Publications 2019

In vivo multiphoton microscopy imaging of melasma
Lentsch G, Balu M, Williams J, Lee S, Harris RM, König K, Ganesan A, Tromberg BJ, Nair N, Santhanam U, Misra M
Pigm Cell&Melanoma Res 32,3(2019)403-411 (DOI: 10.1111/psmr.12756)

Abstract
Melasma is a skin disorder characterized by hyperpigmented patches due to increased melanin production and deposition. In this pilot study we evaluate the potential of multiphoton microscopy (MPM) to characterize non-invasively the melanin content, location, and distribution in melasma and assess the elastosis severity. We employed a clinical MPM tomograph to image in-vivo morphological features in melasma lesions and adjacent normal skin in 12 patients. We imaged dermal melanophages in most dermal melasma lesions and occasionally in epidermal melasma. The melanin volume fraction values measured in epidermal melasma (14±4%) were significantly higher (p<0.05) than the values measured in peri-lesional skin (11±3%). The basal keratinocytes of melasma and perilesions showed different melanin distribution. Elastosis was predominantly more severe in lesions than in perilesions and was associated with changes in melanin distribution of the basal keratinocytes. These results demonstrate that MPM may be a non-invasive im aging tool for characterizing melasma.

Riboflavin Concentrations at the Endothelium During Corneal Cross-Linking in Humans
Seiler TG, Batista A, Frueh BE, König K
Invest Ophthalmol & Visual Science 60(6)(2019)2140 (DOI: 10.1167/lovs.19-26686)

Abstract
Purpose: To determine the riboflavin concentration in the posterior corneal stroma, Descemet’s membrane, and endothelium prior to UV irradiation in corneal cross-linking (CXL) in humans. Methods: Five human deepithelialized cadaver corneas were mounted into artificial anterior chambers. After the establishment of stable physiological hydration, two-photon imaging with a certified multiphoton tomograph was used to determine fluorescence intensity and second harmonic generation signals from collagen throughout each cornea by optical sectioning, with a step size of 2.5 μm. Afterward, 0.1% riboflavin solution was applied to the anterior corneal surface, similar to the standard CXL protocol. To determine the absolute riboflavin concentration immediately before UV irradiation, corneas were measured by two-photon imaging just at the end of the riboflavin imbibition and after riboflavin saturation. Results: The topical application of 0.1% riboflavin results in a riboflavin concentration that decreases to 0.035% in the posterior stroma. Inside Descemet’s membrane and endothelium, the concentration drops further to only approximately 0.015% at the endothelial level. Local riboflavin distribution indicates a predominantly paracellular passive diffusion of riboflavin into the anterior chamber. Conclusion: The experimentally determined riboflavin concentration of 0.015% at the endothelium shows a substantial discrepancy of a factor of 1.7 to the previously theoretically calculated 0.025%. A lower riboflavin concentration at the endothelium may enable higher radiant exposures and further improve the efficacy of CXL.