{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T03:57:42Z","timestamp":1780891062576,"version":"3.54.1"},"reference-count":35,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T00:00:00Z","timestamp":1780704000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Diagnostics"],"abstract":"<jats:p>Macular pigment optical density (MPOD) is widely measured by dual-wavelength autofluorescence imaging (AFI) because the method is noninvasive, image-based, and clinically practical. AFI-derived MPOD and macular pigment optical volume (MPOV) are increasingly used as retinal biomarkers in clinical research, supplementation studies, disease-risk interpretation, and population-based comparisons. However, conventional dual-wavelength AFI assumes that posterior absorbers other than macular pigment (MP), particularly melanin, are negligible or sufficiently stable not to bias the measurement. This assumption may limit the diagnostic and biomarker reliability of AFI-derived MP metrics. This manuscript presents a focused biological, optical, and mathematical analysis of AFI-based MP quantification. The foundational AFI literature, the melanin imaging literature, retinal pigment epithelium (RPE) pigment biology, and related optical modeling concepts were examined to evaluate where melanin enters the AFI signal pathway, how it may confound MPOD and MPOV, and how a melanin-sensitive baseline could improve quantitative specificity within the AFI domain. Conventional dual-wavelength AFI estimates MP indirectly from attenuation of RPE lipofuscin autofluorescence under excitation wavelengths that differ in MP absorption. Because melanin is located in the RPE and choroid, varies with retinal location, age, and pigmentation, and can influence the same excitation and detection pathway, unmeasured melanin can become embedded in the apparent MPOD signal. Under these conditions, reported MPOD and derived MPOV are better understood as model-dependent estimates whose quantitative specificity may vary across subjects, retinal locations, devices, and studies. This has direct implications for diagnostic interpretation, normative databases, cross-subject comparison, supplementation-response studies, and biomarker-based retinal assessment. Melanin correction is not a minor refinement of AFI-based MP quantification. It is likely necessary when AFI-derived MP metrics are intended to be interpreted as quantitatively specific retinal biomarkers rather than conditionally approximate optical estimates. A melanin-corrected AFI framework, based on introducing a melanin-sensitive baseline wavelength outside the principal MP absorption range, offers a path toward more reliable MPOD and MPOV interpretation in clinical, diagnostic, and supplementation-related studies.<\/jats:p>","DOI":"10.3390\/diagnostics16121751","type":"journal-article","created":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T02:32:46Z","timestamp":1780885966000},"page":"1751","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Melanin Correction Is Essential for Quantitative Autofluorescence-Based Measurement of Macular Pigment"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-4761-1990","authenticated-orcid":false,"given":"Mohsen","family":"Sharifzadeh","sequence":"first","affiliation":[{"name":"Department of Electrical and Computer Engineering, Brigham Young University, Provo, UT 84602, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1212","DOI":"10.1364\/JOSAA.18.001212","article-title":"Macular pigment density measured by autofluorescence spectrometry: Comparison with reflectometry and heterochromatic flicker photometry","volume":"18","author":"Delori","year":"2001","journal-title":"J. Opt. Soc. Am. A Opt. Image Sci. Vis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.abb.2004.05.016","article-title":"Autofluorescence method to measure macular pigment optical densities fluorometry and autofluorescence imaging","volume":"430","author":"Delori","year":"2004","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1186\/s13036-018-0124-5","article-title":"Imaging retinal melanin: A review of current technologies","volume":"12","author":"Carroll","year":"2018","journal-title":"J. Biol. Eng."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2373","DOI":"10.1364\/JOSAA.23.002373","article-title":"Nonmydriatic fluorescence-based quantitative imaging of human macular pigment distributions","volume":"23","author":"Sharifzadeh","year":"2006","journal-title":"J. Opt. Soc. Am. A Opt. Image Sci. Vis."