This work was funded by the German Ophthalmologic Society (DOG) grant for doctoral students (MW) and the NIH/NEI 1R01EY027948 (TA).

Exploring Quantitative Autofluorescence in Age-Related Macular Degeneration

<ol>
	<li>Schmitz-Valckenberg, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundus+autofluorescence+imaging.">Fundus autofluorescence imaging.</a> Progress in Retinal and Eye Research. 81, 100893 (2021).</li><li>Bermond, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescent+granules+of+the+human+retinal+pigment+epithelium:+phenotypes,+intracellular+distribution,+and+age-related+topography.">Autofluorescent granules of the human retinal pigment epithelium: phenotypes, intracellular distribution, and age-related topography.</a> Investigative Ophthalmology and Visual Science. 61 (5), 35 (2020).</li><li>Bermond, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescent+organelles+within+the+retinal+pigment+epithelium+in+human+donor+eyes+with+and+without+age-related+macular+degeneration.">Autofluorescent organelles within the retinal pigment epithelium in human donor eyes with and without age-related macular degeneration.</a> Investigative Ophthalmology and Visual Science. 63 (1), 23 (2022).</li><li>Delori, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurements+of+autofluorescence+with+the+scanning+laser+ophthalmoscope.">Quantitative measurements of autofluorescence with the scanning laser ophthalmoscope.</a> Investigative Ophthalmology and Visual Science. 52 (13), 9379-9390 (2011).</li><li>Fleckenstein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> Nature Reviews Disease Primers. 7 (1), 31 (2021).</li><li>Greenberg, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+in+healthy+eyes.">Quantitative fundus autofluorescence in healthy eyes.</a> Investigative Ophthalmology and Visual Science. 54 (8), 5684-5693 (2013).</li><li>Sparrow, J. R., Boulton, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPE+lipofuscin+and+its+role+in+retinal+pathobiology.">RPE lipofuscin and its role in retinal pathobiology.</a> Experimental Eye Research. 80 (5), 595-606 (2005).</li><li>vonder Emde, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+quantitative+autofluorescence+in+intermediate+age-related+macular+degeneration.">Natural history of quantitative autofluorescence in intermediate age-related macular degeneration.</a> Retina. 41 (4), 694-700 (2021).</li><li>Reiter, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+changes+in+quantitative+autofluorescence+during+progression+from+intermediate+to+late+age-related+macular+degeneration.">Longitudinal changes in quantitative autofluorescence during progression from intermediate to late age-related macular degeneration.</a> Retina. 41 (6), 1236-1241 (2021).</li><li>Gliem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+in+early+and+intermediate+age-related+macular+degeneration.">Quantitative fundus autofluorescence in early and intermediate age-related macular degeneration.</a> JAMA Ophthalmology. 134 (7), 817-824 (2016).</li><li>Hussain, R. M., Gregori, N. Z., Ciulla, T. A., Lam, B. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration+therapy:+a+review.">Age-related macular degeneration therapy: a review.</a> Current Opinion in Ophthalmology. 31 (3), 215-221 (2020).</li><li>Ach, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipofuscin+redistribution+and+loss+accompanied+by+cytoskeletal+stress+in+retinal+pigment+epithelium+of+eyes+with+age-related+macular+degeneration.">Lipofuscin redistribution and loss accompanied by cytoskeletal stress in retinal pigment epithelium of eyes with age-related macular degeneration.</a> Investigative Ophthalmology and Visual Science. 56 (5), 3242-3252 (2015).</li><li>Zanzottera, E. C., Messinger, J. D., Ach, T., Smith, R. T., Curcio, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subducted+and+melanotic+cells+in+advanced+age-related+macular+degeneration+are+derived+from+retinal+pigment+epithelium.">Subducted and melanotic cells in advanced age-related macular degeneration are derived from retinal pigment epithelium.</a> Investigative Ophthalmology and Visual Science. 