光學透明化運用在人體離體與活體皮膚之倍頻影像術上的效用與機制

Investigate the Optical Clearing Effects and Mechanisms of Ex Vivo and In Vivo Human Skin with Glycerol Application by Harmonic Generation Microscopy

賴嘉泓(Jia-Hong Lai)

指導教授 ： 孫啟光

國立臺灣大學/電機資訊學院/光電工程學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202000120

摘要

光學清潔法可用來提升光學顯微鏡的成像品質與深度。然而，由於缺乏影像的細節資訊，其運用在人體皮膚上的效果與機制仍未被完全了解。此研究中，我們使用倍頻顯微鏡，藉由結合三倍頻與二倍頻的高解析度影像，研究光學清潔法使用在離體與活體皮膚上的效用與機制。實行光學清潔法後，樣品內部的折射率均勻度提升，造成三倍頻影像強度在上表皮衰減，而二倍頻影像在下表皮的強度變化作為一個可靠的指標，顯示光學散射在上表皮的減少。結合這兩個影像技術，可以獲得更多關於光學清潔法的細節。
在離體皮膚的研究裡，我們將九位受試者分配到四種不同的實驗條件，分別將皮膚樣品浸泡或是表面塗抹上100% 或是 50% 的甘油，研究在不同狀況下的光學清潔效果。在50% 塗抹的實驗條件下，發現角質層扮演一個重要的關鍵，其光學清潔效果在厚角質層的皮膚樣品更為明顯。
過去光學清潔法在活體皮膚的研究中，使用在手臂內側與手掌的結果有很大的差異，在薄角質層的部位，效果非常薄弱。這些過去的活體皮膚研究也與我們的離體皮膚研究也發現一樣的結果。然而，許多的光學療法以及光學診斷都是運用在薄角質層的地方，光學清潔法使用在此類薄角質皮膚上的效果及機制仍需要被研究與了解。在此研究的活體皮膚部分，我們將　50%　甘油塗抹在手前臂內側，我們將八位受試者分配到四種不同的作用時間（15, 30, 90, 180　分鐘），發現光學清潔法的效果在90分鐘以內的效用不明顯，而在180分鐘作用後，在三個受試者中有兩位有明顯的效果，在此類薄角質層皮膚的部位，越長的作用時間，有越高的機率達成光學清潔的效用。

關鍵字

光學清潔法 ； 活體皮膚 ； 離體皮膚 ； 甘油 ； 三倍頻 ； 二倍頻

並列摘要

Imaging depth and quality of the optical microscopy can be enhanced by using the method of the optical clearing. However, the optical clearing effects and mechanisms in human skin remain largely unclear due to the lack of detailed image information. Here we investigate the optical clearing of the ex vivo and in vivo human skin by glycerol application. Harmonic generation microscopy, by combining second and third harmonic generation (THG) modalities, was utilized to retrieve high-resolution skin images. The THG image intensity in the epidermis is decreased due to the optical homogeneity after optical clearing, and the second harmonic generation (SHG) image intensity in the dermis is a beacon to confirm the reduction of the scattering in the epidermis layer. Combining these two image information, the details of the optical clearing effects can be well studied. For ex vivo experiment, nine different volunteers were separated into four different case types to study and unravel the optical clearing mechanisms. The 100% or 50% glycerol is applied on the skin by immersion or topical application. For the case type of 50% glycerol combined with the topical application, the method which can be used for the further in vivo or clinical research, SC acts the most important role. We observed stronger OC effect in thicker SC skin tissues.
For the previous in vivo human optical clearing studies, the effect between the volar forearm and palm showed different results. The optical clearing effect was found less effective in the skin area with thinner SC. The same result was also noted in our ex vivo experiment. However, it is essential to study the optical clearing on the skin with thinner SC, the area where the light therapeutic and dermatological diagnosis often focus on. In this study, we investigated the OC effect on the in vivo volar forearm by using 50% glycerol with topical application. For in vivo experiment, eight different volunteers were separated into four different applying time (15, 30, 90, and 180 minutes) to unravel the optical clearing mechanisms. The results showed that the optical clearing was less effective within 90 minutes application. After 180 minutes application, two of three cases were found the effect of the optical clearing. In the skin area with thinner SC, there was a higher probability to witness the optical clearing effects after a longer applying time.

並列關鍵字

optical clearing ； in vivo human skin ； ex vivo human skin ； glycerol ； third-harmonic generation ； second-harmonic generation

參考文獻

A. Forli, D. Vecchia, N. Binini, F. Succol, S. Bovetti, C. Moretti, F. Nespoli, M. Mahn, C. A. Baker, M. M. Bolton, O. Yizhar, and T. Fellin, “Two-photon bidirectional control and imaging of neuronal excitability with high spatial resolution in vivo,” Cell Rep. 22, 3087-3098 (2018).

T. H. Tsai, S. P. Tai, W. J. Lee, H. Y. Huang, Y. H. Liao, and C. K. Sun, “Optical signal degradation study in fixed human skin using confocal microscopy and higher-harmonic optical microscopy,” Opt. Express 14, 749-758 (2006).

V. V. Tuchin, I. L. Maksimova, D. A. Zimnyakov, I. L. Kon, A. H. Mavlyutov, and A. A. Mishin, “Light propagation in tissues with controlled optical properties,” J. Biomed. Opt. 2, 401-417 (1997).

H. Zhong, Z. Guo, H. Wei, C. Zeng, H. Xiong, Y. He, and S. Liu, “In vitro study of ultrasound and different-concentration glycerol-induced changes in human skin optical attenuation assessed with optical coherence tomography,” J. Biomed. Opt. 15, 036012 (2010).

A. Sdobnov, M. E. Darvin, J. Lademann, and V. Tuchin, “A comparative study of ex vivo skin optical clearing using two-photon microscopy,” J Biophotonics 10, 1115-1123 (2017).