多模态反射荧光实时共聚焦成像系统的研制
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摘要
皮肤组织因容易受到外界紫外线、化学物质的侵蚀,病变的几率非常大。由于美容的因素,皮肤组织的病理取样检查会受到很大限制。因此,共聚焦激光扫描显微镜由于其无创性,实时性,成像分辨率高等优点被皮肤病领域研究人员广泛采纳。常用的共聚焦激光扫描显微镜可以分为两类:反射式共聚焦激光扫描显微镜和荧光式共聚焦激光扫描显微镜。前者,我们可以得到样本组织的结构信息;后者,则可以通过荧光标记获取样本组织的病理学信息。然而,到目前为止绝大多数科研人员只专注于单一模式的共聚焦显微镜的成像研究。仅通过单一的组织学结构信息或病理学信息的简单判断,无疑会对病情的精确诊断产生影响。
本文研究了一种新型的用于皮肤病诊断的多模态反射荧光实时共聚焦成像系统,该系统不但结合了反射荧光两种成像功能,实现了皮肤组织细胞新陈代谢的生化病理成像与皮肤组织细胞结构成像的结合,而且同时实现了两种信号同点实时成像,更重要的是荧光成像针对的是细胞体内的自发荧光(NADH和FAD),无需借助外界荧光探针标记,真正实现了无损成像。通过这两种信号的同点实时成像,可为皮肤科医生同时提供丰富的组织学和病理学影像信息,提高皮肤疾病乃至皮肤癌症的早期诊断率。
本文工作的具体内容包括以下几个方面:
1多模态反射荧光实时共聚焦成像系统硬件部分实现,包括反射光路和荧光光路设计、扫描装置设计、检测装置设计。
2多模态反射荧光实时共聚焦成像系统软件部分实现,包括扫描方式算法、采集方式算法、图像重建算法以及对该系统的一系列优化算法的实现。
3多模态反射荧光实时共聚焦成像系统在皮肤组织成像中的应用,包括表皮结构的反射成像实验、黑色素瘤诊断实验。
Confocal laser scanning microscopy has been widely accepted in dermatological research, due to its advantages in non-invasive, real time, high resolution in vivo imaging. Conventional confocal laser scanning microscopy can be divided into two modes: reflectance mode and fluorescence mode. The reflectance mode demonstrates tissue structural information based on the different refractive indices among the various tissue microstructures. The fluorescence mode can detect tissue pathological information with the excitation of exogenous fluorescent dye to produce contrast. However, up to now most researchers have been focusing on either reflectance or fluorescence confocal imaging separately which can only provide either structural or pathological information of tissue. This greatly affected the accuracy of dermatological diagnosis by lack of enough diagnostic information.
In order to improve diagnostic performance of dermatosis efficiently, we developed a new design of noninvasive simultaneous multimodality fluorescence and reflectance confocal microscope which combines the structural imaging and pathological imaging. Moreover, in the confocal system the fluorescence imaging is aim to autofluorescence substance such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) which exclude the toxic affection from the introduction of exogenous fluorescent dyes. With both imaging modality acquired simultaneously, the combined image revealed the morphological and pathological information of the suspicious site, yielding a possibility of precancer diagnosis of skin.
The study contents in this thesis are demonstrated as follows:
1 The hardware design of multimodality reflectance and fluorescence confocal microscope, including reflectance and fluorescence optical system, scanning system and detecting system.
2 The software design of multimodality reflectance and fluorescence confocal microscope, including scan mode algorithm design, acquisition mode algorithm design and image reconstruction algorithm design.
3 In vivo applications in skin tissue imaging by multimodality reflectance and fluorescence confocal microscope, including epidermis tissue imaging in mouse skin and melanoma imaging diagnosis.