56 (5), 3269-3278 (2015).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperreflective+foci,+optical+coherence+tomography+progression+indicators+in+age-related+macular+degeneration,+include+transdifferentiated+retinal+pigment+epithelium.">Hyperreflective foci, optical coherence tomography progression indicators in age-related macular degeneration, include transdifferentiated retinal pigment epithelium.</a> Investigative Ophthalmology and Visual Science. 62 (10), 34 (2021).</li><li>Zanzottera, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Project+MACULA+retinal+pigment+epithelium+grading+system+for+histology+and+optical+coherence+tomography+in+age-related+macular+degeneration.">The Project MACULA retinal pigment epithelium grading system for histology and optical coherence tomography in age-related macular degeneration.</a> Investigative Ophthalmology and Visual Science. 56 (5), 3253-3268 (2015).</li><li>Sparrow, J. R., Duncker, T., Schuerch, K., Paavo, M., de Carvalho, d. R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+learned+from+quantitative+fundus+autofluorescence.">Lessons learned from quantitative fundus autofluorescence.</a> Progress in Retinal and Eye Research. 74, 100774 (2020).</li><li>Schmitz-Valckenberg, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+the+area+of+increased+autofluorescence+surrounding+geographic+atrophy+and+disease+progression+in+patients+with+AMD.">Correlation between the area of increased autofluorescence surrounding geographic atrophy and disease progression in patients with AMD.</a> Investigative Ophthalmology and Visual Science. 47 (6), 2648-2654 (2006).</li><li>Ach, T., Bermond, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autofluorescence+of+the+human+retinal+pigment+epithelium+in+normal+aging+and+in+age-related+macular+degeneration:+histology+and+clinical+correlation.">Autofluorescence of the human retinal pigment epithelium in normal aging and in age-related macular degeneration: histology and clinical correlation.</a> Klinische Monatsblatter Fur Augenheilkunde. 236 (5), 672-681 (2017).</li><li>Pollreisz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+melanosomes,+lipofuscin,+and+melanolipofuscin+in+human+retinal+pigment+epithelium+using+serial+block+face+scanning+electron+microscopy.">Visualizing melanosomes, lipofuscin, and melanolipofuscin in human retinal pigment epithelium using serial block face scanning electron microscopy.</a> Experimental Eye Research. 166, 131-139 (2018).</li><li>Bernstein, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=meso-zeaxanthin:+The+basic+and+clinical+science+underlying+carotenoid-based+nutritional+interventions+against+ocular+disease.">meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease.</a> Progress in Retinal and Eye Research. 50, 34-66 (2016).</li><li>G&#246;bel, A. P., Fleckenstein, M., Heeren, T. F. C., Holz, F. G., Schmitz-Valckenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+mapping+of+drusen+by+fundus+autofluorescence+and+spectral-domain+optical+coherence+tomography+imaging.">In-vivo mapping of drusen by fundus autofluorescence and spectral-domain optical coherence tomography imaging.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 254 (1), 59-67 (2016).</li><li>Pfau, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+and+dark-adapted+two-color+fundus-controlled+perimetry+in+geographic+atrophy+secondary+to+age-related+macular+degeneration.">Mesopic and dark-adapted two-color fundus-controlled perimetry in geographic atrophy secondary to age-related macular degeneration.</a> Retina. 40 (1), 169-180 (2020).</li><li>vonder Emde, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesopic+and+dark-adapted+two-color+fundus-controlled+perimetry+in+choroidal+neovascularization+secondary+to+age-related+macular+degeneration.">Mesopic and dark-adapted two-color fundus-controlled perimetry in choroidal neovascularization secondary to age-related macular degeneration.</a> Translational Vision Science and Technology. 8 (1), 7 (2018).