Introduction
Fundus autofluorescence (FAF) imaging has become an invaluable imaging technique to monitor and diagnose retinal health and disease.1 2 On a cellular and subcellular level, FAF mainly originates from fluorophores in the outer retina and the retinal pigment epithelium (RPE). In RPE cells, these fluorophores are linked to intracellular granules, which show different autofluorescence (AF) properties: melanolipofuscin and lipofuscin granules with peak excitation in the short wavelength and melanin in melanosomes and melanolipofuscin granules with peak excitation in the near-infrared (IR) spectrum.3–5
In clinical settings, blue (BAF) and green FAF (GAF) imaging heavily relies on the clarity of optical media, such as the cornea, the lens and the vitreous.6 With age, glycation products amass in the lenticular fibres, leading to increased opacity7 causing reduced transmission of external light to the retina. Short wavelengths are most notably affected.8 This phenomenon, known as Rayleigh scattering, explains that the intensity of scattered light is inversely proportional to the wavelength’s fourth power (with blue having the shortest wavelength in the visual spectrum). As a consequence, many FAF studies exclude elderly patients or those with cataract.9–11 However, the criteria for exclusion are not defined and vary based on the investigator, making the cut-off potentially irreproducible. This poses a significant limitation, especially since many eye diseases like age-related macular degeneration predominantly affect the elderly.12 Moreover, this practice raises concerns about patient selection in clinical research, as it systematically excludes a significant portion of the population who are most likely to be affected by these conditions.13
To evaluate to what extent lenticular opacification affects retinal image quality, a reliable assessment is a prerequisite. There are different approaches to quantifying lens opacity, as previously published.14 One is to determine the cataract grade clinically by slit-lamp examination using the Lens Opacities Classification System grading score.15 The cataract is classified subjectively in terms of both its severity and anatomical position, which, however, requires clinical experience of the grader and may introduce grader bias. In contrast, there are also several lens imaging modalities that allow more objective measurements. Scheimpflug photography, in conjunction with densitometric image analysis, is able to measure the amount of light that is back-scattered from the lens.16 Further, using swept-source anterior chamber optical coherence tomography (AC-OCT) imaging, the reflectivity of the lens can be quantified.17 It is also possible to analyse the intensity of the fourth Purkinje image across different wavelengths to accurately determine lens density and spectral transmittance.18 In addition, an alternative is to use fluorophotometry deploying BAF and GAF images to measure lens transmission19 by comparing AF measures of the anterior and posterior parts of the lens. The difference in AF between both parts can be attributed to a loss of exciting and fluorescent light in the lens. Likewise, Charng and colleagues recently described a novel method to measure lens AF (LQAF) intensities by shifting the focus of the AF acquisition to the lens.20 LQAF uses tools previously developed to quantify the AF of the fundus (QAF). An internal reference simultaneously captured during image acquisition enables the comparison of AF intensities across study participants and in the follow-up.
In this study, we investigated the impact of an array of lens opacification and AF measurements on qualitative and quantitative estimates of retinal image quality. Our results serve as a first step towards successful screening of patients for clinical trials where retinal image quality is pivotal. These findings underscore the critical importance of stringent criteria in study participant selection to ensure data accuracy and reliability in such trials.