5.3 50% Glycerol with Topical Application
Chapter 7 Discussion of the Ex Vivo Optical Clearing
Within 90-minutes glycerol application (Case T15, Case T30, and Case I50-T90), most of the volunteers showed no significant difference except Case I50-T30-1.
The optical clearing was less effective within 90 minutes application. This result correlates well with the previous studies on the volar forearm by 60- and 120-minutes topical application [33, 50] and on the dorsal hand by 60-minutes application [33]. The only case showing the OC effect can be attributed to the skin shrinkage, and will be discussed separately below.
However, in a case type of 180-minutes application, two of three volunteers (Case I50-T180-1 and Case I50-T180-3) were found with the optical clearing effect, on the skin area with a thin SC. The thickness of SC was all lower than 20 μm (Table 6.13). This indicates that even with a thin SC, it is with a higher probability to witness the optical clearing after a longer applying time. It is further noted that these two cases show different trends in the HGM image, indicating that the optical clearing mechanisms were different between these two cases.
7.1 Case I50-T180-1
There were enhancements of the SHG intensities in the dermis (Table 6.15), indicating that the light scattering in the epidermal skin was reduced [60, 61]. This is
further evidenced by the fact that the THG intensity at the SC and the epidermis were both decreased (Table 6.14). Decrease of THG intensity is the indication of improved optical homogeneity [61, 68, 69]. A 50 % glycerol is with a higher refractive index (1.40) [72] than extracellular and intracellular fluids (1.34-1.36) [66] . After immersion the skin tissues into anhydrous glycerol, glycerol diffused inside and filled all of the skin tissues to create a higher refractive index matching environment for the scattering particles (such as organelles, protein fibrils, membranes, protein globules, with refractive index 1.39-1.47 [49, 66] ). Our result supports that fact that the 50% glycerol solution diffused into the epidermal skin, created a higher homogeneous environment, and resulted in reduction of the light scattering as well as the THG intensity [60]. The skin became transparent in the superficial skin layers and there were more photons travelling in/out the tissue, so as to enhance the image intensity in the deep skin. The results were similar to our ex vivo study by 100% glycerol immersion. Similar conclusion was also suggested in the study of Wen et al. [29] by introducing thiazone-PEG400 in vivo on rat skin, and in the study of Fox et al. [32] by introducing 100% glycerol with SC removal and low pressure transdermal device on in vivo human skin. The correlated HGM images of Case I50-T180-1 have shown in section 6.2.4.
7.2 Case I50-T180-3
In this case, the THG intensity increased greatly at the bottom of the SC and the middle layer of the viable epidermis (Table 6.14). However, the optical clearing effect was not found in the dermis (Table 6.15). The reason might be that the glycerol solution did not penetrate far enough into the epidermis as the above-mentioned case to introduce an optical clearing effect at the dermis. The results of this volunteer were similar to our another previous ex vivo study by 100% glycerol topical application. The glycerol diffused into the SC and reduced its optical scattering, thus increasing the laser excitation power in layers beneath as well as the back-reflection signals. The relationship of the OCAs diffusion and the optical clearing effect at the deep area was also noted by Tuchin in 2017 [5]
7.3 Case I50-T30-1
In this case, we found the strongest OC effects, and it was the only case the optical clearing effect found with application within 90 minutes. The average THG and SHG intensities were all greatly enhanced (Table 6.4 and Fig. 6.10 in section 6.2.2). The 50%
glycerol diffused into the skin within 30 minutes and this is the only tissue that was found to shrink after the glycerol application (Table 6.13). In our previous ex vivo study, 50%
glycerol was found to be able to dehydrate and shrink the skin. The dehydration and shrinkage of the tissue were both proved to benefit the OC efficacy, and to enhance the
image intensities [49, 70, 71]. It might be the main reason that the optical clearing effect was pronounced in this case. Similar results were also found in our previous ex vivo study by 50% glycerol topical application, and in the study of Zhong et al. by introducing thiazone-PEG400 with ultrasonic on in vivo human skin [15] in which the OCT image intensity increased at all detected depths.