</li><li>Aumann, S., Donner, S., Fischer, J., M&#252;ller, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+(OCT):+principle+and+technical+realization.">Optical coherence tomography (OCT): principle and technical realization.</a> High Resolution Imaging in Microscopy and Ophthalmology: New Frontiers in Biomedical Optics. , 59-85 (2019).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FIJI:+an+open-source+platform+for+biological-image+analysis.">FIJI: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Kleefeldt, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence:+advanced+analysis+tools.">Quantitative fundus autofluorescence: advanced analysis tools.</a> Translational Vision Science and Technology. 9 (8), 2 (2020).</li><li>Hong, I. H., Jung, W. H., Lee, J. H., Chang, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macular+pigment+optical+density+in+the+Korean+population:+a+cross+sectional+study.">Macular pigment optical density in the Korean population: a cross sectional study.</a> Journal of Korean Medical Science. 35 (5), e30 (2020).</li><li>Putnam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+imaging+of+macular+pigment+optical+density+and+spatial+distribution.">Clinical imaging of macular pigment optical density and spatial distribution.</a> Clinical and Experimental Optometry. 100 (4), 333-340 (2017).</li><li>Zweifel, S. A., Spaide, R. F., Curcio, C. A., Malek, G., Imamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reticular+pseudodrusen+are+subretinal+drusenoid+deposits.">Reticular pseudodrusen are subretinal drusenoid deposits.</a> Ophthalmology. 117 (2), 303-312 (2010).</li><li>Curcio, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+drusenoid+deposits+in+non-neovascular+age-related+macular+degeneration:+morphology,+prevalence,+topography,+and+biogenesis+model.">Subretinal drusenoid deposits in non-neovascular age-related macular degeneration: morphology, prevalence, topography, and biogenesis model.</a> Retina. 33 (2), 265-276 (2013).</li><li>Spaide, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+retinal+atrophy+after+regression+of+subretinal+drusenoid+deposits+as+a+newly+recognized+form+of+late+age-related+macular+degeneration.">Outer retinal atrophy after regression of subretinal drusenoid deposits as a newly recognized form of late age-related macular degeneration.</a> Retina. 33 (9), 1800-1808 (2013).</li><li>Reiter, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lens+opacities+and+cataract+severity+on+quantitative+fundus+autofluorescence+as+a+secondary+outcome+of+a+randomized+clinical+trial.">Influence of lens opacities and cataract severity on quantitative fundus autofluorescence as a secondary outcome of a randomized clinical trial.</a> Scientific Reports. 11 (1), 12685 (2021).</li><li>Gliem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+in+pseudoxanthoma+elasticum.">Quantitative fundus autofluorescence in pseudoxanthoma elasticum.</a> Investigative Ophthalmology and Visual Science. 58 (14), 6159-6165 (2017).</li><li>Burke, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+in+recessive+Stargardt+disease.">Quantitative fundus autofluorescence in recessive Stargardt disease.</a> Investigative Ophthalmology and Visual Science. 55 (5), 2841-2852 (2014).</li><li>Armenti, S. T., Greenberg, J. P., Smith, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+for+the+evaluation+of+retinal+diseases.">Quantitative fundus autofluorescence for the evaluation of retinal diseases.</a> Journal of Visualized Experiments. (109), 53577 (2016).</li><li>Pr&#246;bster, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+in+the+developing+and+maturing+healthy+eye.">Quantitative fundus autofluorescence in the developing and maturing healthy eye.</a> Translational Vision Science and Technology. 10 (2), 15 (2021).</li><li>Duncker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fundus+autofluorescence+and+optical+coherence+tomography+in+PRPH2/RDS-+and+ABCA4-associated+disease+exhibiting+phenotypic+overlap.">Quantitative fundus autofluorescence and optical coherence tomography in PRPH2/RDS- and ABCA4-associated disease exhibiting phenotypic overlap.</a> Investigative Ophthalmology and Visual Science. 56 (5), 3159-3170 (2015).</li></ol>

