The Future of Retinal Imaging

Daniel Q. Li; Netan Choudhry


Curr Opin Ophthalmol. 2020;31(3):199-206. 

In This Article

Fluorescence Lifetime Imaging Ophthalmoscopy

FLIO is a novel noninvasive imaging technique based on fundus autofluorescence (FAF) imaging. Unlike intensity-based FAF imaging, in which lipofuscin predominates the resulting density map because of its high concentration, FLIO measures retinal fluorophores by their unique decay lifetime, which is largely independent of the fluorophore's concentration and intensity.[1] This allows for more sensitive detection of weakly fluorescing fluorophores, which would otherwise be masked by lipofuscin in FAF imaging and provides additional information about the structural integrity and metabolic activity of the retina. Although the technique is relatively new, several studies have already characterized FLIO patterns for many retinal diseases and demonstrated potential value in the early detection and more sensitive follow-up observation of subtle retinal changes.

Basic Science

Over the last four decades, imaging of retinal fluorophores has mainly focused on intensity measures, known as autofluorescence intensity (AFI) imaging or FAF. In AFI, the retina is excited using blue-light, and the resulting fluorescence emission is detected by a fundus camera or scanning laser ophthalmoscope to form a spatially distributed brightness map. The predominant fluorophore captured on AFI is lipofuscin, whose accumulation within the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging RPE cells.[2] This is useful in identifying macular diseases, such as age-related macular degeneration (ARMD), Stargardt disease and pattern dystrophies. However, given the overshadowing effect of the strong lipofuscin signal, AFI has difficulty in identifying other retinal molecules with weaker fluorescence emissions. AFI is also unable to detect changes in the chemical microenvironment of fluorophores, which reveal the biochemical processes occurring in the proximity of the fluorophore. These limitations are addressed by the recent advent of FLIO.

FLIO is a novel technique that measures the fluorescence lifetime, which is the duration of fluorescence before the excited electrons return to their ground state. As this duration is unique to specific fluorophores, molecules with overlapping fluorescence emission spectra can be differentiated based on different autofluorescence lifetimes. Changes in fluorescence lifetimes reflect specific changes in the configuration of fluorescent molecules and their molecular microenvironment, which reveal many hidden structural and biochemical changes of the retina not previously seen in other imaging modalities. A previous review by Dysli et al.[3] has compiled a detailed description of the lifetimes of endogenous retinal fluorophores. The most notable groups include lipofuscin, retinal carotenoids (lutein, zeaxanthin and meso-zeaxanthin), redox equivalents [reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and flavin-mononucleiotide], extracellular matrix components (collagen and elastatin), skin pigments (melanin and bilirubin) and pathological fluorophores, such as advanced glycation end-products (AGEs) found in diabetic retinopathy.

Clinical Applications

In healthy eyes, FLIO has been thoroughly studied with a constant lifetime distribution pattern that is reproducible across literature. Specifically, the shortest lifetimes are found at the fovea, intermediate lifetimes are found across the retina and the longest lifetimes are found at the optic disc.[4] More importantly, a wide range of retinal diseases have already been investigated using this technique, as summarized below.

Macular Holes

Unlike OCT, FLIO does not directly visualize layers of the retina; however, it is able to indirectly visualize the location and relocation of macular pigment, which has a short fluorescence lifetime. Accordingly, lifetimes inside a macular hole are prolonged as the neuronal retinal layers containing macular pigment are absent leaving only the RPE, while lifetimes adjacent to the defect are shortened from the dislocated macular pigment that were originally at the fovea.[5] This is useful in assessing the outcome of macular hole closure after surgery: visual acuity correlated significantly with the normalization of fluorescence lifetime at the fovea at 1 month postsurgery. More than indicating anatomical closure, FLIO could show how the original cells relocated after surgery, making it a useful tool in follow-up assessments.

Retinal Artery Occlusion

In both central and branch retinal artery occlusions, prolonged fluorescence lifetimes were measured during the acute ischemia phase.[6] This is hypothesized to be due to a hypoxia-induced shift toward reduced flavin adenine dinucleotide (FADH2) and less protein-bound FAD, which has a short fluorescence lifetime, and increased blockage of autofluorescence from retinal swelling. In the postacute disease stage (>30 days), fluorescence lifetimes normalized despite ischemia-induced atrophy of the inner retinal layers, suggesting little contribution of the inner retinal layers to the fluorescence lifetime signal.