7.4 Conclusion
In this study, we investigated the optical clearing of the human volar forearm. The results showed that the optical clearing was less effective with topical application within 90 minutes. Only one case was found the effect of the optical clearing accompanied with its significant decrements of the skin thickness. For 180 minutes topical 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 after a longer applying time.
In general, topical application by introducing 50% glycerol cannot provide a promising optical clearing effect in the in vivo human skin. For most of the cases, the skin structure and the HGM intensity remained the same after the glycerol application. Optical clearing agents cannot introduce the same optical clearing effect on the skin in vivo as that of ex vivo. We believe the main reason might be the hydration effect, while
dehydration has been proved as one of the most important factors for the optical clearing [49, 52, 71]. The metabolism and blood circulation of the human body prevented the skin from dehydration and thus decreased the optical clearing efficacy. Besides, in our previous ex vivo study, SC played a critical role for the observed THG enhancement. We found signal enhancement in THG imaged epidermal tissues with a SC layer thicker than 27 μm. Skin tissue with a thicker SC layer suffers stronger light scattering in SC so that the OC effect is more pronounced after topical applications. In other previous studies, the optical clearing of in vivo human skin was found effective in in vivo human palm [13, 30]
and finger [31], in which the thickness of SC is thicker than the volar forearm. In this studies, the SC thicknesses of the inner side of forearm were all thinner than 21 μm.
Weaker scattering in the thin SC might make the OC effect less observable after application.
Still, we successfully achieved the optical clearing of the in vivo human skin by using topical application with 50% glycerol after long application time. After the glycerol application, no photodamage or the destruction of the skin structure was observed. This true noninvasive method with low concentration glycerol (50%) is safer than other methods in previous studies which adopted invasive methods that made the skin suppurate, necrose, or develop erythema [14, 48, 51]. Furthermore, in case I50-T180-1, the glycerol diffused into the viable epidermis and enhanced the image intensity at the dermis, proved
the possibility OC effect beyond the SC. We found different effects of the optical clearing in different volunteers, indicating that the optical clearing efficacy strongly depends on human factor and mainly depends on the individual difference of each volunteer.
Chapter 8 Summary
In the previous studies, the mechanisms of the skin optical clearing still not be well-understood. In this study, the mechanisms and effects of the ex vivo and in vivo optical clearing were investigated by HGM which combined both SHG and THG. The optical clearing was first studied by combined these two different image techniques. SHG is a beacon to accumulate the reduction of the scattering. On the other hand, the THG intensity variation strongly based on its homogeneity sensitivity. We investigated the optical clearing by this unprecedented approach with a statistic analysis, to reveal the detail mechanisms of the optical clearing.
In ex vivo experiments, nine different volunteers were separated into four different case types to study the OC. Based on the different refractive indices between 100% and 50% glycerol, the THG intensity variation showed different consequences. We first addressed the optical clearing by using THG. It was also a new approach to provide information of OCAs diffusion. The topical application with the 50% glycerol, which was a safe method for the clinical, showed minimal optical efficacy. In the skin area with a thicker SC, the more enhancement of the THG intensity at the epidermis. The thickness of the skin and the diffusion depth of the glycerol played a critical role in the optical clearing.
Based on the results of the ex vivo experiments, we further studied the in vivo optical clearing, especially at the skin area with a thin SC, volar forearm. In in vivo experiments, eight volunteers were separated into four different applying time to study the OC. In most volunteers, the effect of the optical clearing was not obvious. With and within 90 minutes application, only one case was found the optical clearing with significant shrinkage of the skin. After 180 minutes application, two of three cases were found the effect of the OC.
There was a higher probability to witness the optical clearing after a longer applying time.
Although it was not obvious OC effect in in vivo experiment, we still found OC effect beyond the SC in one of the volunteers. We supposed that the metabolism and blood circulation would decrease the OC efficacy, and it also strongly depended on the individual difference of the volunteers. The in vivo OC in the skin area with a thin SC still needs more exploitation.