Leon von der Emde reports receiving payments from Heidelberg Engineering. Merten Mallwitz reports no financial disclosures. Kenneth R. Sloan also reports no financial disclosures. Frank G. Holz reports consulting/personal payments for Acucela, Alcon (C), Gyroscope Allergan Apellis, Bayer Bioeq/Formycon, CenterVue, Roche/Genentech, Geuder, Ivericbio, NightStarX, Novartis, Optos, Oxurion, Pixium Vision, Stealth BioTherapeutics, Zeiss, and GRADE Reading center. Thomas Ach reports consulting/personal payments for Bayer, Apellis, Roche, and Novartis.

This workflow provides a step-by-step guide to use open-source FIJI plug-in tools to determine and compare AF of AMD-specific lesions. The plug-ins provide easy-to-use templates that do not require any coding knowledge and can be applied by physicians without technical support27. To our knowledge, these tools are one of a kind for lesion-specific AF quantification.
QAF values naturally vary across the retina, with values being higher at the periphery and lower in the macula due to uneven lipofuscin and melanolipofuscin distribution within the retina, the low AF of vessels, and uneven macular pigment distribution. Due to the high variation of naturally occurring QAF levels in the retina, analyzing absolute QAF values of lesions directly is not a promising approach. For example, a hypoautofluorescent lesion in the periphery might still have higher absolute QAF values than physiologic fluorescence levels of the macula. The use of a StandardRetina and the use of z-scores to measure the fluorescence levels of drusen correct for this naturally occurring variance of QAF values.
A z-score is a numerical measurement of a region of interest value&#39;s relationship to the mean in the StandardRetina. It is calculated by subtracting the mean from an individual from the mean from the StandardRetina at the same location, and then dividing the result by the standard deviation. This standardization allows for the comparison of different QAF images, as the z-score indicates how many standard deviations a value differs from the mean. A positive z-score indicates that the value is above the mean, while a negative z-score indicates that it is below the mean.
It is important to note that there may be potential pitfalls that should be considered. While this method accounts for the varying amount of AF levels throughout the fundus, it may still not be the most accurate way to measure and compare an RPE&#39;s AF. Individuals have different levels and topography of macular luteal pigment, and lesions may affect the translucency of the overlying retina as well28,29. It is therefore plausible that the measured reduced AF in areas of SDDs (see representative results) is a consequence of shadowing effects rather than decreased fluorophores in the RPE30,31,32.
We are currently working on a workflow to account for retinal reflectivity, thickness, and quantified macular pigment (using green and blue AF) with linear mixed models. Additionally, so far, QAF uses an age-dependent correction factor to account for lenticular opacification that disregards interindividual differences in the lenticular opacification of participants of a similar age33. We are therefore currently working on a workflow for a personalized correction factor of lenticular autofluorescence and opacification. To reliably extract information of AF from small lesions, adequate test-retest reliability of QAF images is needed. To further differentiate those QAF images where more detailed analysis is viable, we are investigating &#34;QAF image reliability indices&#34; that can predict the test-retest reliability of QAF images. At the current stage, the prudent approach is to acquire duplicate images and investigate the retest reliability of lesion-specific AF.
The presented method of additionally analyzing the iso-hulls of lesions was technically difficult to implement, as iso-hulls of neighboring lesions merge. Areas of merged iso-hulls could be characterized distinctively depending on which lesion is considered. Our solution was to consider all lesions of one type as one lesion and to analyze their periphery as a joint iso-hull. This method, however, drastically reduces the ability to measure the iso-hulls of individual drusen and might be considered a further pitfall of this technique. More technically sophisticated methods to account for merged iso-hulls or suspended reporting of AF in areas of merged iso-hulls could facilitate the analysis of AF in the circumference of lesions in the future.
We used AMD as a model disease for this study. The workflow can be adapted to study lesions in other diseases as well. So far, QAF has been used in many chorioretinal diseases, including recessive Stargardt disease, Bestrophin-1 associated diseases, various forms of retinitis pigmentosa, acute zonal occult outer retinopathy, pseudoxanthoma elasticum, and others17,33,34,35,36,37. As this workflow uses open-source software, we encourage others to duplicate this work in determining lesion-specific AF and expand our knowledge of retinal disorders. In summary, we present a workflow to determine and compare AF levels of different retinal lesions throughout the macula. This workflow paves the way for more in-depth analysis of AF and could facilitate the development of new biomarkers in AMD and beyond.

