In the human skin, the distribution and content of melanin in the epidermis and dermis play important roles in determining the color of the human skin26. After being produced by melanocytes, melanin granules mainly aggregate at the dermal–epidermal junction (DEJ) and disperse in dermal macrophages27. Both HE staining and FeSO4 staining required biopsy and are time-consuming. Therefore, in the field of melanin imaging, especially in the quantitative analysis of melanin granules in skin with dark circles, it is very necessary to apply a noninvasive, accurate, fast and label-free method.
Pump-probe imaging allows specific imaging of pigment molecules and is characterized by low tissue damage and strong penetration depth28. Specifically, in the pump-probe imaging in this study, melanin in dispersed macrophages tends to lack negative signal at zero delay, which may be due to the thermal effects of concentrated melanin absorbing the laser and dissipating in the form of heat or the influence of oxidative degradation28. These results were consistent with FeSO4 staining of melanin, which suggests that pump-probe imaging can be used for melanin imaging in skin tissues of the lower eyelid, distinguishing melanin between epidermal cells and macrophages, and even helping distinguish macrophages and melanocytes in vivo28. Under the same imaging conditions (pump, 1045 nm; probe, 850 nm), which shielded the influence of the Raman spectrum of melanin, the transient absorption spectrum of the lower eyelid skin (Sample) was very similar to that of pure melanin solution (Standard), which further confirmed that the target regions we selected in the sample were indeed melanin granules.
In terms of correlation analysis, the MCIs of the two melanin imaging methods (pump-probe imaging and FeSO4 staining) showed a high consistency, indicating that the pump-probe imaging method is reliable for the quantitative analysis of melanin. In addition, the MCIs of both melanin imaging methods showed a negative correlation with the L* value, which means that the darker the skin of the lower eyelid, i.e. the lower L*, the higher the proportion of melanin in pump-probe and FeSO4 staining. Moreover, the linear fit of L* and pump-probe imaging showed nearly the same slope with the linear fit of L* and FeSO4 staining, which further indicates that the pump-probe method has high reproducibility in melanin imaging and high accuracy in the quantitative analysis of melanin. This is an interesting observation because, so far, only a few studies have examined the relationship between the quantitative index of melanin and the L* value.
Pump-probe imaging and statistical analysis revealed that the density of melanin granules in the pigmented group was significantly higher than that in the normal group and the vascular group (in both epidermis and dermis), which as Watanabe et al.29 found by anti-S100 protein and Masson-Fontana silver staining, periorbital pigmentation was mainly characterized by a relative increase in the number of skin melanocytes and melanin content. In addition, when there is little or no subcutaneous tissue, the color of the orbicularis oculi muscle will be revealed, or the number of microvessels in the skin of the lower eyelid is higher than that in the cheeks, which will cause the purple shadowing of the skin of the lower eyelid4,30. This is because the skin in the vascular group is thinner than that in the normal group; thus, melanin is more easily deposited and the MCI-1 and MCI-2 values were slightly higher than those of the normal group.
In pump-probe imaging, the MFI of melanin granules in the pigmented group was significantly higher than that in the normal group and vascular group. The main reason is the significantly increased melanin content of the pigmented group when compared with the other two groups. Therefore, significant differences were noted in the number of photons emitted by the instantaneous absorption of the melanin in a certain area of pump-probe imaging. Interestingly, the difference in MFI-1 and MFI-2 between groups is consistent with that in MCI-1 and MCI-2, as Antoniou et al.31 used multiphoton tomography to observe different skin depths and types of melanin, that is, fluorescence intensity is correlated with melanin concentration. In summary, MCI and MFI are reliable indicators of identifying the difference between the skin with dark circles and normal skin, assessing treatment response, and tracking the development of dark circles.
The advantages of abundant molecular contrast signals and high spatial resolution make pump-probe microscopic imaging an ideal method to investigate melanocytosis15. Multiple microscopy methods have been used for the analysis of melanin content (such as diffuse reflectance spectroscopy, optical coherence tomography and confocal microscopy). However, none of the above methods can distinguish different melanin species. Pump-probe microscopy, on the other hand, takes advantage of the difference in electron transfer rate within melanin molecules, which is the only method that can specifically distinguish different species. Although this study did not use the distortion of species, this method is the best means to study melanin.
In this study, pump-probe imaging was used in evaluating dark circles for the first time, and the results confirmed the linear correlation between the pump-probe imaging and the L* value. However, the L* value needs to be obtained under strict and unified photographic conditions, and relevant software was required for correction. In addition, the results of this study confirmed that pump-probe imaging could accurately analyze the melanin content and fluorescence intensity in dark circles. Not only did the imaging conditions stable with very few interference factors, but also could characterize melanin of different depths, which could guide the depth of clinical treatment (such as choosing a laser with appropriate depth and model). Therefore, the results of this study provide some insights into the study of melanin deposition-related diseases in plastic surgery and dermatology.
This study aimed to verify the feasibility of pump-probe imaging for quantitative analysis of the melanin granules in the lower eyelid skin through ex vivo experiments. The experimental results confirmed the accuracy of pump-probe imaging in melanin and the possibility of classification in dark circles. In terms of clinical applications, only certain parameters need to be adjusted based on the current imaging system and the corresponding adapter at different parts of the body needs to be developed, the non-invasive application of pump-probe imaging could be theoretically implemented. Figure 7 shows the clinical application of in vivo pump-probe imaging and classification-evaluation-monitoring system. This technology could characterize melanin of different depths, which could guide the depth of clinical treatment, such as choosing a laser with an appropriate model. Therefore, this study laid the foundation for the application of pump-probe imaging in the diagnosis and treatment of dark circles. With the development and diversification of hyperspectral imaging instrument miniaturization, clinicians may then classify skin disorders, evaluate treatment responses, and monitor disease progression based on pump-probe images of pigmentation lesions.
Figure 7
In vivo pump-probe imaging and classification-evaluation-monitoring system.