Overall, in this study, the optical clearing effects were obvious in the ex vivo experiments. We studied many different skin areas to reveal the mechanisms of the OC.
The OC was effective at different skin areas ex vivo. However, we didn’t study the OC effect at the same site continuously. It was challenging to observe the effect of the OC continuously at one same skin site, especially for the human in vivo with such a long experiment time. The next step of this research is to observe the OC continuously at the
same skin site. The most detail information of the OC, and its relationship between the image intensity variation and the OCAs diffusion can be better revealed and studied.
References
1. 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).
2. 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).
3. 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).
4. 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).
5. 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).
6. Z. Mao, D. Zhu, Y. Hu, X. Wen, and Z. Han, “Influence of alcohols on the optical clearing effect of skin in vitro,” J. Biomed. Opt. 13, 1-6 (2008).
7. T. Yu, X. Wen, V. V. Tuchin, Q. Luo, and D. Zhu, “Quantitative analysis of dehydration in porcine skin for assessing mechanism of optical clearing,” J.
Biomed. Opt. 16, 095002 (2011).
8. V. Genin, D. Tuchina, A. J. Sadeq, E. Genina, V. Tuchin, and A. Bashkatov, “Ex vivo
investigation of glycerol diffusion in skin tissue,” Journal of Biomedical Photonics
& Engineering 2, 010303 (2016).
9. O. Vargas, E. K. Chan, J. K. Barton, H. G. Rylander, and A. J. Welch, “Use of an agent to reduce scattering in skin,” Lasers Surg. Med. 24, 133-141 (1999).
10. M. Kohl, M. Cope, M. Essenpreis, and D. Böcker, “Influence of glucose concentration on light scattering in tissue-simulating phantoms,” Opt. Lett. 19, 2170-2172 (1994).
11. J. S. Maier, S. A. Walker, S. Fantini, M. A. Franceschini, and E. Gratton, “Possible correlation between blood glucose concentration and the reduced scattering coefficient of tissues in the near infrared,” Opt. Lett. 19, 2062-2064 (1994).
12. H. Liu, B. Beauvoit, M. Kimura, and B. Chance, “Dependence of tissue optical properties on solute-induced changes in refractive index and osmolarity,” J.
Biomed. Opt. 1, 200-211 (1996).
13. R. K. Wang, X. Xu, Y. He, S. G. Proskurin, R. K. Wang, J. B. Elder, J. C. Hebden, A. V.
Priezzhev, and V. V. Tuchin, "Optical clearing of in vivo human skin with hyperosmotic chemicals investigated by optical coherence tomography and near-infrared reflectance spectroscopy," presented at the Proc. SPIE 5486,129-135 (2004).
14. V. V. Tuchin, S. P. Chernova, N. y. V. Kuznetsova, A. B. Pravdin, and V. V. Tuchin,
"Dynamics of optical clearing of human skin in vivo," presented at the Proc. SPIE 4162,227-235 (2000).
15. H. Zhong, Z. Guo, H. Wei, L. Guo, C. Wang, Y. He, H. Xiong, and S. Liu,
“Synergistic effect of ultrasound and thiazone-peg 400 on human skin optical clearing in vivo,” Photochem. Photobiol. 86, 732-737 (2010).
16. X. Xu, and Q. Zhu, “Feasibility of sonophoretic delivery for effective skin optical
clearing,” IEEE Trans. Biomed. Eng. 55, 1432-1437 (2008).
17. V. V. Tuchin, G. B. Altshuler, A. A. Gavrilova, A. B. Pravdin, D. Tabatadze, J. Childs, and I. V. Yaroslavsky, “Optical clearing of skin using flash lamp-induced enhancement of epidermal permeability,” Lasers Surg. Med. 38, 824-836 (2006).
