Abstract
Imaging depth and quality of optical microscopy can be enhanced by optical clearing. Here we investigate the optical clearing of the ex vivo human skin by 50% glycerol topical application, which is allowed for cosmetic usage. Harmonic generation microscopy, by combining second and third harmonic generation (THG) modalities, was utilized to examine the clearing effect. The THG image intensity is sensitive to the improved optical homogeneity after optical clearing, and the second harmonic generation (SHG) image intensity in the dermis could serve as a beacon to confirm the reduction of the scattering in the epidermis layer. As a result, our study supports the OC effect through 50% glycerol topical application. Our study further indicates the critical role of stratum corneum shrinkage for the observed SHG and THG signal recovery.
Highlights
Imaging depth and quality of optical microscopy can be enhanced by optical clearing
In order to overcome the depth limitation and to enhance the image quality, researchers have attempted to reduce the scattering of tissue turbidity by matching the refractive index using various osmotic agents, a procedure known as optical clearing (OC)
OC of human skin have been studied by many different optical imaging techniques such as near-infrared (NIR) s pectrophotometer[9,10,11,12,23,29,36], confocal microscopy[13,14], multi-photon microscopy[8,15,35], second harmonic generation (SHG) microscopy[8], and optical coherence tomography (OC T)[5,12,17,18,26,29,30,34,36,37] in the past few decades, the effects and mechanisms of optical clearing in human skin with glycerol topically applied are not fully understood and are with inconsistent results, especially for the case of glycerol at low concentration
Summary
Optical clearing effect of 100% glycerol immersion. First, we discuss the results of Case E100-I90, which was under the most frequently studied condition by using 100% glycerol immersion of 90 min. For 100% glycerol immersion cases, THG intensity decreased in epidermis while increased or remain the same in dermis, indicating that refractive index matching is the dominant effect in. With its lower refractive index (1.398)[63] and lower dehydration power compared to 100% glycerol, we found that the 50% glycerol was able to decrease the scattering in the tissue, evidenced by the improved transparency after immersion, and by the significantly increased SHG intensity in dermis (Fig. 6). The epidermal THG signal was increased after 50% glycerol application for all samples with a SC layer thicker than 27 μm, including cases E50-I90-2, E50-T90-2, and E50-T90-4 This might be due to the fact that the skin tissue with a thick SC layer suffers stronger light scattering so that the OC effect is more pronounced. It is noted that in a previous paper, decreased SHG intensity was
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