},{"key":"ref_5","first-page":"145","article-title":"Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes","volume":"27","author":"Weiter","year":"1986","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3556","DOI":"10.1167\/iovs.06-0122","article-title":"Near-infrared autofluorescence imaging of the fundus: Visualization of ocular melanin","volume":"47","author":"Keilhauer","year":"2006","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_7","unstructured":"Sharifzadeh, M. (2024). A Method for Accurate Measurement of Macular Pigment Optical Density Using Autofluorescence Imaging with Melanin Consideration. (Patent Application No. 18\/757,450; International Application No. PCT\/US25\/61130)."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1167\/tvst.9.8.40","article-title":"Evaluation of Macular Pigment Optical Density in Healthy Eyes Based on Dual-Wavelength Autofluorescence Imaging in South Indian Population","volume":"9","author":"Srinivasan","year":"2020","journal-title":"Transl. Vis. Sci. Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1097\/IAE.0000000000000893","article-title":"Reproducibility of Macular Pigment Optical Density Measurement by Two-Wavelength Autofluorescence in a Clinical Setting","volume":"36","author":"You","year":"2016","journal-title":"Retina"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1167\/tvst.8.6.41","article-title":"Standardizing the Assessment of Macular Pigment Using a Dual-Wavelength Autofluorescence Technique","volume":"8","author":"Bernstein","year":"2019","journal-title":"Transl. Vis. Sci. Technol."},{"key":"ref_11","first-page":"583","article-title":"Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical, and ultrastructural studies","volume":"17","author":"Feeney","year":"1978","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1016\/j.ophtha.2015.12.026","article-title":"Incidence of Age-Related Macular De-generation in a Multi-Ethnic United States Population: The Multi-Ethnic Study of Atherosclerosis","volume":"123","author":"Fisher","year":"2016","journal-title":"Ophthalmology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"716","DOI":"10.1016\/j.visres.2009.10.014","article-title":"The value of measurement of macular carotenoid pigment optical densities and distributions in age-related macular degeneration and other retinal disorders","volume":"50","author":"Bernstein","year":"2010","journal-title":"Vis. Res."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Nagai, N., Minami, S., Suzuki, M., Shinoda, H., Kurihara, T., Sonobe, H., Watanabe, K., Uchida, A., Ban, N., and Tsubota, K. (2020). Macular pigment optical density and photoreceptor outer segment length as predisease biomarkers for age-related macular degenera-tion. J. Clin. Med., 9.","DOI":"10.3390\/jcm9051347"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2449","DOI":"10.1007\/s00417-024-06437-6","article-title":"Comparing 2-dimensional macular pigment optical density with objective and subjective perimetry and visual acuity in age-related macular degeneration","volume":"262","author":"Rai","year":"2024","journal-title":"Graefe\u2019s Arch. Clin. Exp. Ophthalmol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1937","DOI":"10.1167\/iovs.19-26566","article-title":"Impact of Drusen Volume on Quantitative Fundus Autofluorescence in Early and Inter-mediate Age-Related Macular Degeneration","volume":"60","author":"Reiter","year":"2019","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2284","DOI":"10.1167\/iovs.16-20510","article-title":"Macular Pigment Density and Quantitative Fundus Autofluorescence in Young Healthy Subjects","volume":"58","author":"Eandi","year":"2017","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.abb.2010.06.019","article-title":"Dose-dependent response of serum lutein and macular pigment optical density to sup-plementation with lutein esters","volume":"504","author":"Bone","year":"2010","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"100039","DOI":"10.1016\/j.xops.2021.100039","article-title":"Macular Pigment Response to Lutein, Zeaxanthin, and Meso-zeaxanthin Supplementation in Open-Angle Glaucoma: A Randomized Controlled Trial","volume":"1","author":"Loughman","year":"2021","journal-title":"Ophthalmol. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2244","DOI":"10.1093\/advances\/nmab071","article-title":"The Effect of Lutein\/Zeaxanthin Intake on Human Macular Pigment Optical Density: A Systematic Review and Meta-Analysis","volume":"12","author":"Wilson","year":"2021","journal-title":"Adv. Nutr."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"100216","DOI":"10.1016\/j.advnut.2024.100216","article-title":"Effect of Antioxidant Supplementation on Macular Pigment Optical Density and Visual Functions: A Systematic Review and Network Meta-Analysis of Randomized Controlled Trials","volume":"15","author":"Hu","year":"2024","journal-title":"Adv. Nutr."