18. C. Liu, Z. Zhi, V. V. Tuchin, Q. Luo, and D. Zhu, “Enhancement of skin optical clearing efficacy using photo-irradiation,” Lasers Surg. Med. 42, 132-140 (2010).
19. I. V. Meglinskii, E. A. Genina, D. Y. Churmakov and V. V. Tuchin, “Study of the possibility of increasing the probing depth by the method of reflection confocal microscopy upon immersion clearing of near-surface human skin layers,”
Quantum Electronics 32, 875-882 (2002).
20. A. Majdzadeh, Z. Wu, H. Lui, D. I. McLean, and H. Zeng, “Investigation of optically cleared human skin in combined multiphoton and reflectance confocal microscopy,” Optics in the Life Sciences, NM4C.6 (2015).
21. E. Song, Y. Ahn, J. Ahn, S. Ahn, C. Kim, S. Choi, R. M. Boutilier, Y. Lee, P. Kim, and H. Lee, “Optical clearing assisted confocal microscopy of ex vivo transgenic mouse skin,” Optics & Laser Technology 73, 69-76 (2015).
22. R. Cicchi, F. S. Pavone, D. Massi, and D. D. Sampson, “Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents,” Opt. Express 13 (2005).
23. V. Hovhannisyan, P. S. Hu, S. J. Chen, C. S. Kim, and C. Y. Dong, “Elucidation of the mechanisms of optical clearing in collagen tissue with multiphoton imaging,” J.
Biomed. Opt. 18, 046004 (2013).
24. B. Choi, N. Kollias, H. Zeng, H. W. Kang, B. J. F. Wong, J. F. Ilgner, G. J. Tearney, K.
W. Gregory, L. Marcu, M. C. Skala, P. J. Campagnola, A. Mandelis, M. D. Morris, R.
Samatham, N. K. Wang, and S. L. Jacques, "Investigation of the effect of hydration
on dermal collagen in ex vivo human skin tissue using second harmonic generation microscopy," presented at the Photonic Therapeutics and Diagnostics XII (2016).
25. T. Son, and B. Jung, “Cross-evaluation of optimal glycerol concentration to enhance optical tissue clearing efficacy,” Skin Res. Technol. 21, 327-332 (2015).
26. R. K. Wang, X. Xu, V. V. Tuchin, and J. B. Elder, “Concurrent enhancement of imaging depth and contrast for optical coherence tomography by hyperosmotic agents,” J. Opt. Soc. Am. B 18, 948-953 (2001).
27. C. G. Rylander, O. F. Stumpp, T. E. Milner, N. J. Kemp, J. M. Mendenhall, K. R. Diller, and A. J. Welch, “Dehydration mechanism of optical clearing in tissue,” J. Biomed.
Opt. 11, 041117 (2006).
28. K. V. Larin, M. G. Ghosn, A. N. Bashkatov, E. A. Genina, N. A. Trunina, and V. V.
Tuchin, “Optical clearing for oct image enhancement and in-depth monitoring of molecular diffusion,” IEEE Journal of Selected Topics in Quantum Electronics 18, 1244-1259 (2012).
29. X. Wen, S. L. Jacques, V. V. Tuchin, and D. Zhu, “Enhanced optical clearing of skin in vivo and optical coherence tomography in-depth imaging,” J. Biomed. Opt. 17, 066022 (2012).
30. X. Guo, Z. Guo, H. Wei, H. Yang, Y. He, S. Xie, G. Wu, X. Deng, Q. Zhao, and L. Li,
“In vivo comparison of the optical clearing efficacy of optical clearing agents in human skin by quantifying permeability using optical coherence tomography,”
Photochem. Photobiol. 87, 734-740 (2011).
31. S. G. Proskurin, and I. V. Meglinski, “Optical coherence tomography imaging depth enhancement by superficial skin optical clearing,” Laser Physics Letters 4, 824-826 (2007).