Fundus autofluorescence (FAF) imaging provides a noninvasive mapping of naturally and pathologically occurring fluorophores of the ocular fundus1. The most common blue (488 nm excitation) autofluorescence (AF) excites lipofuscin and melanolipofuscin granules of the retinal pigment epithelium (RPE)2,3,4. The distribution and increase/decrease of granules play a central role in normal aging and various retinal diseases, including age-related macular degeneration (AMD)5.
A further development of FAF, quantitative fundus autofluorescence (QAF), now allows the accurate determination of topographically resolved retinal AF intensities4,6. By incorporating a reference into the optical pathway of the FAF imaging device, AF intensities can be compared between devices, time points, and subjects. This technique has resulted in a paradigm shift with regard to a presumed pathogenetic factor in AMD, which for a long time was speculated to be due to excessive lipofuscin accumulation in RPE cells7. Histologic and clinical quantification of AF, however, has revealed a decrease in AF in AMD (due to the redistribution and loss of autofluorescent lipofuscin and melanolipofuscin granules), instead of the proposed increase in AF8,9,10.
Monitoring AF has clinical implications. Von der Emde et al. and others have showed that AF is not only decreased but also further decreases in the course of AMD in high-risk, intermediate AMD eyes8,9. Additionally, histological studies suggest that most AMD-affected RPE cells show a characteristic behavior with granule aggregation and extrusion prior to RPE cell loss via subduction, sloughing, migration, or atrophy13,14,15,16. This further indicates that AF loss might be a trigger or a surrogate signal of impending disease progression.
QAF studies so far have only evaluated AF globally at the posterior pole-using prefabricated grid polar coordinate systems (e.g., QAF8/Delori Grid)17. Using prefabricated grids to measure AF results in multiple AF values on predetermined areas per eye of a subject. Investigating AF values in this way might miss local changes in areas with pathologically altered AF, for example, in AMD atop or close to drusen or subretinal drusenoid deposits (SDDs). Drusen, and to a higher degree SDDs, are associated with a high risk of developing late AMD and vision loss. Drusen in particular have a typical cycle of increasing in size over many years and may deteriorate rapidly prior to atrophy. It is conceivable that, for example, global AF decreases in AMD, but increases or is even further reduced in and around these specific disease-related focal lesions.
Different local AF patterns could also have prognostic relevance for disease progression. For example, autofluorescence levels might be used to assess whether drusen are increasing in size or are already in regression to atrophy. It has already been shown that altered AF perilesional patterns in geographic atrophy largely impact atrophy progression over time18. Additionally, local autofluorescence patterns could reveal further details into the health of the RPE. Oftentimes, the optical coherence tomography (OCT) shows hyperreflectance into the choriocapillaris, although the RPE layer appears intact. A multimodal approach combining local QAF values and OCT might help differentiate lesions with a high risk of RPE disruption and impending atrophy.
One reason spatially resolved analyses in studies have not been performed is because the most commonly used manufacturer&#39;s software does not provide a tool for these types of analysis. AF properties of different lesions dependent on the AMD disease stage could further explain the pathogenesis of AMD. Therefore, a tool to measure regional, lesion-specific AF would be desirable. To accurately compare lesions that are located throughout the retina, the workflow needs a way to account for varying degrees of AF in the human fundus19. Most centrally, AF is characteristically lower due to the shadowing effects of macular pigment and differing granule counts20,21.
AF reaches its peak at ~9&#176; (distance to the fovea in all directions) and decreases to a greater extent peripherally4. Therefore, if one were to compare absolute values of AF levels from soft drusen (located at the fovea and parafovea in low AF areas) and SDDs (located paracentrally in high AF areas), the results would not be comparable22. Inspired by the work by Pfau et al. and the concept of sensitivity loss (correcting sensitivity measured in AMD for the hill of vision [declining retinal sensitivity with distance to the fovea] of healthy controls) for fundus controlled perimetry, AF is compared to standardized AF values throughout the macula23,24. The results are reported as z-scores (numerical measurement of a region of interest value&#39;s relationship to the mean).
The purpose of this study is to evaluate the use of a new tool for measuring local QAF levels in different types of lesions in patients with AMD. This tool is designed to measure autofluorescence levels of lesions identified on OCT scans. This enables the assessment of local autofluorescence levels in lesions, such as soft drusen or SDDs, and allows for tracking of AF changes from lesions over time. The potential utility of this tool is to enable a new structural biomarker that estimates the health of the RPE and may have prognostic value for the investigated lesions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BatchStandardRetina plugin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>FIJI (Image J)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>Mark_Bscans_OCT plugin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>Microspft office</td>
			<td>Microsoft</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>QAF_xml_reader plugin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>Register_OCT_2 plugin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>Spectralis</td>
			<td>Heidelberg Engineering</td>
			<td>n.a.</td>
			<td>QAF extension</td>
		</tr>
		<tr>
			<td>StandardRetina plugin</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
	</tbody>
</table>

