exposed animals, Akkermansia, a genus known for its protective impact on the intestinal barrier was depleted to hardly detectable levels. In SiO2 nanoparticles-treated animals, several genera were significantly reduced, including probiotics such as Enterococcus. From the analysis of 231 plasma metabolites, we found 18 metabolites to be significantly altered in Ag-or SiO2 nanoparticles-treated rats. For most of these metabolites, an association with gut microbiota has been reported previously. Strikingly, both nanoparticle-treatments led to a significant reduction of gut microbiota-derived indole-3-acetic acid in plasma. This ligand of the arylhydrocarbon receptor is critical for regulating immunity, stem cell maintenance, cellular differentiation and xenobiotic-metabolizing enzymes. CONCLUSIONS: The combined profiling of intestinal microbiome and plasma metabolome may serve as an early and sensitive indicator of gut microbiome changes induced by orally administered nanoparticles; this will help to recognize potential adverse effects of these changes to the host.

LONGITUDINAL FLUORESCENCE LIFETIME IMAGING OPHTHALMOSCOPY ANALYSIS IN PATIENTS WITH MACULAR TELANGIECTASIA TYPE 2 (MacTel).

PURPOSE: Fluorescence lifetime imaging ophthalmoscopy (FLIO) shows characteristic patterns in macular telangiectasia Type 2 (MacTel). This study investigates FLIO changes over time to better understand disease progression. METHODS: Thirty-three patients with MacTel (age 60 ± 15 years) were followed at the Moran Eye Center with a prototype Heidelberg Engineering FLIO. The mean follow-up time was 19 ± 8 months (range 6-34 months). Fundus autofluorescence was excited at 473 nm, and FLIO lifetimes were recorded in in short (498-560 nm) and long (560-720 nm) spectral wavelengths channels. RESULTS: Autofluorescence lifetimes imaging ophthalmoscopy lifetimes from the MacTel area prolonged significantly over time (subfield T1, baseline: short spectral channel 210 ± 54 ps, long spectral channel 269 ± 58 ps; follow-up: short spectral channel 225 ± 59 ps, P < 0.001, long spectral channel 282 ± 64 ps, P < 0.01). The average 12-months prolongation of FLIO lifetimes was 9 ps (short spectral channel) and 8 ps (long spectral channel). Autofluorescence lifetimes changes correlated positively with ellipsoid zone loss and negatively with changes in retinal thickness. CONCLUSION: Autofluorescence lifetimes in MacTel slowly prolong over time, and temporal patterns progress to full rings. Detailed knowledge about FLIO changes will aid in understanding disease development and progression.

Ocular Carotenoid Status in Health and Disease.

Retinal carotenoids are dietary nutrients that uniquely protect the eye from light damage and various retinal pathologies. Their antioxidative properties protect the eye from many retinal diseases, such as age-related macular degeneration. As many retinal diseases are accompanied by low carotenoid levels, accurate noninvasive assessment of carotenoid status can help ophthalmologists identify the patients most likely to benefit from carotenoid supplementation. This review focuses on the different methods available to assess carotenoid status and highlights disease-related changes and potential nutritional interventions.

FLUORESCENCE LIFETIME IMAGING OPHTHALMOSCOPY (FLIO) PATTERNS IN CLINICALLY UNAFFECTED CHILDREN OF MACULAR TELANGIECTASIA TYPE 2 (MACTEL) PATIENTS.

PURPOSE: Macular telangiectasia Type 2 (MacTel) is an inherited retinal disease following an autosomal dominant pattern with late onset and reduced penetrance. Fluorescence lifetime imaging ophthalmoscopy (FLIO) enhances diagnosis by showing distinct changes in MacTel. This study investigates FLIO-associated changes in clinically unaffected family members. METHODS: Eighty-one patients with MacTel (61 ± 12 years), 33 clinically healthy children under age 40 years of these MacTel patients (MacTel-C; 31 ± 6 years), 27 other family members (children over age 40 years, siblings, and parents) and 30 controls were investigated with the Heidelberg FLIO. All subjects underwent multimodal conventional imaging, including optical coherence tomography, blue-light reflectance, fluorescein angiography, and macular pigment imaging. RESULTS: All 81 patients with MacTel showed typical FLIO patterns. Of the 33 investigated MacTel-C with completely normal eye examinations and conventional imaging, 12 (36%) show FLIO patterns consistent with early MacTel. CONCLUSION: Prolonged FLIO lifetimes in the parafoveal area within the short spectral channel, especially temporally, are MacTel-specific. Fluorescence lifetime imaging ophthalmoscopy detects these lifetime patterns in over one-third of clinically unaffected MacTel-C. Although further studies will be necessary to determine the specificity of FLIO, it may help diagnose MacTel before conventional imaging modalities show changes or patients experience visual disturbances. Early detection may facilitate future gene discovery studies and interventional trials.

