Non-Invasive Examination of Plant Surfaces by Opto-Electronic Means--Using Russet as a Prime Example.

(1) BACKGROUND: Many disorders and diseases of agricultural produce change the physical features of surfaces of plant organs; in terms of russet, e.g., of apple or pear, affected fruit peel becomes rough and brown in color, which is associated with changes in light reflection; (2) OBJECTIVE AND METHODS: The objective of the present project was an interdisciplinary approach between horticultural science and engineering to examine two new innovative technologies as to their suitability for the non-destructive determination of surfaces of plant organs, using russet as an example, and (a) an industrial luster sensor (type CZ-H72, Keyence, Japan) and (b) a new type of a three-dimensional (3D) color microscope (VHX 5000); (3) RESULTS: In the case of russet, i.e., suberinization of the fruit peel, peel roughness increased by ca. 2.5-fold from ca. 20 µm to ca. 50 µm on affected fruit sections when viewed at 200× magnification. Russeted peel showed significantly reduced luster, with smaller variation than russet-devoid peel with larger variation; (4) CONCLUSION: These results indicate that both sensors are suitable for biological material and their use for non-contact, non-invasive detection of surface disorders on agricultural produce such as russet may be a very powerful tool for many applications in agriculture and beyond in the future.

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.

Spectral fundus autofluorescence peak emission wavelength in ageing and AMD.

PURPOSE: To investigate the spectral characteristics of fundus autofluorescence (FAF) in AMD patients and controls. METHODS: Fundus autofluorescence spectral characteristics was described by the peak emission wavelength (PEW) of the spectra. Peak emission wavelength (PEW) was derived from the ratio of FAF recordings in two spectral channels at 500-560 nm and 560-720 nm by fluorescence lifetime imaging ophthalmoscopy. The ratio of FAF intensity in both channels was related to PEW by a calibration procedure. Peak emission wavelength (PEW) measurements were done in 44 young (mean age: 24.0 ± 3.8 years) and 18 elderly (mean age: 67.5 ± 10.2 years) healthy subjects as well as 63 patients with AMD (mean age: 74.0 ± 7.3 years) in each pixel of a 30° imaging field. The values were averaged over the central area, the inner and the outer ring of the ETDRS grid. RESULTS: There was no significant difference between PEW in young and elderly controls. However, PEW was significantly shorter in AMD patients (ETDRS grid centre: 571 ± 26 nm versus 599 ± 17 nm for elderly controls, inner ring: 596 ± 17 nm versus 611 ± 11 nm, outer ring: 602 ± 16 nm versus 614 ± 11 nm). After a mean follow-up time of 50.8 ± 10.8 months, the PEW in the patients decreased significantly by 9 ± 19 nm in the inner ring of the grid. Patients, showing progression to atrophic AMD in the follow up, had significantly (p ≤ 0.018) shorter PEW at baseline than non-progressing patients. CONCLUSIONS: Peak emission wavelength (PEW) is related to AMD pathology and might be a diagnostic marker in AMD. Possibly, a short PEW can predict progression to retinal and/or pigment epithelium atrophy.

Influence of Lens Fluorescence on Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) Fundus Imaging and Strategies for Its Compensation.

Purpose: To explore the contribution of crystalline lens fluorescence to fluorescence lifetimes measured with fluorescence lifetime imaging ophthalmoscopy (FLIO) and to propose a computational model to reduce the lens influence. Methods: FLIO, which detects autofluorescence decay over time in a short-wavelength spectral channel (SSC, 498-560 nm) and a long-wavelength spectral channel (LSC, 560-720 nm), was performed on 32 patients before and after cataract extraction. The mean autofluorescence lifetime (τ m ) of the fundus was determined from a three-exponential fit of the postoperative fluorescence decays. The preoperative measurements were fit with series of exponential functions in which one fluorescence component was time-shifted in order to represent lens fluorescence. Results: Postoperatively, τ m was 185 ± 22 ps in the SSC and 209 ± 34 ps in the LSC at the posterior pole. These values were best reproduced by fitting the postoperative measurements with a three-exponential model with a time-shifted third fluorescence component (SSC, 203 ± 45 ps; LSC, 215 ± 29 ps), whereas disregarding time-shifted lens fluorescence resulted in significantly (P < 0.001) longer τ m values (SSC, 474 ± 206 ps; LSC, 215 ± 29 ps). The fluorescence of the cataract lens contributed to the total fluorescence by 54.2 ± 10.6% (SSC) and 29.5 ± 9.9% (LSC). Conclusions: Cataract lens fluorescence greatly alters fluorescence lifetimes measured at the fundus by FLIO, resulting in an overestimation of the lifetimes; however, this may be compensated for considerably by taking lens influence into account in the fitting model. Translational Relevance: This study investigates cataract fluorescence in FLIO and a mathematical model for compensation of this influence.

Corrigendum: Pulsed Electrical Stimulation of the Human Eye Enhances Retinal Vessel Reaction to Flickering Light.

[This corrects the article DOI: 10.3389/fnhum.2019.00371.].

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.