a workflow to quantitatively determine age-related macular degeneration lesion-specific variations in fundus autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear.
This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids.
AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.&#160;This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Author Spotlight: Understanding Age-Related Macular Degeneration Pathophysiology with QAF Workflow 

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence

So far, quantitative autofluorescence or QAF, analysis was limited to prefabricated grids analyzing global autofluorescence of the posterior pole of the eye. Our protocol allows the measurement and comparison of autofluorescence intensities of specific regions of interest. QAF revealed reduced autofluorescence intensities in age-related macular degeneration.As a matter of fact, this important finding contradicted the historical view of pathological lipofuscin accumulations in the AMD. Today, the clinical findings can nicely be confirmed by new histological data on the retinal pigment epithelium at the cellular level. The characteristic autofluorescence levels of the ocular fundus display strong regional differences.There seems to be a strong relationship between autofluorescence and rod photoreceptor density. For an accurate comparison of autofluorescence, one needs to account for regional differences, which is challenging. We could show for the first time the typical AMD lesions like drusen show further reduced autofluorescence intensities in addition to the generally reduced autofluorescence levels in these patients.We are currently focusing on spectral analysis of the emitted light from fluorophores from the retinal pigment epithelium in both in vivo and ex vivo settings. With this approach, we hope to get better information on the physiology and pathophysiology of photoreceptors and the retinal pigment epithelium in healthy and deceased eyes.