ABSENCE OF MACULAR DEGENERATION IN A PATIENT WITH ACERULOPLASMINEMIA.

PURPOSE: To describe the clinical, histological, electrophysiologic, and multimodal imaging findings in a 76-year-old patient with aceruloplasminemia with low genetic risk of age-related macular degeneration (AMD). METHODS: Clinical examination as well as multimodal imaging including fundus photography, optical coherence tomography, fluorescence lifetime imaging ophthalmoscopy imaging, and full-field and multifocal electroretinography were performed on one patient with aceruloplasminemia. The ceruloplasmin gene was sequenced to confirm a known mutation. Single nucleotide polymorphism genotyping of known AMD risk alleles was performed to characterize the AMD risk profile of the patient. Prussian blue staining in postmortem retinal sections was used to confirm iron accumulation. RESULTS: A homozygous mutation in the ceruloplasmin gene was detected at position c.395-1 G>A. The clinical assessment and imaging of the patient did not show any findings of AMD. Fundus examination revealed yellow flecks in the midperiphery with notable absence of macular drusen or geographic atrophy. Genotyping for AMD risk alleles revealed a low AMD risk profile. Histopathologic analysis confirms iron accumulation in retinal pigment epithelial cells. CONCLUSION: In contrast to a previous report, these findings suggest that neither aceruloplasminemia nor iron accumulation was sufficient to cause AMD in this patient.

Extending Peripheral Retinal Vascularization in Retinopathy of Prematurity Through Regulation of VEGF Signaling.

PURPOSE: Experimental studies provide evidence that regulation of VEGF receptor-2 signaling in endothelial cells orders cell divisions and extends developmental angiogenesis while inhibiting pathologic intravitreal angiogenesis and has relevance to retinopathy of prematurity (ROP). We tested the hypothesis that intravitreal anti-VEGF would extend vascularization into peripheral avascular retina in human type 1 ROP compared with controls. DESIGN: Retrospective, nonrandomized treatment comparison. METHODS: The study was conducted at an academic institution, with the study population comprising all premature infants screened for ROP from January 2019 through December 2022. The experimental group included type 1 ROP treated with bilateral bevacizumab (0.25 mg) and had adequate fundus imaging by a certified ophthalmic photographer at 2 examinations: within 2 weeks of treatment and 1-3 weeks later. A control group included gestational age- and birthweight-matched infants with ROP less severe than type 1 ROP. The main outcome measure was extent of temporal retinal vasculature measured by a masked analyst between treated and control eyes. Paired and nonpaired t tests were used. RESULTS: Of 382 screened infants, 34 developed type 1 ROP; 11 comprised the experimental group and 11 the control group. At baseline, there was a trend toward shorter temporal vascular extent in treatment compared with control groups (3667±547 vs 4262±937 pixels, 95% CI -1277, 88; P = .084) but no difference between groups at follow-up (P = .945). Vascular extension was significantly greater in the treatment than control (872±521 vs 253±151 pixels, 95% CI 262, 977; P = .003), showing catch-up growth. CONCLUSIONS: This clinical evidence supports laboratory-based studies that regulation of VEGF using an intravitreal anti-VEGF agent increases developmental angiogenesis into the peripheral avascular retina while inhibiting pathologic intravitreal angiogenesis in ROP.

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

Fluorescence lifetime imaging ophthalmoscopy: autofluorescence imaging and beyond.

