Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent...Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation.High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration(AMD);however,this has not been confirmed in histology so far.Here,based on our previous dataset of RPE granule characteristics,we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence(AF)intensities.Methods:RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy(HR-SIM)and laser scanning microscopy(LSM).Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules.In addition,total AF intensity per cell and granule density(number of granules per cell area)were determined.For the final analysis,RPE cells with total granule number below 5th or above the 95th percentile,or a total AF intensity±1.5 standard deviations above or below the mean were included,and compared to the average RPE cell at the same location.Data are presented as mean±standard deviation.Results:Within 420 RPE cells examined,42 cells were further analyzed due to extremes regarding total granule numbers.In addition,20 RPE cells had AF 1.5 standard deviations below,28 RPE cells above the mean local AF intensity.Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity.RPE cells with high granule content have nearly twice(1.8 times)as many granules as an average RPE cell.Conclusions:In normal eyes,outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells,suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level.The AF of a cell is related to the co展开更多
Background:Soft drusen and basal linear deposit(BLinD)are two forms of the same extracellular lipid rich material that together make up an Oil Spill on Bruch’s membrane(BrM).Drusen are focal and can be recognized cli...Background:Soft drusen and basal linear deposit(BLinD)are two forms of the same extracellular lipid rich material that together make up an Oil Spill on Bruch’s membrane(BrM).Drusen are focal and can be recognized clinically.In contrast BLinD is thin and diffusely distributed,and invisible clinically,even on highest resolution OCT,but has been detected on en face hyperspectral autofluorescence(AF)imaging ex vivo.We sought to optimize histologic hyperspectral AF imaging and image analysis for recognition of drusen and sub-RPE deposits(including BLinD and basal laminar deposit),for potential clinical application.Methods:Twenty locations specifically with drusen and 12 additional locations specifically from fovea,perifovea and mid-periphery from RPE/BrM flatmounts from 4 AMD donors underwent hyperspectral AF imaging with 4 excitation wavelengths(λex 436,450,480 and 505 nm),and the resulting image cubes were simultaneously decomposed with our published non-negative matrix factorization(NMF).Rank 4 recovery of 4 emission spectra was chosen for each excitation wavelength.Results:A composite emission spectrum,sensitive and specific for drusen and presumed sub-RPE deposits(the SDr spectrum)was recovered with peak at 510-520 nm in all tissues with drusen,with greatest amplitudes at excitationsλ_(ex)436,450 and 480 nm.The RPE spectra of combined sources Lipofuscin(LF)/Melanolipofuscin(MLF)were of comparable amplitude and consistently recapitulated the spectra S1,S2 and S3 previously reported from all tissues:tissues with drusen,foveal and extra-foveal locations.Conclusions:A clinical hyperspectral AF camera,with properly chosen excitation wavelengths in the blue range and a hyperspectral AF detector,should be capable of detecting and quantifying drusen and sub-RPE deposits,the earliest known lesions of AMD,before any other currently available imaging modality.展开更多
Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieot...Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH c展开更多
文摘Background:Cells of the retinal pigment epithelium(RPE)accumulate different kinds of granules(lipofuscin,melanolipofuscin,melanosomes)within their cell bodies,with lipofuscin and melanolipofuscin being autofluorescent after blue light excitation.High amounts of lipofuscin granules within the RPE have been associated with the development of RPE cell death and age-related macular degeneration(AMD);however,this has not been confirmed in histology so far.Here,based on our previous dataset of RPE granule characteristics,we report the characteristics of RPE cells from human donor eyes that show either high or low numbers of intracellular granules or high or low autofluorescence(AF)intensities.Methods:RPE flatmounts of fifteen human donors were examined using high-resolution structured illumination microscopy(HR-SIM)and laser scanning microscopy(LSM).Autofluorescent granules were analyzed regarding AF phenotype and absolute number of granules.In addition,total AF intensity per cell and granule density(number of granules per cell area)were determined.For the final analysis,RPE cells with total