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Obana, A., Tanito, M., Gohto, Y., Okazaki, S., Gellermann, W., and Bernstein, P.S. (2015). Changes in Macular Pigment Optical Density and Serum Lutein Concentration in Japanese Subjects Taking Two Different Lutein Supplements. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0139257"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1167\/iovs.65.3.13","article-title":"Personalized Lens Correction Improves Quantitative Fundus Autofluorescence Analysis","volume":"65","author":"Rennen","year":"2024","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"12685","DOI":"10.1038\/s41598-021-92309-6","article-title":"Influence of lens opacities and cataract severity on quantitative fundus autofluorescence as a secondary outcome of a randomized clinical trial","volume":"11","author":"Reiter","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1167\/tvst.11.10.23","article-title":"Impact of the Aging Lens and Posterior Capsular Opacification on Quantitative Autofluorescence Imaging in Age-Related Macular Degeneration","volume":"11","author":"Berlin","year":"2022","journal-title":"Transl. Vis. Sci. Technol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e001628","DOI":"10.1136\/bmjophth-2023-001628","article-title":"Impact of lens autofluorescence and opacification on retinal imaging","volume":"9","author":"Rennen","year":"2024","journal-title":"BMJ Open Ophthalmol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3259","DOI":"10.1016\/j.visres.2007.09.002","article-title":"Macular pigment, photopigments, and melanin: Distributions in young subjects determined by four-wavelength reflectometry","volume":"47","author":"Bone","year":"2007","journal-title":"Vis. Res."},{"key":"ref_28","unstructured":"Sharifzadeh, M. (2025). Systems and Methods for Melanin- and Macular-Pigment\u2013Corrected Quantification of Retinal Lipofuscin Using Dual-Excitation Autofluorescence Imaging. (Patent Application No. 63\/980,399)."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.survophthal.2008.10.004","article-title":"Fundus autofluorescence and progression of age-related macular degeneration","volume":"54","author":"Fleckenstein","year":"2009","journal-title":"Surv. Ophthalmol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.exer.2014.01.016","article-title":"Studying melanin and lipofuscin in RPE cell culture models","volume":"126","author":"Boulton","year":"2014","journal-title":"Exp. Eye Res."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Masri, A., Armanazi, M., Inouye, K., Geierhart, D.L., Davey, P.G., and Vasudevan, B. (2024). Macular Pigment Optical Density as a Measurable Modifiable Clinical Biomarker. Nutrients, 16.","DOI":"10.20944\/preprints202407.0822.v1"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2290","DOI":"10.1016\/j.ophtha.2012.06.014","article-title":"Effect of Lutein and Zeaxanthin on Macular Pigment and Visual Function in Patients with Early Age-related Macular Degeneration","volume":"119","author":"Ma","year":"2012","journal-title":"Ophthalmology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.1001\/jamaophthalmol.2017.0929","article-title":"The Role of Macular Pigment Density Measures in Future Clinical Studies of Age-Related Macular Degeneration","volume":"135","author":"Lindblad","year":"2017","journal-title":"JAMA Ophthalmol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"927","DOI":"10.1167\/iovs.10-5664","article-title":"Effect of cataract in evaluation of macular pigment optical density by autofluorescence spectrometry","volume":"52","author":"Sasamoto","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"8174","DOI":"10.1167\/iovs.11-7522","article-title":"Effects of Lutein Supplementation on Macular Pigment Optical Density and Visual Acuity in Patients with Age-Related Macular Degeneration","volume":"52","author":"Weigert","year":"2011","journal-title":"Investig. Ophthalmol. Vis. Sci."}],"container-title":["Diagnostics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/12\/1751\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T03:00:06Z","timestamp":1780887606000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2075-4418\/16\/12\/1751"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,6]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["diagnostics16121751"],"URL":"https:\/\/doi.org\/10.3390\/diagnostics16121751","relation":{},"ISSN":["2075-4418"],"issn-type":[{"value":"2075-4418","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,6]]}}}