Fluorescence lifetime imaging ophthalmoscopy, FLIO, has gained large interest in the scientific community in the recent years. It is a noninvasive imaging modality that has been shown to provide additional information to conventional imaging modalities. The FLIO device is based on a Heidelberg Engineering Spectralis system. Autofluorescence lifetimes are excited at 473 nm and recorded in two spectral wavelength channels, a short spectral channel (SSC, 498-560 nm) and a long spectral channel (LSC, 560-720 nm). Typically, mean autofluorescence lifetimes in a 30° retinal field are investigated. FLIO shows a clear benefit for imaging different retinal diseases. For example, in age-related macular degeneration (AMD), ring patterns of prolonged FLIO lifetimes 1.5-3.0 mm from the fovea can be appreciated. Macular telangiectasia type 2 (MacTel) shows a different pattern, with prolonged FLIO lifetimes within the typical MacTel zone. In Stargardt disease, retinal flecks can be appreciated even before they are visible with other imaging modalities. Early hydroxychloroquine toxicity appears to be detectable with FLIO. This technique has more potential that has yet to be discovered. This review article focuses on current knowledge as well as pitfalls of this technology. It highlights clinical benefits of FLIO imaging in different ophthalmic and systemic diseases, and provides an outlook with perspectives from the authors.

Comparing Fluorescence Lifetime Imaging Ophthalmoscopy in Atrophic Areas of Retinal Diseases.

Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a non-invasive imaging modality to investigate the human retina. This study compares FLIO lifetimes in different degenerative retinal diseases. Methods: Included were eyes with retinal pigment epithelium (RPE) and/or photoreceptor atrophy due to Stargardt disease (n = 66), pattern dystrophy (n = 18), macular telangiectasia type 2 (n = 49), retinitis pigmentosa (n = 28), choroideremia (n = 26), and geographic atrophy (n = 32) in age-related macular degeneration, as well as 37 eyes of 37 age-matched healthy controls. Subjects received Heidelberg Engineering FLIO, autofluorescence intensity, and optical coherence tomography imaging. Amplitude-weighted mean FLIO lifetimes (τm) were calculated and analyzed. Results: Retinal FLIO lifetimes show significant differences depending on the disease. Atrophic areas in geographic atrophy and choroideremia showed longest mean FLIO lifetimes. τm values within areas of RPE and outer nuclear layer atrophy were significantly longer than within areas with preserved outer nuclear layer (P < 0.001) or non-atrophic areas (P < 0.001). Conclusions: FLIO is able to contribute additional information regarding differences in chronic degenerative retinal diseases. Although it cannot replace conventional autofluorescence imaging, FLIO adds to the knowledge in these diseases and may help with the correct differentiation between them. This may lead to a more in-depth understanding of the pathomechanisms related to atrophy and types of progression. Translational Relevance: Differences between atrophic retinal diseases highlighted by FLIO may indicate separate pathomechanisms leading to atrophy and disease progression.

Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) in Patients with Choroideremia.

Purpose: To provide a detailed characterization of choroideremia (CHM) using fluorescence lifetime imaging ophthalmoscopy (FLIO) and to provide a deeper understanding of disease-related changes and progression. Methods: Twenty-eight eyes of 14 patients with genetically confirmed CHM (mean age, 28 ± 14 years) and 14 age-matched healthy subjects were investigated in this study. FLIO images of a 30° retinal field were collected at the Moran Eye Center using a Heidelberg Engineering FLIO device. FLIO lifetimes were recorded in short spectral channels (SSC; 498-560 nm) and long spectral channels (LSC; 560-720 nm), and mean autofluorescence lifetimes (τm) were calculated. Optical coherence tomography (OCT) scans were recorded for each patient. Three patients were re-imaged after a year. Results: Patients with CHM exhibit specific FLIO lifetime patterns. Prolonged FLIO lifetimes (around 600-700 ps) were found in the peripheral macula corresponding to atrophy in OCT imaging. In the central macula, τm was unrelated to autofluorescence intensity. Some areas of persistent retinal pigment epithelial islands had prolonged FLIO lifetimes, whereas other areas of hypofluorescence had short FLIO lifetimes. At 1-year follow-up, FLIO lifetimes were significantly prolonged within atrophic areas (P < 0.05). Conclusions: FLIO shows distinct patterns in patients with CHM, indicating lesions of atrophy and areas of preserved function in the presence or absence of findings in fundus autofluorescence intensity images. FLIO may provide differentiated knowledge about pathophysiology and atrophy progression in CHM compared to conventional imaging modalities. Translational Relevance: FLIO shows distinctive lifetime patterns that potentially identify areas of function, atrophy, and disease progression in patients with CHM.

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.