32. M. A. Fox, D. G. Diven, K. Sra, A. Boretsky, T. Poonawalla, A. Readinger, M.
Motamedi, and R. J. McNichols, “Dermal scatter reduction in human skin: A method using controlled application of glycerol,” Lasers Surg. Med. 41, 251-255 (2009).
33. X. X. Q. Zhu, “Sonophoretic delivery for contrast and depth improvement in skin optical coherence tomography,” IEEE Journal of Selected Topics in Quantum Electronics 14, 56 - 61 (2008).
34. E. A. Genina, N. S. Ksenofontova, A. N. Bashkatov, G. S. Terentyuk, and V. V. Tuchin,
“Study of the epidermis ablation effect on the efficiency of optical clearing of skin in vivo,” Quantum Electronics 47, 561-566 (2017).
35. A. Y. Sdobnov, V. V. Tuchin, J. Lademann, and M. E. Darvin, “Confocal raman microscopy supported by optical clearing treatment of the skin—influence on collagen hydration,” J. Phys. D: Appl. Phys. 50 (2017).
36. R. F. Rushmer, K. J. K. Buettner, J. M. Short, and G. F. Odland, “The skin,” Science 154, 343-348 (1966).
37. M. A. Lampe, A. L. Burlingame, J. Whitney, M. L. Williams, B. E. Brown, E. Roitman, and P. M. Elias, “Human stratum corneum lipids: Characterization and regional variations,” J. Lipid Res. 24, 120-130 (1983).
38. R. R. Wickett, and M. O. Visscher, “Structure and function of the epidermal barrier,” Am. J. Infect. Control 34, S98-S110 (2006).
39. M. B. Brown, G. P. Martin, S. A. Jones, and F. K. Akomeah, “Dermal and transdermal drug delivery systems: Current and future prospects,” Drug Deliv. 13, 175-187 (2006).
40. G. J. Tearney, M. E. Brezinski, J. F. Southern, B. E. Bouma, M. R. Hee, and J. G.
Fujimoto, “Determination of the refractive index of highly scattering human
tissue by optical coherence tomography,” Opt. Lett. 20, 2258-2260 (1995).
41. J. W. Fluhr, M. Gloor, L. Lehmann, S. Lazzerini, F. Distante, and E. Berardesca,
“Glycerol accelerates recovery of barrier function in vivo,” Acta Derm. Venereol.
79, 418-421 (1999).
42. D. L. Bissett, and J. F. Mcbride, “Skin conditioning with glycerol,” J. Soc. Cosmet.
Chem. 35, 345-350 (1984).
43. R. L. Evans, G. A. Turner, S. Bates, T. Robinson, D. Arnold, R. E. Marriott, P. D. A.
Pudney, E. Y. M. Bonnist, and D. Green, “Human axillary skin condition is improved following incorporation of glycerol into the stratum corneum from an antiperspirant formulation,” Arch. Dermatol. Res. 309, 739-748 (2017).
44. C. E. Rommel, C. Dierker, L. Schmidt, S. Przibilla, G. von Bally, B. Kemper, and J.
Schnekenburger, “Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index,” J. Biomed. Opt. 15, 041509 (2010).
45. S. Plotnikov, V. Juneja, A. B. Isaacson, W. A. Mohler, and P. J. Campagnola, “Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle,” Biophys. J. 90, 328-339 (2006).
46. B. Choi, L. Tsu, E. Chen, T. S. Ishak, S. M. Iskandar, S. Chess, and J. S. Nelson,
“Determination of chemical agent optical clearing potential using in vitro human skin,” Lasers Surg. Med. 36, 72-75 (2005).
47. G. Vargas, K. F. Chan, S. L. Thomsen, and A. J. Welch, “Use of osmotically active agents to alter optical properties of tissue: Effects on the detected fluorescence signal measured through skin,” Lasers Surg. Med. 29, 213-220 (2001).
48. D. S. Jäger, Z. Mao, D. Liu, Z. Han, X. Wen, P. Shum, Q. Luo, and D. Zhu, "Influence of glycerol with different concentrations on skin optical clearing and
morphological changes in vivo," presented at the Proc. SPIE 7278(2008).