Age-related Macular Degeneration

Fluorescence lifetimes are generally prolonged in patients with both exudative and nonexudative AMD compared with aged-matched healthy eyes, likely because of the accumulation of bisretinoids in the RPE.[7,8] Individual soft drusen and large drusen associated with pigment epithelial bulging did not display specific fluorescence lifetimes. Subretinal deposits that are hyperreflective on OCT and hyperfluoroscent on AFI featured short fluorescence lifetimes, likely because of high concentrations of visual cycle by-products. Intraretinal deposits at the level of the photoreceptors featured longer lifetimes, likely reflecting connective tissue remodeling at the highly altered RPE-photoceptor band. In geographic atrophy in AMD, areas of total retinal atrophy exhibited long lifetimes, possibly because of the contribution of fluorophores from the underlying choroid and connective tissue components.[9] Furthermore, FLIO allows sensitive demarcation and visualization of the geographic atrophy marginal zones, which are of particular interest in analyzing the progression of disease and monitoring novel treatment approaches (Figure 1).

Figure 1.

Fluorescence lifetime ophthalmoscopy (FLIO) imaging of the left eye of two aged individuals: 75 y.o. normal male (top row) and 77 y.o. male with age-related macular macular degeneration and reticular pseudodrusen. Left images show standard blue-light autofluorescence images, which show evidence of pseudodrusen in the lower left image. Middle panels show pseudo color FLIO short spectral images and right panels show the long spectral channel. Lifetimes are significantly longer (more blue than red) in the AMD eye, particularly in regions of reticular pseudodrusen in the long spectral channel image.

Diabetic Retinopathy

FLIO can be used to detect the formation of AGEs in diabetes, which have a prolonged fluorescence lifetime. This has been demonstrated in patients with non-proliferative diabetic retinopathy, as well as patients with type 2 diabetes before they are diagnosed with diabetic retinopathy.[10,11]

Central Serous Chorioretinopathy

In central serous chorioretinopathy, FLIO showed shortened fluorescence lifetimes in the areas of active lesions, correlating with elongated outer photoreceptor segments, likely because of the accumulation of visual cycle by-products.[12] The presence or absence of subretinal fluid did not influence lifetimes. In the chronic disease stage, secondary retinal changes, such as scar formation and retinal atrophy, exhibited prolonged lifetimes consistent with findings from FLIO in AMD with geographic atrophy.

Macular Telangiectasia Type 2

FLIO images have demonstrated very distinct patterns in macular telangiectasia type 2 (MacTel Type 2). In early stages, lifetime prolonged in a crescent shape temporal to the fovea. This can be distinguished very well from AMD-related patterns, opening new possibilities for reliable early diagnosis of MacTel.[13] In later stages, the entire MacTel area shifted toward longer lifetimes in an oval-shaped manner surrounding the disease zone, likely because of the enhancement from MacTel-related macular pigment changes, which correlate with disease severity.[14]

Stargardt Disease

In Stargardt disease, retinal deposits with short fluorescence lifetimes were detected before they were visible in the intensity image.[15] Over time, they appeared as hyperautofluorescent spots and shifted to long fluorescence lifetimes, suggesting a change in the composition of these retinal deposits.

Alzheimer's Diseae

Preliminary studies of this condition have demonstrated increased fluorescence lifetimes in patients with Alzheimer's disease, correlating with levels of amyloid-B and tau-protein in cerebrospinal fluid (CSF), as well as ganglion cell layer and inner plexiform layer thickness on OCT.[16,17] The authors of these studies suggest that FLIO can serve as a simple, noninvasive diagnostic tool for Alzheimer's disease, though these findings need to be validated in future longitudinal study.

Future Directions

FLIO represents a powerful tool for early detection of subtle retinal changes and can highlight fluorophores in 'silent' areas on conventional intensity-based autofluorescence imaging. It is anticipated that FLIO may be used to monitor patients' responses to therapies in clinical trials, such as gene therapy for hereditary retinal dystrophies. Analyzing retinal fluorescence lifetimes, FLIO may also show value in the detection of early systemic metabolic conditions, such as diabetes, dyslipidemia and neurodegenerative diseases. Currently, information from FLIO is based on individual pilot studies and the complex analysis required for individual lifetime components means the technique may not be suitable for everyday clinical use at this stage. However, with further refinement, including standardized and automated lifetimes, FLIO has the potential to be an important adjunct to the more commonly used multimodal imaging technologies.