Abstract
Pump-probe microscopy is an emerging technique that provides detailed chemical information of absorbers with sub-micrometer spatial resolution. Recent work has shown that the pump-probe signals from melanin in human skin cancers correlate well with clinical concern, but it has been difficult to infer the molecular origins of these differences. Here we develop a mathematical framework to describe the pump-probe dynamics of melanin in human pigmented tissue samples, which treats the ensemble of individual chromophores that make up melanin as Gaussian absorbers with bandwidth related via Frenkel excitons. Thus, observed signals result from an interplay between the spectral bandwidths of the individual underlying chromophores and spectral proximity of the pump and probe wavelengths. The model is tested using a dual-wavelength pump-probe approach and a novel signal processing method based on gnomonic projections. Results show signals can be described by a single linear transition path with different rates of progress for different individual pump-probe wavelength pairs. Moreover, the combined dual-wavelength data shows a nonlinear transition that supports our mathematical framework and the excitonic model to describe the optical properties of melanin. The novel gnomonic projection analysis can also be an attractive generic tool for analyzing mixing paths in biomolecular and analytical chemistry.
Highlights
Melanin is a complex biopolymer with a number of interesting physical and chemical properties that has gathered much interest from both the biomedical and materials science communities[1]
Note that ground-state bleaching produces an increase in the transmitted probe intensity, whereas excited state absorption reduces the intensity; further, pump-probe convention is that lossy signals are positive
The lifetimes of the excited states are set by the chromophore population that was selected by the pump, in our physical model the endmembers are a function of the pump wavelength, λpu, and independent of the probe wavelength, λpr
Summary
Melanin is a complex biopolymer with a number of interesting physical and chemical properties that has gathered much interest from both the biomedical and materials science communities[1]. To overcome some of these limitations, femtosecond transient absorption and nonlinear optical microscopy techniques were combined[5] This novel approach, called pump-probe microscopy, provides contrast between different types of melanins based on excited state ultrafast photodynamics, with subcellular spatial resolution[6]; pump-probe microscopy is sensitive to the pigment’s ion content, aggregate size, oxidative stress, and type (eu-/pheo-melanin)[7]. This method has found use in analyzing pigmented lesions for melanoma detection and staging in excised human biopsy specimens[8,9,10,11] and animal models in vivo[12,13]. Our model predicts that when multiple pump-probe wavelength pairs are combined these differences in the rates of progress produce a nonlinear transition–this behavior is verified experimentally
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