Pulsed Electrical Stimulation of the Human Eye Enhances Retinal Vessel Reaction to Flickering Light.

Recent studies indicate therapeutic benefits of electrical stimulation in cases of specific ophthalmic diseases that are associated with dysfunctional ocular microcirculation. This suggests effects of electrical stimulation on vascular functions. In the present study, we investigated the effects of electrical stimulation on retinal vessel reactions using dynamic vessel analysis (DVA). Eighty healthy subjects were randomly assigned to one of three groups receiving electrical stimulation with different current intensities: 400 µA (n = 26); 800 µA (n = 27); 1200 µA (n = 27). The electrode montage for electrical stimulation consisted of a ring-shaped active electrode surrounding one eye and a square return electrode at the occiput. Rectangular, monophasic, positive current pulses were applied at 10 Hz for a duration of 60 s per stimulation period. DVA was used to observe the stimulation-induced reactions of retinal vessel diameters in response to different provocations. In three DVA measurements, three stimulus conditions were investigated: flicker light stimulation (FLS); electrical stimulation (ES); simultaneous electrical and flicker light stimulation (ES+FLS). Retinal vasodilation caused by these stimuli was compared using paired t-test. The subjects receiving electrical stimulation with 800 µA showed significantly increased retinal vasodilation for ES+FLS compared to FLS (p < 0.05). No significant differences in retinal vessel reactions were found between ES+FLS and FLS in the 400 and 1200 µA groups. No retinal vasodilation was observed for ES for all investigated current intensities. The results indicate that positive pulsed electrical stimulation of an adequate intensity enhances the flicker light-induced retinal vasodilation.

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.

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.

Monitoring foveal sparing in geographic atrophy with fluorescence lifetime imaging ophthalmoscopy - a novel approach.

PURPOSE: To investigate fundus autofluorescence (FAF) lifetimes in geographic atrophy (GA) with a focus on macular pigment (MP) and foveal sparing. METHODS: The study included 35 eyes from 28 patients (mean age 79.2 ± 8.0 years) with GA. A 30° retinal field, centred at the macula, was investigated using fluorescence lifetime imaging ophthalmoscopy (FLIO). The FLIO technology is based on a Heidelberg Engineering Spectralis system. Decays of FAF were detected in a short (498-560 nm, SSC) and long (560-720 nm, LSC) spectral channel. The mean fluorescence lifetime, τm , was calculated from a three-exponential approximation of the FAF decays. Macular optical coherence tomography (OCT) scans as well as fundus photography were recorded. RESULTS: Review of FLIO data reveals specific patterns of significantly prolonged τm in regions of GA (SSC 616 ± 343 ps, LSC 615 ± 154 ps) as compared to non-atrophic regions. Large τm differences between the fovea and atrophic areas correlate with better visual acuity (VA). Shorter τm at the fovea than within other non-atrophic regions indicates sparing, which was identified in 16 eyes. Seventy per cent of patients treated with lutein supplementation showed foveal sparing, whereas the rate among non-supplemented patients was 22%. CONCLUSION: Using FLIO, we present a novel way to detect foveal sparing, investigate MP, and analyse variability of τm in different foveal regions (including the prognostic valuable border region) in GA. These findings support the potential utility of FLIO in monitoring disease progression. The findings also highlight the possibly protective effect of lutein supplementation, with implication in recording the presence and distributional pattern of MP.

Arsenic-induced APL differentiation in cerebrospinal fluid.

Although new approaches have dramatically improved, the treatment of acute promyelocytic leukemia (APL) involvement of the central nervous system (CNS) confers a bad prognosis in the disease. Here, we report a patient who was diagnosed with relapsed APL preferentially involving the CNS. Treatment with arsenic trioxide led to impressive morphological changes in CNS cellularity consistent with the induction of a differentiation syndrome. Since arsenic trioxide could be identified in the CNS, we provide evidence that the drug can cross the blood-brain barrier and can be used for treatment of extramedullary APL.

Hydrogen peroxide modulates energy metabolism and oxidative stress in cultures of permanent human Müller cells MIO-M1.

In this study the influence of hydrogen peroxide (H2 O2 ) on the redox state, NADH protein binding, and mitochondrial membrane potential in Müller cells is investigated. Cultures of permanent human Müller cells MIO-M1 were exposed to H2 O2 in 75 µM and 150 µM concentration for two hours. Fluorescence emission spectra and lifetimes were measured by two-photon microscopy (excitation wavelength: 740 nm) at the mitochondria which were identified in the microscopic images by their fluorescence properties (spectra and intensity). Two hours of H2 O2 exposure did not impair viability of MIO-M1 cells in culture. Whereas the ratio of flavine- to NADH fluorescence intensity did not change under either H2 O2 concentration, the mean lifetime was significantly different between controls, not exposed to H2 O2 , and the 150 µM H2 O2 exposure (972 ± 63 ps vs. 1152 ± 64 ps, p = 0.014). One hour after cessation of the H2 O2 exposure, the value retuned to that of the control (983 ± 36 ps). A hyperpolarization of the mitochondrial membrane under 150 µM H2 O2 was found. These findings suggest a shift form free to protein-bound NADH in mitochondria as well as a hyperpolarization of their inner membrane which could be related to an impairment of Müller cell function despite their preserved viability. Exposure of human Müller cells to hydrogen peroxide for two hours results in a reversible change of protein binding of mitochondrial NADH upon unchanged redox ratio. The mitochondrial membrane potential is increased during exposure.