本文研究了一种新型的用于皮肤病诊断的多模态反射荧光实时共聚焦成像系统,该系统不但结合了反射荧光两种成像功能,实现了皮肤组织细胞新陈代谢的生化病理成像与皮肤组织细胞结构成像的结合,而且同时实现了两种信号同点实时成像,更重要的是荧光成像针对的是细胞体内的自发荧光(NADH和FAD),无需借助外界荧光探针标记,真正实现了无损成像。通过这两种信号的同点实时成像,可为皮肤科医生同时提供丰富的组织学和病理学影像信息,提高皮肤疾病乃至皮肤癌症的早期诊断率。
本文工作的具体内容包括以下几个方面:
1多模态反射荧光实时共聚焦成像系统硬件部分实现,包括反射光路和荧光光路设计、扫描装置设计、检测装置设计。
2多模态反射荧光实时共聚焦成像系统软件部分实现,包括扫描方式算法、采集方式算法、图像重建算法以及对该系统的一系列优化算法的实现。
3多模态反射荧光实时共聚焦成像系统在皮肤组织成像中的应用,包括表皮结构的反射成像实验、黑色素瘤诊断实验。
Confocal laser scanning microscopy has been widely accepted in dermatological research, due to its advantages in non-invasive, real time, high resolution in vivo imaging. Conventional confocal laser scanning microscopy can be divided into two modes: reflectance mode and fluorescence mode. The reflectance mode demonstrates tissue structural information based on the different refractive indices among the various tissue microstructures. The fluorescence mode can detect tissue pathological information with the excitation of exogenous fluorescent dye to produce contrast. However, up to now most researchers have been focusing on either reflectance or fluorescence confocal imaging separately which can only provide either structural or pathological information of tissue. This greatly affected the accuracy of dermatological diagnosis by lack of enough diagnostic information.
In order to improve diagnostic performance of dermatosis efficiently, we developed a new design of noninvasive simultaneous multimodality fluorescence and reflectance confocal microscope which combines the structural imaging and pathological imaging. Moreover, in the confocal system the fluorescence imaging is aim to autofluorescence substance such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) which exclude the toxic affection from the introduction of exogenous fluorescent dyes. With both imaging modality acquired simultaneously, the combined image revealed the morphological and pathological information of the suspicious site, yielding a possibility of precancer diagnosis of skin.
The study contents in this thesis are demonstrated as follows:
1 The hardware design of multimodality reflectance and fluorescence confocal microscope, including reflectance and fluorescence optical system, scanning system and detecting system.
2 The software design of multimodality reflectance and fluorescence confocal microscope, including scan mode algorithm design, acquisition mode algorithm design and image reconstruction algorithm design.
3 In vivo applications in skin tissue imaging by multimodality reflectance and fluorescence confocal microscope, including epidermis tissue imaging in mouse skin and melanoma imaging diagnosis.
引文
[1]霍霞,吕建勋.激光共聚焦显微镜与光学显微镜之比较.激光生物学报, 2001, 10(1): 76-79.
[2] J. Young, F. Roberts. A flying-spot microscope. Nature, 1951, 167: 231.
[3] M. Minsky, "US Patent 3013467, Microscopy apparatus," (United States, 1961).
[4] T. Wilson, C. Sheppard. Theory and practice of scanning optical microscopy. Academic New York, 1984.
[5] T. Wilson. Three-dimensional imaging in confocal systems. Journal of Microscopy, 1989, 153(2): 161-169.
[6] G. Brakenhoff, H. Van der Voort, E. Van Spronsen, et al. Three-dimensional imaging in fluorescence by confocal scanning microscopy. Journal of Microscopy, 1989, 153(2): 151-159.
[7] S. Paddock. Confocal laser scanning microscopy. Biotechniques, 1999, 27(5): 992-1007.
[8] J. White, W. Amos. Confocal microscopy comes of age. Nature, 1987, 328: 183-184.
[9] R. Tsien, A. Waggoner, "Handbook of biological confocal microscopy," (Plenum Press, New York, 1995).
[10] S. Inoué. Foundations of confocal scanned imaging in light microscopy. Handbook of biological confocal microscopy, 1995: 1-18.
[11] BIL, "Confocal Microscopy" (University of Illinois at Urbana-Champaign), retrieved http://biophotonics.illinois.edu/technology/confocal/.
[12] M. Rajadhyaksha, R. Anderson, R. Webb. Video-rate confocal scanning laser microscope for imaging human tissues in vivo. Applied Optics, 1999, 38: 2105-2115.