Viewing the output 
To adequately analyze and draw conclusions from the results, it is important to understand the output file of Mark_Bscans_OCT. The first three columns are labeled after the case ID, the laterality of the file, and the imaging modality that was chosen. The fourth column is referred to by mode and is labeled z-score. Note that as of writing this text, Mark BScans can only calculate all lesions in one go; the rows refer to iso-hulls, whose distances from the outer edge of the lesion are specified in the lower and upper columns of the spreadsheet. Iso-hulls measure AF in z-scores (in case of QAF) in a specified circumference surrounding the lesion. Note that the minimum value of a pixel in an iso-hull can be found in the columns labeled min, the columns labeled median, max, mean, and stdev, respectively contain the median, maximum, mean, and standard deviation of the mean of the pixel values in aniso-hull. The column n contains the total number of pixels in an iso-hull. Figure 1 shows a singular marked soft drusen of an 84-year-old male patient with intermediate age-related macular degeneration (iAMD).
Figure 2&#160;shows the left eye of a representative patient with SDDs marked with the QAF-Workflow tool (Figure 3). SDDs in this patient were associated with reduced AF (z-score = -0.4 &#177; 0.2). Similarly, the iso-hulls around the SDD demonstrated reduced AF (e.g., closest iso-hull = -0.3 &#177; 0.3) compared to the StandardRetina. A plausible explanation for this phenomenon might be shadowing effects (reduced translucency) of SDD lesions on the RPE. The use of SDDs was exemplary. The tool enables the assessment of local AF levels in other lesions, such as drusen, as well. Furthermore, the tool allows for tracking AF changes from lesions over time.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65238/65238fig01.jpg" /> 
Figure 1: A singular marked soft drusen of an 84-year-old male patient with intermediate age-related macular degeneration (iAMD). (A) The QAF image of a left eye with the marked drusen. (B) Close-up of the drusen: brown center representing the marked drusen and colored bands representing the surrounding iso-hulls. The table below depicts the output file. QAF drusen values are compared to corresponding QAF values of the corresponding eccentricity from the StandardRetina. This results in z-scores that represent deviation from the mean of unaffected areas. The blue box shows from left to right: the case ID, laterality of the eye, modality used, and the desired output (in this case, z-scores). Columns within the orange box show the boundaries of the measured area in millimeters (lower = lower bound, upper = upper bound). The green box labels the columns showing the QAF measurements. From left to right, these contain the minimum, median, maximum, number of pixels, mean, and standard deviation of the mean. Each row represents an iso-hull, rows within the blue box represent values within the lesion, and rows within the purple box show the iso-hulls surrounding each lesion (from top to bottom with increasing distance to the lesion). Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/65238/65238fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65238/65238fig02.jpg" /> 
Figure 2: Marked SDDs in a QAF image of an 80-year-old female patient with early AMD. (A) SDDs can be seen in the QAF image. The same QAF image is shown with imprinted annotations of SDDs. (B) Around each marked lesion, the iso-hulls are depicted with color-coding (light green, dark green, and red). (C) A magnified version of the blue rectangle. The outer edge of each SDD is marked in blue. Abbreviations: QAF = quantitative autofluorescence; AMD = age-related macular degeneration; SDD = subretinal drusenoid deposit. Scale bar = 1 mm. <a href="https://www.jove.com/files/ftp_upload/65238/65238fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65238/65238fig03.jpg" /> 
Figure 3: Workflow to determine AF of lesions. This figure visualizes the software plug-ins needed to determine lesion-specific AF. (A) Image shows a color-coded QAF image that can be used to visualize the distribution of QAF values, but should not be used for further analysis. (B) A QAF image in the foreground, with the infrared image from the SD-OCT scan in the background is shown. This is supposed to visualize the alignment using vessel bifurcations. This can be done using the Register_OCT_2 plug-in. (C)&#160;A StandardRetina which is used to measure the z-score values of lesions. StandardRetinas can be created using StandardRetina/BatchStandardRetina.(D) A BScan with blue arrows pointing to SDDs, which are highlighted by yellow lines (note: lesions are always marked below the RPE independent of the location in the z direction) is depicted. (E) All marked lesions are seen imprinted on a QAF image (see Figure 1). The last two steps are done using the Mark_BScans_OCT plug-in. Abbreviations: AF = autofluorescence; QAF = quantitative autofluorescence; SDD = subretinal drusenoid deposit; IR = infrared; RPE = retinal pigment epithelium; SD-OCT = spectral domain optical coherence tomography. <a href="https://www.jove.com/files/ftp_upload/65238/65238fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Understanding Age-Related Macular Degeneration Pathophysiology with QAF Workflow at JoVE.com

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment ...

a-workflow-to-quantitatively-determine-age-related-macular

Research

JoVE Journal

Medicine

Quantitative Autofluorescence (QAF) Image Acquisition

To begin, get the participant seated comfortably in front of the quantitative autofluorescence, or qAF device to ensure accurate image acquisition. Have the participant's chin and forehead pressed against the chin and headrest. Adjust the height of the chin rest until the lateral eyelid angle is at the same height as the red marking.Then rotate the wheel on the recording device until the small vessels are in focus to ensure the image is focused in the near infrared modus. Zoom in on the eye by moving the camera forward until the image corners are evenly illuminated. As a rule of thumb, adjust the focus to the spherical equivalent.Reduce the focus before qAF imaging by one or two diopters as blue QAF uses a shorter wavelength, and switch to the modus of the qAF device from the near infrared to the qAF mode. Readjust and upscale the illumination and fine-tune the focus of the image until the small vessels closest to the fovea are in focus and the image is brightly illuminated without red dots. To bleach the photoreceptor pigment in the field of view of the camera, wait for at least 30 seconds in the qAF mode before image acquisition.To capture images, press Image Acquisition on the touchpad of the imaging device. Capture more than one qAF image in case of blinking or sudden eye movements during acquisition.

