![]() ![]() This resulted in images with some hundred photons per pixel only. However, the lack of an image registration algorithm limited the time available for recording an image without motion artifacts to a few seconds. They fiber-coupled a mode-locked argon-ion laser to a scanning ophthalmoscope (cLSO, Carl Zeiss, Jena, Germany) and used TCSPC for fluorescence detection. first applied fluorescence lifetime imaging to the human retina in vivo. Īs ocular fundus autofluorescence was reported to be a possible indicator of retinal diseases, Schweitzer et al. Another milestone in fluorescence microscopy was the introduction of Förster resonance energy transfer (FRET) enabling the detection of interaction between labeled molecules. Whereas FLIM of intrinsic fluorophores gives detailed information on cell metabolism and may detect malignant changes, the development of genetically expressed fluorescent proteins resulted in further progress in structural as well as functional imaging. Specifically, two-photon excitation microscopy, using an inherently pulsed fluorescence excitation source, was used for FLIM investigations. Fluorescence lifetime imaging microscopy (FLIM) evolved based on two different techniques: (1) full field illumination and the use of gated or streak cameras, an approach pursued in frequency domain technique and (2) the time-domain method in combination with confocal scanning laser microscopy. However, only the availability of short pulse lasers and the introduction of time correlated single photon counting (TCSPC) made fluorescence lifetime measurements sufficiently sensitive for the detection of intrinsic fluorophores in living tissue. In the 1920s, time resolution was improved to 10 −8 s which enabled the first fluorescence lifetime measurements. In 1859 Edmond Bequerel developed the so called phosphoroscope with a time resolution of 10 −4 s. Īlthough fluorescence lifetime measurement is considered a relatively new technique in biomedical imaging (see Berezin and Achilefu for review ), it has been discovered in the nineteenth century already. FLIO is specific for fluorophores as well as their embedding matrix and offers high temporal resolution. developed fluorescence lifetime ophthalmoscopy (FLIO), a method to measure the fluorescence decay time. In order to distinguish fluorophores, Schweitzer et al. ![]() Specific fluorescence patterns were assigned to sub-types of AMD and to their progression rate, but these characteristic patterns did not reveal any information about the present fluorophores which might be of pathogenetic relevance. FAF was used to describe the progression of geographic atrophy of the RPE, and different patterns of FAF distribution were found. As lipofuscin, which accumulates in the retinal pigment epithelium (RPE) and is involved in the pathogenesis of age-related macular degeneration (AMD), was found to be a major retinal fluorophore, subsequent FAF studies addressed this disease. ![]() Delori were the first to record fundus autofluorescence (FAF) spectra from single retinal locations and first images of FAF were recorded by von Rückmann et al. Whereas sodium fluorescein, a fluorescent tracer to image and assess retinal vasculature and its integrity, is used since the 1960s, the intrinsic fluorescence of the human retina was first reported in the 1980s. FLIO data acquired on patients with age-related macular degeneration (AMD), diabetic retinopathy, macular dystrophies, and other diseases are discussed in regard to the additional contrast and information provided in comparison to standard intensity-based autofluorescence images. In the subsequent sections, selected results obtained in clinical studies conducted in Bern, Salt Lake City and Jena are presented. In this chapter the historical and technical background of FLIO technology is described first, followed by the description how the technique was integrated into a modified Spectralis system. The fluorophores are excited by picosecond laser pulses and the fluorescence emission is detected using time correlated single photon counting (TCSPC) technology. The system is based on a confocal scanning laser ophthalmoscope with an implemented real-time eye tracking system. The decay time of the fluorescence is a characteristic parameter for fluorescent molecules and their environment, and therefore FLIO is a promising tool to detect and assess varying metabolic states of different areas in the retina. Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging technology, which enables the time-resolved in-vivo measurement of fluorescence emitted by endogenous fluorophores within the retina. ![]()
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