granule number below 5th or above the 95th percentile,or a total AF intensity±1.5 standard deviations above or below the mean were included,and compared to the average RPE cell at the same location.Data are presented as mean±standard deviation.Results:Within 420 RPE cells examined,42 cells were further analyzed due to extremes regarding total granule numbers.In addition,20 RPE cells had AF 1.5 standard deviations below,28 RPE cells above the mean local AF intensity.Melanolipofuscin granules predominate in RPE cells with low granule content and low AF intensity.RPE cells with high granule content have nearly twice(1.8 times)as many granules as an average RPE cell.Conclusions:In normal eyes,outliers regarding autofluorescent granule load and AF intensity signals are rare among RPE cells,suggesting that granule deposition and subsequent AF follows intrinsic control mechanisms at a cellular level.The AF of a cell is related to the co
文摘Background:Soft drusen and basal linear deposit(BLinD)are two forms of the same extracellular lipid rich material that together make up an Oil Spill on Bruch’s membrane(BrM).Drusen are focal and can be recognized clinically.In contrast BLinD is thin and diffusely distributed,and invisible clinically,even on highest resolution OCT,but has been detected on en face hyperspectral autofluorescence(AF)imaging ex vivo.We sought to optimize histologic hyperspectral AF imaging and image analysis for recognition of drusen and sub-RPE deposits(including BLinD and basal laminar deposit),for potential clinical application.Methods:Twenty locations specifically with drusen and 12 additional locations specifically from fovea,perifovea and mid-periphery from RPE/BrM flatmounts from 4 AMD donors underwent hyperspectral AF imaging with 4 excitation wavelengths(λex 436,450,480 and 505 nm),and the resulting image cubes were simultaneously decomposed with our published non-negative matrix factorization(NMF).Rank 4 recovery of 4 emission spectra was chosen for each excitation wavelength.Results:A composite emission spectrum,sensitive and specific for drusen and presumed sub-RPE deposits(the SDr spectrum)was recovered with peak at 510-520 nm in all tissues with drusen,with greatest amplitudes at excitationsλ_(ex)436,450 and 480 nm.The RPE spectra of combined sources Lipofuscin(LF)/Melanolipofuscin(MLF)were of comparable amplitude and consistently recapitulated the spectra S1,S2 and S3 previously reported from all tissues:tissues with drusen,foveal and extra-foveal locations.Conclusions:A clinical hyperspectral AF camera,with properly chosen excitation wavelengths in the blue range and a hyperspectral AF detector,should be capable of detecting and quantifying drusen and sub-RPE deposits,the earliest known lesions of AMD,before any other currently available imaging modality.
基金Canadian Institute for Health Researchgrant number:MOP74600+3 种基金Canadian Foundation for Innovationgrant number:N°9b84Groupe de Recherche Universitaire sur Mdicament grant to AC,FRSQ-NSFCgrant number:N°5540
文摘Objective：To study the mitochondrial redox state in experimental animals to sensitively detect early signs of mitochondrial function in pathophysiologieal conditions, such as isehemia. Methods： Fluorescence of nieotinamide adenine dinucleotide （phosphate） , or NAD（P）H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply of cardiomyocytes, is studied for non-invasive fluorescent probing of the mitochondrial function. Examination of NAD （P）H fluorescence in living cardiomyocytes following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting （TCSPC） , is based on the simultaneous measurement of the fluorescence spectra and lifetime. Results ： The dynamic characteristics of NAD （P） H fluorescence decay in living rat cardiomyocytes show that at least a 3-exponential decay model, with 0.4 - 0.7 ns, 1.2 - 1.9 ns and 8.0 - 13.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescenee （AF）. Decay-associated spectra （DSA） revealed the presence of 4 spectrally-distinct populations of NADH molecules in eardiomyocytes with spectral maximum at 470 nm for short-lifetime pool for the first time, and emission peaks at 450 nm, 470 nm and 490 nm for intermediate and long-lifetime pools. Increased mitochondrial NADH content ratio by ketone bodies enhanced the AF intensity, without the significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF and shortened the average fluorescence lifetime. Dinitrophenol （DNP）, an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF,broadened the spectral shoulder at 520 nm and increased the average lifetime. These effects, comparable to the changes in the concentration and in the rate of dehydrogenation of NADH in vitro, were also examined under ischemia-mimetic conditions. Conclusion： Our findings anticipate a contribution of both conformational NADH c