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.

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.

Combination of confocal principle and aperture stop separation improves suppression of crystalline lens fluorescence in an eye model.

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique to detect changes in the human retina. The autofluorescence decay over time, generated by endogenous fluorophores, is measured in vivo. The strong autofluorescence of the crystalline lens, however, superimposes the intensity decay of the retina fluorescence, as the confocal principle is not able to suppress it sufficiently. Thus, the crystalline lens autofluorescence causes artifacts in the retinal fluorescence lifetimes determined from the intensity decays. Here, we present a new technique to suppress the autofluorescence of the crystalline lens by introducing an annular stop into the detection light path, which we call Schweitzer's principle. The efficacy of annular stops with an outer diameter of 7 mm and inner diameters of 1 to 5 mm are analyzed in an experimental setup using a model eye based on fluorescent dyes. Compared to the confocal principle, Schweitzer's principle with an inner diameter of 3 mm is able to reduce the simulated crystalline lens fluorescence to 4%, while 42% of the simulated retina fluorescence is preserved. Thus, we recommend the implementation of Schweitzer's principle in scanning laser ophthalmoscopes used for fundus autofluorescence measurements, especially the FLIO device, for improved image quality.

FLIMX: A Software Package to Determine and Analyze the Fluorescence Lifetime in Time-Resolved Fluorescence Data from the Human Eye.

Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a new technique for measuring the in vivo autofluorescence intensity decays generated by endogenous fluorophores in the ocular fundus. Here, we present a software package called FLIM eXplorer (FLIMX) for analyzing FLIO data. Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel. We also expand existing decay models (multi-exponential, stretched exponential, spectral global analysis, incomplete decay) to account for the layered structure of the eye and present a method to correct for the influence of the crystalline lens fluorescence on the retina fluorescence. Subsequently, the Holm-Bonferroni method is applied to FLIO measurements to allow for group comparisons between patients and controls on the basis of fluorescence lifetime parameters. The performance of the new approaches was evaluated in five experiments. Specifically, we evaluated static and adaptive binning in a diabetes mellitus patient, we compared the different decay models in a healthy volunteer and performed a group comparison between diabetes patients and controls. An overview of the visualization capabilities and a comparison of static and adaptive binning is shown for a patient with macular hole. FLIMX's applicability to fluorescence lifetime imaging microscopy is shown in the ganglion cell layer of a porcine retina sample, obtained by a laser scanning microscope using two-photon excitation.

Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy.

The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included n the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τ(i), amplitudes α(i), and relative contributions Q(i) were statistically compared between corresponding groups in two spectral channels (490 < ch1 < 560 nm, 560 < ch2 < 700 nm). The change in single fluorophores was estimated by applying the Holm­Bonferroni method and by calculating differences in the sum histograms of lifetimes. Median and mean of the histograms of τ(2), τ(3), and α(3) in ch1 show the greatest differences between phakic diabetic patients and age-matched controls (p < 0.000004). The lack of pixels with a τ(2) of ∼360 ps, the increased number of pixels with τ(2) > 450 ps, and the shift of τ(3) from ∼3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine inucleotide at the fundus. AGE also accumulated in the crystalline lens.

Repeatability of autofluorescence lifetime imaging at the human fundus in healthy volunteers.

PURPOSE: We aim to evaluate the repeatability of a new fluorescence lifetime imaging (FLIM) technique which measures time-resolved autofluorescence to assess metabolism of the retina. MATERIALS AND METHODS: We performed FLIM with two spectral channels (channel 1: 490-560 nm and channel 2: 560-700 nm) on 10 healthy volunteers, with 10 replicates per volunteer. From the 30° fundus FLIM images, we selected three regions: the fovea, the optic disc and the papillo-macular bundle. For each channel in these regions, we determined an average multi-exponential approximation with three components, and the six resulting parameters, α1-α3 (amplitudes) and τ1-τ3 (fluorescence lifetimes), were analyzed in terms of the coefficient of variation (CV). RESULTS: Repeatability was highest in the papillo-macular bundle, followed by the fovea and the optic disc. Repeatability was higher in channel 1 (mean CV of 7.9%) than in channel 2 (mean CV of 17.7%). The average CV for the diagnostically most relevant channel 1 and the most relevant parameters was as follows: τ1 (5.5%) and τ2 (4.7%) in the papillo-macular bundle, and τ1 (6.8%) and τ2 (6.9%) in the fovea. CONCLUSIONS: We demonstrated repeatability of FLIM measurement results within acceptable ranges of variation. Based on the detailed coefficients of variation, we derived recommendations for parameter ranges suitable for diagnostic applications.