Study on melanin enhanced third harmonic generation in a live cell model

Abstract

Melanin dominates the endogenous contrasts of in vivo third harmonic generation (THG) imaging of human skin. A recent study investigated the THG in melanin solution and a linear relationship between melanin concentration and THG intensity was reported, in contrast to the expected nonlinear relationship. Since melanin hydrocolloid solution is very different from the skin tissue, here we report our study on the origin of the melanin-enhanced THG by using a live cell model. Different from the previous conclusion, our live cell study has indicated an initial nonlinear process where the THG intensity was enhanced according to the 3.5th power of melanin mass density (MMD). When the MMD is higher than 11 mg/ml, a transition from the resonance-enhanced THG to the high-order hyper-Rayleigh scattering process occurs. This saturation phenomenon of the virtual-transition-based THG nonlinear process is attributed to the multi-melanosome-induced scattering within the sub-femtoliter focal volume.

Fig. 1.

Nonlinear emission spectra excited by a femtosecond Cr:forsterite laser of (a) natural melanosomes collected from cultured B16-F10 cells, and of (b) the synthetic melanin dispersed in 1M NaOH solution.

Fig. 2.

Simultaneous endogenous a. Epi-THG image and b. Epi-TPEF image of B16-F10 live cells. c. The corresponding bright field image. d. A demonstration of ROI selection. e. Statistics of 41 combined TPEF and THG images. Mean values and standard errors are shown. Error bars show standard error. Epi-THG and Epi-TPEF are represented by magenta and yellow pseudo-colors.

Fig. 3.

Averaged TPEF pixel value of artificial melanin samples with different melanin mass densities. A strong linear relation (r-square = 0.99) between the measured TPEF intensity and melanin mass density can be found (shown as the orange line). The highly linear relationship verifies our assumption that TPEF is proportional to the melanin mass density and a simple linear equation $\text{TPEF}=68\times \text{MMD}+391$ can be used to estimate the melanin mass density from TPEF pixel values.

Fig. 4.

a. Statistics results illustrating the correlation between melanin mass density and THG enhancement ratio. b. A log-log plot of (a) with MMD below 11 mg/ml. The orange line is the linear regression. c. Data and the regression curves of two phases are plotted in the same graph. The intersection of two regression curves is at 11.00 mg/ml melanin mass density with a 5.93 THG enhanced ratio.