Autofluorescence Lifetimes Measured with Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) Are Affected by Age, but Not by Pigmentation or Gender.

Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a novel modality to investigate the human retina. This study aims to characterize the effects of age, pigmentation, and gender in FLIO. Methods: A total of 97 eyes from 97 healthy subjects (mean age 37 ± 18 years, range 9-85 years) were investigated in this study. This study included 47 (49%) females and 50 males. The pigmentation analysis was a substudy including 64 subjects aged 18 to 40 years (mean age 29 ± 6 years). These were categorized in groups A (darkly pigmented, 8), B (medium pigmented, 20), and C (lightly pigmented, 36). Subjects received Heidelberg Engineering FLIO and optical coherence tomography imaging. Retinal autofluorescence lifetimes were detected in two spectral channels (short spectral channel [SSC]: 498-560 nm; long spectral channel [LSC]: 560-720 nm), and amplitude-weighted mean fluorescence lifetimes (τm) were calculated. Additionally, autofluorescence lifetimes of melanin were measured in a cuvette. Results: Age significantly affected FLIO lifetimes, and age-related FLIO changes in the SSC start at approximately age 35 years, whereas the LSC shows a consistent prolongation with age from childhood. There were no gender- or pigmentation-specific significant differences of autofluorescence lifetimes. Conclusions: This study confirms age-effects in FLIO but shows that the two channels are affected differently. The LSC appears to show the lifelong accumulation of lipofuscin. Furthermore, it is important to know that neither gender nor pigmentation significantly affect FLIO lifetimes. Translational Relevance: This study helps to understand the FLIO technology better, which will aid in conducting future clinical studies.

Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) in Eyes With Pigment Epithelial Detachments Due to Age-Related Macular Degeneration.

Purpose: To investigate fluorescence lifetime imaging ophthalmoscopy (FLIO) in neovascular AMD and pigment epithelial detachments (PEDs). Methods: A total of 46 eyes with PEDs (>350 µm) as well as age-matched healthy controls were included in this study. We found 28 eyes showed neovascular AMD (nvAMD), and 17 had nonneovascular (dry) AMD (dAMD). The Heidelberg Engineering FLIO excited fluorescence at 473 nm. Fluorescence decays were detected in two spectral channels (498-560 nm; 560-720 nm) to determine fluorescence lifetimes of endogenous fluorophores in their specific spectral emission ranges. Mean fluorescence lifetimes (τm) were investigated. Multimodal imaging was reviewed by two ophthalmologists who circumscribed and classified PEDs as either serous (n = 4), hemorrhagic (n = 4), fibrovascular (n = 16), drusenoid (n = 17), or mixed (n = 5). Blood samples from a healthy subject and a patient with PED were investigated in a quartz cuvette. Results: Eyes with nvAMD show similar FLIO patterns to dAMD: ring-shaped prolongations of τm 3 to 6 mm from the fovea. Different PED-forms show characteristic τm, while serous and hemorrhagic PEDs exhibit shortened τm, drusenoid PEDs show prolonged τm, and τm in fibrovascular PEDs is variable. Areas corresponding to sub-/intraretinal fluid display shortened τm. Ex vivo studies of blood also show short τm. Conclusions: The previously described dAMD-related FLIO pattern is also present in nvAMD. Short τm in serous, fibrovascular, and hemorrhagic PEDs as well as sub/intraretinal fluid may disrupt this pattern. FLIO appears to differentiate between PEDs, hemorrhage, and fluid. Additionally, ex vivo studies of human blood help to better interpret FLIO images.

Bleaching effects and fluorescence lifetime imaging ophthalmoscopy.

