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

Klemm M, Schweitzer D, Peters S, Sauer L, Hammer M, Haueisen J - PLoS ONE (2015)

Bottom Line: Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, POB 100565, 98694, Ilmenau, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Results of the Holm-Bonferroni method applied to FLIO measurements to allow for group comparisons between diabetes patients and controls.The normalized histograms of the fluorescence amplitudes α and lifetimes τ in both spectral channels are obtained from a multi-exponential approximation using three exponential functions, for controls (blue) and diabetes patients (red). Histogram classes with significant differences, according to the Holm-Bonferroni method, are colored in light gray. The class with the highest significance level (the smallest p value) is indicated in dark gray. Only the fluorescence lifetimes showed significant differences, except for τ2 in spectral channel 2. For the class with the highest significance level, the corresponding receiver operating characteristic curve (orange) is shown next to the histogram. The cut-off point as best trade-offs between true positive rate and false positive rate is colored in light blue. The AUC is given under the ROC curve.
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pone.0131640.g010: Results of the Holm-Bonferroni method applied to FLIO measurements to allow for group comparisons between diabetes patients and controls.The normalized histograms of the fluorescence amplitudes α and lifetimes τ in both spectral channels are obtained from a multi-exponential approximation using three exponential functions, for controls (blue) and diabetes patients (red). Histogram classes with significant differences, according to the Holm-Bonferroni method, are colored in light gray. The class with the highest significance level (the smallest p value) is indicated in dark gray. Only the fluorescence lifetimes showed significant differences, except for τ2 in spectral channel 2. For the class with the highest significance level, the corresponding receiver operating characteristic curve (orange) is shown next to the histogram. The cut-off point as best trade-offs between true positive rate and false positive rate is colored in light blue. The AUC is given under the ROC curve.

Mentions: As stated above, the motivation of this experiment is to show the ability of our analysis chain to provide group comparison for groups of patients or volunteers because of the high inter-individual variability. Normalized histograms comparing the diabetes patients and controls for all of the fluorescence amplitudes and lifetimes, as well as for both spectral channels, are shown in Fig 10. A shift to longer fluorescence lifetimes in the diabetes patients is clearly visible in all fluorescence lifetimes and both spectral channels. Significantly different populated histogram classes were found for all of the fluorescence lifetimes, except for τ2 in spectral channel 2. No significant differences were observed for the fluorescence amplitudes. For each histogram class with the highest significance level, the corresponding receiver operating characteristic (ROC) curve is given next to the histogram. The area under the ROC curve (AUC), as a measure of the ROC curve’s accuracy, achieves the largest value of 0.85 for fluorescence lifetime τ1 in spectral channel 2. Thus, its cut-off point as best trade-offs between true positive rate and false positive rate would result in the best achievable diabetes detection using only the FLIO data from this experiment.


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

Klemm M, Schweitzer D, Peters S, Sauer L, Hammer M, Haueisen J - PLoS ONE (2015)

Results of the Holm-Bonferroni method applied to FLIO measurements to allow for group comparisons between diabetes patients and controls.The normalized histograms of the fluorescence amplitudes α and lifetimes τ in both spectral channels are obtained from a multi-exponential approximation using three exponential functions, for controls (blue) and diabetes patients (red). Histogram classes with significant differences, according to the Holm-Bonferroni method, are colored in light gray. The class with the highest significance level (the smallest p value) is indicated in dark gray. Only the fluorescence lifetimes showed significant differences, except for τ2 in spectral channel 2. For the class with the highest significance level, the corresponding receiver operating characteristic curve (orange) is shown next to the histogram. The cut-off point as best trade-offs between true positive rate and false positive rate is colored in light blue. The AUC is given under the ROC curve.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507995&req=5

pone.0131640.g010: Results of the Holm-Bonferroni method applied to FLIO measurements to allow for group comparisons between diabetes patients and controls.The normalized histograms of the fluorescence amplitudes α and lifetimes τ in both spectral channels are obtained from a multi-exponential approximation using three exponential functions, for controls (blue) and diabetes patients (red). Histogram classes with significant differences, according to the Holm-Bonferroni method, are colored in light gray. The class with the highest significance level (the smallest p value) is indicated in dark gray. Only the fluorescence lifetimes showed significant differences, except for τ2 in spectral channel 2. For the class with the highest significance level, the corresponding receiver operating characteristic curve (orange) is shown next to the histogram. The cut-off point as best trade-offs between true positive rate and false positive rate is colored in light blue. The AUC is given under the ROC curve.
Mentions: As stated above, the motivation of this experiment is to show the ability of our analysis chain to provide group comparison for groups of patients or volunteers because of the high inter-individual variability. Normalized histograms comparing the diabetes patients and controls for all of the fluorescence amplitudes and lifetimes, as well as for both spectral channels, are shown in Fig 10. A shift to longer fluorescence lifetimes in the diabetes patients is clearly visible in all fluorescence lifetimes and both spectral channels. Significantly different populated histogram classes were found for all of the fluorescence lifetimes, except for τ2 in spectral channel 2. No significant differences were observed for the fluorescence amplitudes. For each histogram class with the highest significance level, the corresponding receiver operating characteristic (ROC) curve is given next to the histogram. The area under the ROC curve (AUC), as a measure of the ROC curve’s accuracy, achieves the largest value of 0.85 for fluorescence lifetime τ1 in spectral channel 2. Thus, its cut-off point as best trade-offs between true positive rate and false positive rate would result in the best achievable diabetes detection using only the FLIO data from this experiment.

Bottom Line: Specifically, we introduce a new adaptive binning approach as an optimal tradeoff between the spatial resolution and the number of photons required per pixel.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Engineering and Informatics, Technische Universität Ilmenau, POB 100565, 98694, Ilmenau, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus