More so than almost any other field in medicine, noninvasive imaging has been critical for progress in the diagnosis and treatment of retinal disease. From the exquisite hand drawings of pioneers of ophthalmology after funduscopy was invented in 1851 by Helmholz to the first photographic records of macular structure to fluorescein angiography to optical coherence tomography, each imaging advance has led to new insights into the physiology and pathophysiology of macular and retinal disorders.
Imaging of the intrinsic autofluorescence of the human macula was first systematically studied by Delori et al1 in the 1970s, but it remained largely a research technique until high-sensitivity detectors were incorporated into digital fundus cameras and scanning laser ophthalmoscopes in the 1990s and 2000s. It soon became clear that patterns of fluorescence originating largely from lipofuscin in the retinal pigment epithelium could have great utility in diagnosing and monitoring progression of a variety of macular diseases such as age-related macular degeneration, Stargardt disease, and pattern dystrophies. Topographic mapping of autofluorescence intensity (AFI) distributions is now routinely performed in medical retina evaluations, but with continued improvements in detector technology, it has become clear that a wealth of additional new information can be gleaned from the fluorophores of the retina. The most notable advance has been the ability to distinguish between fluorophores either spectrally or by monitoring differences in kinetics of photon emission, a technique known as fluorescence lifetime imaging ophthalmoscopy (FLIO). Fluorescence lifetime imaging ophthalmoscopy is still an uncommon technique, however, due to the current rarity of the prototype instruments that are in use in just a handful of sites in Europe and the United States. In this issue of Retina, the group led by Professors Wolf and Zinkernagel in Bern, Switzerland, nicely demonstrates the value of FLIO for monitoring the progression of Stargardt disease over a period ranging from 3 to 45 months.2
Fluorescence originates when a chemical compound absorbs a photon of light and promotes an electron to a higher energy state. The excited electron can then transition to a relatively stable slightly lower energy state where it eventually decays to its ground state through emission of a red-shifted photon, typically over a period of picoseconds or nanoseconds (fluorescence) or much longer times (phosphorescence). Fluorescent compounds have characteristic absorption and emission spectra that can be quantified by exciting the chemicals with various wavelengths of light and through the use of high-resolution spectrophotometers. Because incorporation of a high-resolution spectrometer is generally not feasible in a clinical imaging instrument, the emitted photons are usually filtered through spectral windows and detected on a charge-coupled device array or photon counters. This is the basis of the AFI imaging devices currently on the market based either on fundus cameras or scanning laser ophthalmoscopes. These devices provide high-resolution images of distributions and relative intensities of retinal fluorophores, but they allow for only minimal discrimination between various fluorophores. This is where FLIO manifests its utility. By substituting the conventional continuous-wave blue laser of a scanning laser ophthalmoscope with a pulsed picosecond 473-nm laser and by using 2 high-resolution time-gated photon counters tuned to a short spectral channel (498–560 nm) and a long spectral channel (560–720 nm), investigators can now begin to distinguish between various fluorophores depending on their chemical properties and metabolic environments. Typical FLIO images of a normal macula show shortest lifetimes (usually depicted in orange-red color) at the fovea originating from the weak intrinsic fluorescence of the macular carotenoid pigment, intermediate lifetimes (usually depicted in yellow-green) from lipofuscin fluorescence, and long lifetimes (usually depicted in blue) from collagen and elastin of the optic nerve and blood vessels (Figure 1).
Fluorescence lifetime imaging was first implemented on tissue sections and live cells in the early 1990s and was known as fluorescence lifetime imaging microscopy (FLIM).3 In 2002, Professor Dietrich Schweitzer in Jena, Germany, adapted the technique to image the living human fundus at eye-safe laser exposure levels,4 and in 2012, Heidelberg Engineering (Heidelberg, Germany) released several prototype FLIO instruments for clinical research that incorporated their scanning laser ophthalmoscope technology and infrared eye tracking to yield 256 × 256-pixel FLIO images with about 90 to 120 seconds of light exposure per eye. Since that time, the Jena, Bern, Ilmenau, and Utah groups have been the most productive, with numerous publications on the value of FLIO for imaging age-related macular degeneration, inherited retinal and macular diseases, diabetic retinopathy, and macular telangiectasia Type 2 (MacTel).5–10 Fluorescence lifetime imaging ophthalmoscopy not only allows for the discrimination between fluorophores in lesions that appear identical in conventional AFI images, but they also can highlight fluorophores in “silent” areas of conventional macular AFI images in age-related macular degeneration and MacTel that may be indicative of future risk for visual loss from these late-onset diseases.
The accompanying article by Solberg et al2 clearly demonstrates the value of FLIO for monitoring the progression of Stargardt disease. This group had already reported that the flecks of Stargardt disease can display either long or short FLIO lifetimes relative to background fluorophores, and they suspected that the short lifetime flecks represent newer lesions.11 In the recent study, they followed 12 members of their original cohort with FLIO for a mean of 29.2 months. They found in 75.1% of the eyes that short lifetime flecks did indeed progress to long lifetime flecks, and that new onset flecks always exhibit short lifetimes initially. They speculate that longitudinal changes in fluorescence lifetimes could represent changes in lipofuscin and bis-retinoid composition with time. These results suggest that FLIO could play an important role in monitoring disease progression in natural history studies and interventional trials.
Although FLIO has a bright future as an addition to the wide array of imaging options for medical retina, there are still a number of challenges ahead. First, the instrumentation is expensive to build and is not widely available. Heidelberg Engineering is working on standardizing the equipment and increasing access to retina specialists to promote further clinical research and eventual approval for clinical use. Second, there is still much to learn about what FLIO is actually imaging. Fluorophores of the retina and retinal pigment epithelium are not yet comprehensively characterized, and in vitro behavior does not always predict FLIO properties in complex biological tissues. This article on Stargardt disease and forthcoming ones on the value of FLIO for detecting retinal changes in drug toxicity, uveitis, inherited retinal disease, and systemic disease from the groups that currently have FLIO instruments should pave the way for widespread incorporation of FLIO into retinal practice.
1. Delori FC, Dorey CK, Staurenghi G, et al. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Invest Ophthalmol Vis Sci 1995;36:718–729.
2. Solberg YD, Escher P, Berger L, et al. Retinal flecks in Stargardt disease reveal characteristic fluorescence lifetime transition over time. Retina 2019.
3. Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML. Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A 1992;89:1271–1275.
4. Schweitzer D, Kolb A, Hammer M, Anders R. Time-correlated measurement of autofluorescence. A method to detect metabolic changes in the fundus. Ophthalmologe 2002;99:774–779.
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6. Schweitzer D, Deutsch L, Klemm M, et al. Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy. J Biomed Opt 2015;20:61106.
7. Sauer L, Gensure RH, Andersen KM, et al. Patterns of fundus autofluorescence lifetimes in eyes of individuals with nonexudative age-related macular degeneration. Invest Ophthalmol Vis Sci 2018;59:AMD65–AMD77.
8. Sauer L, Gensure RH, Hammer M, Bernstein PS. Fluorescence lifetime imaging ophthalmoscopy: a novel way to assess macular telangiectasia type 2. Ophthalmol Retina 2018;2:587–598.
9. Dysli C, Fink R, Wolf S, Zinkernagel MS. Fluorescence lifetimes of drusen in age-related macular degeneration. Invest Ophthalmol Vis Sci 2017;58:4856–4862.
10. Dysli C, Quellec G, Abegg M, et al. Quantitative analysis of fluorescence lifetime measurements of the macula using the fluorescence lifetime imaging ophthalmoscope in healthy subjects. Invest Ophthalmol Vis Sci 2014;55:2106–2113.
11. Dysli C, Wolf S, Hatz K, Zinkernagel MS. Fluorescence lifetime imaging in Stargardt disease: potential marker for disease progression. Invest Ophthalmol Vis Sci 2016;57:832–841.