[13] D. Gareau, G. Merlino, C. Corless, et al. Noninvasive imaging of melanoma with reflectance mode confocal scanning laser microscopy in a murine model. Journal of Investigative Dermatology, 2007, 127(9): 2184-2190.
[14] R. Hoffman. Green fluorescent protein imaging of tumor growth, metastasis, andangiogenesis in mouse models. The Lancet Oncology, 2002, 3(9): 546-556.
[15]赵启韬,苗俊英.激光共聚焦显微镜在生物医学研究中的应用.北京生物医学工程, 2003, 22(1): 52-54.
[16] B. Matsumoto, I. Hale, T. Kramer. Theory and applications of confocal microscopy. Analytical morphology: theory, applications, and protocols, 1997: 231-244.
[17]李楠,尹岭,苏振伦.激光扫描共聚焦显微术(北京:人民军医出版社, 1997).
[18]许险峰.活细胞钙动态的共聚焦扫描显微镜检测技术.激光生物学报, 2002, 11(4): 296-300.
[19]朱珊珊,黄志江.激光扫描共聚焦显微镜在生命科学研究中的应用.国外医学:麻醉学与复苏分册, 2005, 26(2): 118-119.
[20]雷国华,吴建春.荧光定量测量方法在共聚焦显微镜上的应用.第一军医大学学报, 1998, 18(1): 47-51.
[21] B. Armstrong, A. Kricker. The epidemiology of UV induced skin cancer. Journal of Photochemistry & Photobiology, B: Biology, 2001, 63(1-3): 8-18.
[22]刘华绪,郑志忠,任秋实.基于光学共聚焦原理的皮肤在体三维成像系统及应用.中华皮肤科杂志, 2006, 39(10): 616-619.
[23] R. MacKie, C. Fleming, A. McMahon, et al. The use of the dermatoscope to identify early melanoma using the three-colour test. British Journal of Dermatology, 2002, 146(3): 481-484.
[24] F. Urbach. Ultraviolet radiation and skin cancer of humans. Journal of Photochemistry & Photobiology, B: Biology, 1997, 40(1): 3-7.
[25] M. Rajadhyaksha, S. González, J. M. Zavislan, et al. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. Journal of Investigative Dermatology, 1999, 113(3): 293-303.
[26] A. Gerger, S. Koller, T. Kern, et al. Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors. Journal of Investigative Dermatology, 2005, 124(3): 493-498.
[27] P. Daukantas. Using optics to detect skin cancer. Optics and Photonics News, 2007, 18(11): 28-33.
[28] A. Vogel, V. Venugopalan. Mechanisms of pulsed laser ablation of biological tissues. Chemical Reviews, 2003, 103(2): 577-644.
[29] A. Carlson, L. Coghlan, A. Gillenwater, et al. Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue. Journal of Microscopy, 2007, 228(1): 11-24.
[30] R. Drezek, C. Brookner, I. Pavlova, et al. Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia. Photochemistry and photobiology, 2001, 73(6): 636-641.
[31] Y. Wu, P. Xi, J. Qu, et al. Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue. Optics Express, 2005, 13(2): 382-388.
[32] Y. Wu, P. Xi, J. Qu, et al. Depth-resolved fluorescence spectroscopy reveals layered structure of tissue. Optics Express, 2004, 12(14): 3218-3223.
[33] S. Chang, N. Marin, M. Follen, et al. Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia. Journal of Biomedical Optics, 2006, 11: 024008.
[34]周展超,郑家润.脉冲激光在皮肤病学中的应用.国外医学:皮肤性病学分册, 2002, 28(3): 145-148.
[35] Thorlabs, THORLABS V20, DL5032-001-830nm 30mW Sanyo Laser Diode (2009) 1046.
[36] RGBLase, "Temperature stabilized semiconductor laser module."
[37] G. Wagnieres, W. Star, B. Wilson. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochemistry and photobiology, 1998, 68(5): 603-632.
[38] Chroma, "Products specification", retrieved http://www.chroma.com/.