This study investigates the influence of photopigment bleaching on autofluorescence lifetimes in the fundus in 21 young healthy volunteers. Three measurements of 30° retinal fields in two spectral channels (SSC: 498-560 nm, LSC: 560-720 nm) were obtained for each volunteer using fluorescence lifetime imaging ophthalmoscopy (FLIO). After dark-adaptation by wearing a custom-made lightproof mask for 30 minutes, the first FLIO-measurement was recorded (dark-adapted state). Subsequently, the eye was bleached for 1 minute (luminance: 3200 cd/m2), followed by a second FLIO-measurement (bleached state). Following an additional 10 minute dark adaptation using the mask, a final FLIO-measurement was recorded (recovered state). Average values of the fluorescence lifetimes were calculated from within different areas of a standardized early treatment diabetic retinopathy study (ETDRS) grid (central area, inner and outer rings). The acquisition time in the bleached state was significantly shortened by approximately 20%. The SSC did not show any significant changes in fluorescence lifetimes with photopigment bleaching, only the LSC showed small but significant bleaching-related changes in the fluorescence lifetimes τ1 and τ2 from all regions, as well as the mean fluorescence lifetime in the central area. The fluorescence lifetime differences caused by bleaching were by far less significant than pathological changes caused by eye diseases. The magnitudes of fluorescence lifetime changes are <10% and do not interfere with healthy or disease related FLIO patterns. Thus, we conclude that bleaching is not a relevant confounder in current clinical applications of FLIO.

Imaging of Hydroxychloroquine Toxicity with Fluorescence Lifetime Imaging Ophthalmoscopy.

PURPOSE: To investigate the impact of retinal toxicity from hydroxychloroquine (HCQ) on fundus autofluorescence lifetimes using fluorescence lifetime imaging ophthalmoscopy (FLIO). DESIGN: Cross-sectional study. PARTICIPANTS: Twenty-four eyes of 12 patients with definite HCQ toxicity, 31 eyes of 16 clinically normal patients at high risk of developing HCQ toxicity (taking HCQ longer than 5 years), and 16 eyes of 8 clinically normal patients at low risk of developing HCQ toxicity (taking HCQ fewer than 5 years), as well as 22 age-matched healthy subjects. METHODS: Fluorescence lifetime images of a 30° retinal field centered at the fovea were collected at the Moran Eye Center, Salt Lake City, Utah. A prototype Heidelberg Engineering Spectralis-based FLIO was used to detect autofluorescence lifetimes in short (SSC; 498-560 nm) and long (LSC; 560-720 nm) spectral channels. Mean fluorescence lifetimes were calculated. OCT scans and macular pigment measures were also recorded. Additionally, the autofluorescence lifetimes of HCQ were measured in a cuvette. MAIN OUTCOME MEASURES: Mean autofluorescence lifetimes (τm). RESULTS: All patients with HCQ toxicity showed significantly prolonged FLIO lifetimes in regions of damage, typically in a bulls-eye distribution corresponding to toxic lesions in the retina (SSC: lesion, 400 ps; unremarkable retina, 294 ps; P < 0.001; LSC: lesion, 404 ps; unremarkable retina, 316 ps; P < 0.001). Some clinically normal patients at high risk (9 of 16) and at low risk (2 of 8) of developing HCQ toxicity also showed prolonged FLIO lifetimes in the parafoveal region, whereas age-matched healthy subjects did not. HCQ at a concentration of 46 mM exhibited long autofluorescence lifetimes of around 1100 ps in either spectral channel. CONCLUSIONS: Fluorescence lifetime imaging ophthalmoscopy seems to detect retinal toxicity from HCQ at very early stages and could be a novel method to detect retinal toxicity before irreversible damage is manifest.

Fundus autofluorescence beyond lipofuscin: lesson learned from ex vivo fluorescence lifetime imaging in porcine eyes.

Fundus autofluorescence (FAF) imaging is a well-established method in ophthalmology; however, the fluorophores involved need more clarification. The FAF lifetimes of 20 post mortem porcine eyes were measured in two spectral channels using fluorescence lifetime imaging ophthalmoscopy (FLIO) and compared with clinical data from 44 healthy young subjects. The FAF intensity ratio of the short and the long wavelength emission (spectral ratio) was determined. Ex vivo porcine fundus fluorescence emission is generally less intense than that seen in human eyes. The porcine retina showed significantly (p<0.05) longer lifetimes than the retinal pigment epithelium (RPE): 584 ± 128 ps vs. 121 ± 55 ps 498-560 nm, 240 ± 42 ps vs. 125 ± 20 ps at 560-720 nm. Furthermore, the lifetimes of the porcine RPE were significantly shorter (121 ± 55 ps and 125 ± 20 ps) than those measured from human fundus in vivo (162 ± 14 ps and 179 ± 13 ps, respectively). The fluorescence emission of porcine retina was shifted towards a shorter wavelength compared to that of RPE and human FAF. This data shows the considerable contribution of fluorophores in the neural retina to total FAF intensity in porcine eyes.