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C. Framme, G. Schuele, J. Roider, F.G. Holz, R. Birngruber, R. Brinkmann; Temperature Dependence of A2–E Fluorescence in vitro and Detection of Fundus Autofluorescence During Selective RPE Laser Treatment (SRT) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):266.
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Purpose: A2–E is a dominant fluorophore of RPE lipofuscin granules. In an in–vitro setup we determined the temperature–dependent changes of the A2–E fluorescence with the aim of assessing the potential value of such measurements for determining retinal temperature by fundus autofluorescence (AF) measurements during laser treatment. Since the green treatment laser beam could also be used for AF excitation, such online measurements were possible during selective RPE treatment (SRT). Methods: A2–E was biosynthesized and diluted in DMSO to 1µM. Fluorescence measurements were performed with a photo–spectrometer under various temperatures ranging from 20°C to 75°C. AF was excited at 467nm, and emission was detected around 632nm. SRT was carried out by use of a frequency–doubled Nd:YLF laser (wavelength: 527nm; pulse duration: 1.7µs; repetition rate: 500 and 100Hz; number of pulses: 100 and 30; single pulse energy: 50µJ to 130µJ) in in–vitro samples (porcine RPE; retinal spot size: 160µm) and during patient treatment (retinal spot size: 176µm). During irradiation, fluorescence light from the RPE was decoupled from the laser light inside the slit–lamp and detected by a photodiode at wavelengths above 550nm. Results: In vitro, A2–E fluorescence intensity showed a linear decrease concomitant with temperature increment at about 1% per 1°C and was completely reversible. The intensity of AF decreased over the number of applied pulses during SRT, and this trend was more pronounced in porcine RPE samples than during human treatment. In patients the decay of AF intensity was greater using 500Hz than 100Hz repetition rate indicating temperature dependence due to different amounts of energy deposition within the tissue. Conclusions: If the A2–E temperature–dependent fluorescence in–vitro is transferable to human AF, the AF decay could be related to the temperature increase within the tissue during laser treatment. Thus it may be possible to apply an AF–based online detection device for noninvasive determination of fundus temperature during in vivo laser treatment. This might be of clinical relevance for interventional strategies such as photodynamic therapy (PDT) and transpupillary thermotherapy (TTT).
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