Near-Infrared Excited State Dynamics of Melanins: The Effects of Iron Content, Photo-Damage, Chemical Oxidation, and Aggregate Size

Abstract

Ultrafast pump–probe measurements can discriminate the two forms of melanin found in biological tissue (eumelanin and pheomelanin), which may be useful for diagnosing and grading melanoma. However, recent work has shown that bound iron content changes eumelanin’s pump–probe response, making it more similar to that of pheomelanin. Here we record the pump–probe response of these melanins at a wider range of wavelengths than previous work and show that with shorter pump wavelengths the response crosses over from being dominated by ground-state bleaching to being dominated by excited-state absorption. The crossover wavelength is different for each type of melanin. In our analysis, we found that the mechanism by which iron modifies eumelanin’s pump–probe response cannot be attributed to Raman resonances or differences in melanin aggregation and is more likely caused by iron acting to broaden the unit spectra of individual chromophores in the heterogeneous melanin aggregate. We analyze the dependence on optical intensity, finding that iron-loaded eumelanin undergoes irreversible changes to the pump–probe response after intense laser exposure. Simultaneously acquired fluorescence data suggest that the previously reported “activation” of eumelanin fluorescence may be caused in part by the dissociation of metal ions or the selective degradation of iron-containing melanin.

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“…At the other extreme (~θ = 2.5; i.e., primarily a negative contribution from PC1) the excited state dynamics are dominated by ground state bleaching. This behavior has been observed in eumelanin with high levels of metal content, small aggregate size, and also in synthetic pheomelanin [20]. The elevation angle shows minor deviations, capturing more subtle changes in the pigments' dynamics.…”

“…1(d)). The first endmember, around θ = 0 (signifying a positive contribution from PC1), is dominated by excited state absorption: this type of transient behavior has been observed for eumelanin void of metals, or with large molecular weight aggregates (>100 kDa) [20]. At the other extreme (~θ = 2.5; i.e., primarily a negative contribution from PC1) the excited state dynamics are dominated by ground state bleaching.…”

“…The elevation angle shows minor deviations, capturing more subtle changes in the pigments' dynamics. These have not been documented using melanin standards, but have been observed in tissue [14,20], and further justify the use of an unsupervised quantitative approach. (We note that a small contribution to the elevation angle may result from small shifts in the pumpprobe time-delay calibration.)…”

“…The center wavelengths of the pulses are 730 nm (pump) and 810 nm (probe)-this combination has been found to provide good contrast between different types of melanins [20]. The pulses are focused onto the sample using a 20X objective (0.8NA) with less than 0.65 mW total power to avoid any possible effects that may alter the melanin pigment chemistry (e.g, photobleaching) [20].…”

“…Previously [14,23,24], we assumed that the pump-probe signals resulted solely from a linear combination of eu-and pheo-melanin (based on similarities to sepia eumelanin and synthetic pheomelanin), which permits quantification of the transient signals using an inverse approach. More recent work, however, has generated a richer picture: pump-probe imaging is sensitive to many more properties of melanin, including type (eu-and pheo-melanin), oxidation, aggregate size, and metal content [20]. While such sensitivity is valuable in capturing detailed molecular information, it presents a significant challenge in modeling the behavior, particularly since different physical properties can exhibit similar dynamics.…”