49. X. Xu, and R. K. Wang, “The role of water desorption on optical clearing of biotissue: Studied with near infrared reflectance spectroscopy,” Med. Phys. 30, 1246-1253 (2003).
50. M. H. Khan, B. Choi, S. Chess, K. M. Kristen, J. McCullough, and J. S. Nelson,
“Optical clearing of in vivo human skin: Implications for light-based diagnostic imaging and therapeutics,” Lasers Surg. Med. 34, 83-85 (2004).
51. E. I. Galanzha, V. V. Tuchin, A. V. Solovieva, T. V. Stepanova, Q. Luo, and H. Cheng,
“Skin backreflectance and microvascular system functioning at the action of osmotic agents,” J Phys D Appl Phys 36, 1739-1746 (2003).
52. X. Wen, Z. Mao, Z. Han, V. V. Tuchin, and D. Zhu, “In vivo skin optical clearing by glycerol solutions: Mechanism,” J Biophotonics 3, 44-52 (2010).
53. Y. Gu, Y. Tang, X. Li, Q. Luo, L. Deng, D. Zhu, T. Yu, and W. Feng, "In vivo monitoring optical clearing process of skin using two-photon microscopy," presented at the Proc. SPIE 10820 (2018).
54. X. Liu, and B. Chen, “In vivo experimental study on the enhancement of optical clearing effect by laser irradiation in conjunction with a chemical penetration enhancer,” Applied Sciences 9, 542 (2019).
55. D. Zhu, J. Wang, Z. Zhi, X. Wen, and Q. Luo, “Imaging dermal blood flow through the intact rat skin with an optical clearing method,” J. Biomed. Opt. 15, 026008 (2010).
56. J. Squier, and M. Müller, “High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging,” Rev. Sci. Instrum. 72, 2855-2867 (2001).
57. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, “Nonlinear scanning laser
microscopy by third harmonic generation,” Appl. Phys. Lett. 70, 922-924 (1997).
58. C. K. Sun, C. T. Kao, M. L. Wei, S. H. Chia, F. X. Kärtner, A. Ivanov, and Y. H. Liao,
“Slide-free imaging of hematoxylin-eosin stained whole-mount tissues using combined third-harmonic generation and three-photon fluorescence microscopy,” Journal of Biophotonics 12, e201800341 (2019).
59. C. K. Sun, S. W. Chu, S. P. Tai, S. Keller, A. Abare, U. K. Mishra, and S. P. Denbaars,
“Mapping piezoelectric-field distribution in gallium nitride with scanning second-harmonic generation microscopy,” Scanning 23, 182-192 (2001).
60. J. H. Lai, Y. H. Liao, and C. K. Sun, “Investigating the optical clearing effects and mechanisms in ex vivo human skin by harmonic generation microscopy,”
Subittmed to Biomedical Optical Express (2020), Manuscript ID: 385788.
61. S. Y. Chen, S. U. Chen, H. Y. Wu, W. J. Lee, Y. H. Liao, and C. K. Sun, “In vivo virtual biopsy of human skin by using noninvasive higher harmonic generation microscopy,” IEEE Journal of Selected Topics in Quantum Electronics 16, 478-492 (2010).
62. Y. H. Liao, Y. H. Su, Y. T. Shih, W. S. Chen, S. H. Jee, and C. K. Sun, “In vivo third-harmonic generation microscopy study on vitiligo patients,” J. Biomed. Opt. 25, 014504 (2019).
63. M. R. Tsai, Y. H. Cheng, J. S. Chen, Y. S. Sheen, Y. H. Liao, and C. K. Sun, “Differential diagnosis of nonmelanoma pigmented skin lesions based on harmonic
63. M. R. Tsai, Y. H. Cheng, J. S. Chen, Y. S. Sheen, Y. H. Liao, and C. K. Sun, “Differential diagnosis of nonmelanoma pigmented skin lesions based on harmonic