[39] Go4Fiber, "Inline fixed value attenuator."
[40]傅雅静,郭丽洁,高建,等.虚拟显微镜技术及其在医学领域的应用.解剖科学进展, 2007, 13(3): 283-285.
[41] K. Maitland, H. Shin, H. Ra, et al. Single fiber confocal microscope with a two-axis gimbaled MEMS scanner for cellular imaging. Optics Express, 2006, 14(19): 8604-8612.
[42] Cambridge-Technology, "Model 6215H optical scanner."
[43]赵毅,卢秉恒.振镜扫描系统的枕形畸变校正算法.中国激光, 2003, 30(3): 216-218.
[44] Cambridge-Technology, "The MicroMax? Model 677XX."
[45]丁伟雄,杨定安,宋晓光.步进电机的控制原理及其单片机控制实现.煤矿机械, 2005, (6): 127-129.
[46] Haydon, "28H-47-05-113混合式直线步进电机", retrieved http://www.haydonkerk.com.cn/.
[47] Haydon, "DCM4010细分驱动器使用手册."
[48] Hamamatsu, "APD module C5460 series," (2007).
[49] Hamamatsu, "Compact side-on PMT photo sensor modules H9305 series," (2003).
[50]胡春华,王忠林.荧光检测中光电倍增管的增益电压控制.中国科技信息, 2007, (18): 94-95.
[51] D. Schweitzer, A. Kolb, M. Hammer, et al., "Method and apparatus for determining fluorophores on objects, especially on the living ocular fundus," United States US6371615B1 (2002).
[52] Hamamatsu, "Wide bandwidth amplifier C6438-01," (1999).
[53] National-Instruments, "High-Speed M Series Multifunction DAQ 16-Bit, up to 1.25 MS/s, up to 32 Analog Inputs," (2005).
[54] T. Klinger, Image processing with LabVIEW and IMAQ Vision (Prentice Hall, 2003).
[55] D. Chou, B. Bower, A. Wax. Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy. Applied optics, 2005, 44(11): 2013-2018.
[56] Q. Nguyen, P. Tsai, D. Kleinfeld. MPScope: a versatile software suite for multiphoton microscopy. Journal of neuroscience methods, 2006, 156(1-2): 351-359.
[57] V. Bansal, S. Patel, P. Saggau. High-speed addressable confocal microscopy for functional imaging of cellular activity. Journal of Biomedical Optics, 2006, 11: 034003.
[58] ImageJ, "T1 Head", retrieved http://rsb.info.nih.gov/ij/download/sample-images.zip.
[59] L. Meyer, N. Otberg, W. Sterry, et al. In vivo confocal scanning laser microscopy: comparison of the reflectance and fluorescence mode by imaging human skin. Journal of Biomedical Optics, 2006, 11: 044012.
[60] M. Rajadhyaksha, "Confocal reflectance microscopy: diagnosis of skin cancer without biopsy," in Fourth Annual Symposium on Frontiers of Engineering, (National Academy of Engineering, 1998), 24.
[61] E. Melanoma, N. Consensus. Diagnosis and treatment of early melanoma. Journal of the American Medical Association, 1992, 268(10): 1314-1319.
[62] P. Calzavara-Pinton, C. Longo, M. Venturini, et al. Reflectance confocal microscopy for in vivo skin imaging. Photochemistry and photobiology, 2008, 84(6): 1421-1430.
[2] J. Young, F. Roberts. A flying-spot microscope. Nature, 1951, 167: 231.
[3] M. Minsky, "US Patent 3013467, Microscopy apparatus," (United States, 1961).
[4] T. Wilson, C. Sheppard. Theory and practice of scanning optical microscopy. Academic New York, 1984.
[5] T. Wilson. Three-dimensional imaging in confocal systems. Journal of Microscopy, 1989, 153(2): 161-169.
[6] G. Brakenhoff, H. Van der Voort, E. Van Spronsen, et al. Three-dimensional imaging in fluorescence by confocal scanning microscopy. Journal of Microscopy, 1989, 153(2): 151-159.
