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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

Fluorescence intensity and average fluorescence lifetime of the ganglion cell layer in a porcine retina ex vivo sample.The 256 x 256 pixels images (34 x34 μm2/pixel) of the fluorescence intensity before binning (left) and the average fluorescence lifetime τm (right) of the ganglion cell layer in a porcine retina sample are shown in two spectral channels (top: 500–560 nm; bottom: 560–700 nm). Adaptive binning with a threshold of 10,000 photons per pixel was applied. A multi-exponential model with two exponential functions was used to determine the fluorescence lifetimes. The length of the white bar is 20 μm.
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pone.0131640.g011: Fluorescence intensity and average fluorescence lifetime of the ganglion cell layer in a porcine retina ex vivo sample.The 256 x 256 pixels images (34 x34 μm2/pixel) of the fluorescence intensity before binning (left) and the average fluorescence lifetime τm (right) of the ganglion cell layer in a porcine retina sample are shown in two spectral channels (top: 500–560 nm; bottom: 560–700 nm). Adaptive binning with a threshold of 10,000 photons per pixel was applied. A multi-exponential model with two exponential functions was used to determine the fluorescence lifetimes. The length of the white bar is 20 μm.

Mentions: The fluorescence intensity and the average fluorescence lifetime τm for spectral channels 1 and 2 are shown in Fig 11. In spectral channel 1, the ganglion cells possess much shorter average fluorescence lifetimes (700 ps–900 ps) than their surroundings. In spectral channel 2, the cell bodies also possess shorter average fluorescence lifetimes (450 ps–650 ps) than their surroundings while the cell nuclei possess the shortest average fluorescence lifetimes (350 ps–450 ps).


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)

Fluorescence intensity and average fluorescence lifetime of the ganglion cell layer in a porcine retina ex vivo sample.The 256 x 256 pixels images (34 x34 μm2/pixel) of the fluorescence intensity before binning (left) and the average fluorescence lifetime τm (right) of the ganglion cell layer in a porcine retina sample are shown in two spectral channels (top: 500–560 nm; bottom: 560–700 nm). Adaptive binning with a threshold of 10,000 photons per pixel was applied. A multi-exponential model with two exponential functions was used to determine the fluorescence lifetimes. The length of the white bar is 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131640.g011: Fluorescence intensity and average fluorescence lifetime of the ganglion cell layer in a porcine retina ex vivo sample.The 256 x 256 pixels images (34 x34 μm2/pixel) of the fluorescence intensity before binning (left) and the average fluorescence lifetime τm (right) of the ganglion cell layer in a porcine retina sample are shown in two spectral channels (top: 500–560 nm; bottom: 560–700 nm). Adaptive binning with a threshold of 10,000 photons per pixel was applied. A multi-exponential model with two exponential functions was used to determine the fluorescence lifetimes. The length of the white bar is 20 μm.
Mentions: The fluorescence intensity and the average fluorescence lifetime τm for spectral channels 1 and 2 are shown in Fig 11. In spectral channel 1, the ganglion cells possess much shorter average fluorescence lifetimes (700 ps–900 ps) than their surroundings. In spectral channel 2, the cell bodies also possess shorter average fluorescence lifetimes (450 ps–650 ps) than their surroundings while the cell nuclei possess the shortest average fluorescence lifetimes (350 ps–450 ps).

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