Quantitative Autofluorescence (QAF) Image Processing for Analysis

To access the open source plugins for Quantitative Autofluorescence or QAF analyses, in the software Fiji. Click on Manage Update sites to add the displayed creative computation update site to the pre-existing update sites. To install the Spectralis pipeline plugin, download the plugins and restart Fiji.The different Spectralis plugins will be located under the plugins dropdown menu in Spectralis or Spectralis batch. The Spectralis QAF XML export files stored in red, green, blue, or RGB format are limited to a measured AF value scale of zero to 255 and include standard and black calibration regions. To produce a QAF image, open the plugins dropdown menu.Click Spectralis, select QAF XML reader, and discard the opening screen. A new window showing the prompt, choose a directory containing a spectral XML QAF export will appear. Select the directory and click select.In the window QAF parameters enter the reference calibration factor or RCF included in the image information, and the patient's age when the image was taken. After clicking okay, when a pop-up labeled map to eight bit appears, enter the minimum QAF or QAF min value, and maximum QAF or QAF max value for a color-coded QAF image. To add one image at a time under plugins, select Spectralis followed by add to standard retina_OCT, and dismiss the opening screen.When a window displays, choose a directory containing a Spectralis OCT XML export. Select the folder and click select to open the BScan. After the OCT volume loads and the prompt, choose a directory containing registered en Face images appears.Select the appropriate folder and click select. At this stage, three windows will pop up. One labeled en Face stack, displaying the stacked images from the selected folder.The second labeled BScan stack, displaying the OCTB scan, and the middle or third one labeled choose modality. Select a modality from the en Face stack. As a new window with the prompt, choose a directory containing the standard retina pops up, select or create the directory containing standard retina.Examine the new standard retina, scroll and move the cursor to view the mean, and standard deviation for that specific location. Click on the button Accept, to either add the latest photo to the standard retina, or discard it. To add multiple images at a time using Batch_QAF_ standard retina first, prepare a manifest.txt file in the same folder as the case IDs. The relative path from the location of the dot txt file to the OCT and the enFace stack must be listed, and separated by a tab space without any additional white space before and after the names. To scroll through the BScan stack, drag the bar at the bottom to the left or right, or select the BScan frame, and use the left and right arrow keys on the keyboard.An overview of the current BScan stack area, is provided by the red line on the enFace stack window, and on the top left of the BScan window where the BScan number is displayed. To mark a region, select mark in the draw line segment in BScan window, and start by right clicking and dragging the mouse cursor to the end of the lesion. Press the plus key to zoom in, and the minus key to zoom out.Finally, click okay to mark the region of interest. The QAF image of an eye of an 84 year old patient with intermediate age related macular degeneration, showed a singular marked soft drusen. A closeup of the area showed a brown center representing the marked drusen, and colored bands representing the surrounding ISO holes.The output file contained the case ID, the laterality of the file, and the chosen imaging modality. Each row referred to an ISO hole, whose distances from the outer edge of the lesion were specified in the lower, and upper columns of the spreadsheet. The QAF measurements contained the minimum median, and maximum values of a pixel in an ISO hole, the number of pixels, the mean pixel value, and the standard deviation of the mean.The QAF image of an eye of an 80 year old patient with early age-related macular degeneration, showed subretinal, drusenoid deposits. Around each marked lesion, the ISO holes were depicted with color coding.

733 Views.  University of Bonn.  This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Exploring Quantitative Autofluorescence in Age-Related Macular Degeneration

Summary