[7] S. Paddock. Confocal laser scanning microscopy. Biotechniques, 1999, 27(5): 992-1007.
[8] J. White, W. Amos. Confocal microscopy comes of age. Nature, 1987, 328: 183-184.
[9] R. Tsien, A. Waggoner, "Handbook of biological confocal microscopy," (Plenum Press, New York, 1995).
[10] S. Inoué. Foundations of confocal scanned imaging in light microscopy. Handbook of biological confocal microscopy, 1995: 1-18.
[11] BIL, "Confocal Microscopy" (University of Illinois at Urbana-Champaign), retrieved http://biophotonics.illinois.edu/technology/confocal/.
[12] M. Rajadhyaksha, R. Anderson, R. Webb. Video-rate confocal scanning laser microscope for imaging human tissues in vivo. Applied Optics, 1999, 38: 2105-2115.
[13] D. Gareau, G. Merlino, C. Corless, et al. Noninvasive imaging of melanoma with reflectance mode confocal scanning laser microscopy in a murine model. Journal of Investigative Dermatology, 2007, 127(9): 2184-2190.
[14] R. Hoffman. Green fluorescent protein imaging of tumor growth, metastasis, andangiogenesis in mouse models. The Lancet Oncology, 2002, 3(9): 546-556.
[15]赵启韬,苗俊英.激光共聚焦显微镜在生物医学研究中的应用.北京生物医学工程, 2003, 22(1): 52-54.
[16] B. Matsumoto, I. Hale, T. Kramer. Theory and applications of confocal microscopy. Analytical morphology: theory, applications, and protocols, 1997: 231-244.
[17]李楠,尹岭,苏振伦.激光扫描共聚焦显微术(北京:人民军医出版社, 1997).
[18]许险峰.活细胞钙动态的共聚焦扫描显微镜检测技术.激光生物学报, 2002, 11(4): 296-300.
[19]朱珊珊,黄志江.激光扫描共聚焦显微镜在生命科学研究中的应用.国外医学:麻醉学与复苏分册, 2005, 26(2): 118-119.
[20]雷国华,吴建春.荧光定量测量方法在共聚焦显微镜上的应用.第一军医大学学报, 1998, 18(1): 47-51.
[21] B. Armstrong, A. Kricker. The epidemiology of UV induced skin cancer. Journal of Photochemistry & Photobiology, B: Biology, 2001, 63(1-3): 8-18.
[22]刘华绪,郑志忠,任秋实.基于光学共聚焦原理的皮肤在体三维成像系统及应用.中华皮肤科杂志, 2006, 39(10): 616-619.
[23] R. MacKie, C. Fleming, A. McMahon, et al. The use of the dermatoscope to identify early melanoma using the three-colour test. British Journal of Dermatology, 2002, 146(3): 481-484.
[24] F. Urbach. Ultraviolet radiation and skin cancer of humans. Journal of Photochemistry & Photobiology, B: Biology, 1997, 40(1): 3-7.
[25] M. Rajadhyaksha, S. González, J. M. Zavislan, et al. In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology. Journal of Investigative Dermatology, 1999, 113(3): 293-303.
[26] A. Gerger, S. Koller, T. Kern, et al. Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors. Journal of Investigative Dermatology, 2005, 124(3): 493-498.
[27] P. Daukantas. Using optics to detect skin cancer. Optics and Photonics News, 2007, 18(11): 28-33.
[28] A. Vogel, V. Venugopalan. Mechanisms of pulsed laser ablation of biological tissues. Chemical Reviews, 2003, 103(2): 577-644.
[29] A. Carlson, L. Coghlan, A. Gillenwater, et al. Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue. Journal of Microscopy, 2007, 228(1): 11-24.
[30] R. Drezek, C. Brookner, I. Pavlova, et al. Autofluorescence Microscopy of Fresh Cervical-Tissue Sections Reveals Alterations in Tissue Biochemistry with Dysplasia. Photochemistry and photobiology, 2001, 73(6): 636-641.
[31] Y. Wu, P. Xi, J. Qu, et al. Depth-resolved fluorescence spectroscopy of normal and dysplastic cervical tissue. Optics Express, 2005, 13(2): 382-388.
[32] Y. Wu, P. Xi, J. Qu, et al. Depth-resolved fluorescence spectroscopy reveals layered structure of tissue. Optics Express, 2004, 12(14): 3218-3223.
[33] S. Chang, N. Marin, M. Follen, et al. Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia. Journal of Biomedical Optics, 2006, 11: 024008.
[34]周展超,郑家润.脉冲激光在皮肤病学中的应用.国外医学:皮肤性病学分册, 2002, 28(3): 145-148.
[35] Thorlabs, THORLABS V20, DL5032-001-830nm 30mW Sanyo Laser Diode (2009) 1046.
[36] RGBLase, "Temperature stabilized semiconductor laser module."
[37] G. Wagnieres, W. Star, B. Wilson. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochemistry and photobiology, 1998, 68(5): 603-632.
[38] Chroma, "Products specification", retrieved http://www.chroma.com/.
[39] Go4Fiber, "Inline fixed value attenuator."
[40]傅雅静,郭丽洁,高建,等.虚拟显微镜技术及其在医学领域的应用.解剖科学进展, 2007, 13(3): 283-285.
[41] K. Maitland, H. Shin, H. Ra, et al. Single fiber confocal microscope with a two-axis gimbaled MEMS scanner for cellular imaging. Optics Express, 2006, 14(19): 8604-8612.
[42] Cambridge-Technology, "Model 6215H optical scanner."
[43]赵毅,卢秉恒.振镜扫描系统的枕形畸变校正算法.中国激光, 2003, 30(3): 216-218.
[44] Cambridge-Technology, "The MicroMax? Model 677XX."
[45]丁伟雄,杨定安,宋晓光.步进电机的控制原理及其单片机控制实现.煤矿机械, 2005, (6): 127-129.
[46] Haydon, "28H-47-05-113混合式直线步进电机", retrieved http://www.haydonkerk.com.cn/.
[47] Haydon, "DCM4010细分驱动器使用手册."
[48] Hamamatsu, "APD module C5460 series," (2007).
[49] Hamamatsu, "Compact side-on PMT photo sensor modules H9305 series," (2003).
[50]胡春华,王忠林.荧光检测中光电倍增管的增益电压控制.中国科技信息, 2007, (18): 94-95.
[51] D. Schweitzer, A. Kolb, M. Hammer, et al., "Method and apparatus for determining fluorophores on objects, especially on the living ocular fundus," United States US6371615B1 (2002).
[52] Hamamatsu, "Wide bandwidth amplifier C6438-01," (1999).
[53] National-Instruments, "High-Speed M Series Multifunction DAQ 16-Bit, up to 1.25 MS/s, up to 32 Analog Inputs," (2005).
[54] T. Klinger, Image processing with LabVIEW and IMAQ Vision (Prentice Hall, 2003).
[55] D. Chou, B. Bower, A. Wax. Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy. Applied optics, 2005, 44(11): 2013-2018.
[56] Q. Nguyen, P. Tsai, D. Kleinfeld. MPScope: a versatile software suite for multiphoton microscopy. Journal of neuroscience methods, 2006, 156(1-2): 351-359.
[57] V. Bansal, S. Patel, P. Saggau. High-speed addressable confocal microscopy for functional imaging of cellular activity. Journal of Biomedical Optics, 2006, 11: 034003.
[58] ImageJ, "T1 Head", retrieved http://rsb.info.nih.gov/ij/download/sample-images.zip.
[59] L. Meyer, N. Otberg, W. Sterry, et al. In vivo confocal scanning laser microscopy: comparison of the reflectance and fluorescence mode by imaging human skin. Journal of Biomedical Optics, 2006, 11: 044012.
[60] M. Rajadhyaksha, "Confocal reflectance microscopy: diagnosis of skin cancer without biopsy," in Fourth Annual Symposium on Frontiers of Engineering, (National Academy of Engineering, 1998), 24.
[61] E. Melanoma, N. Consensus. Diagnosis and treatment of early melanoma. Journal of the American Medical Association, 1992, 268(10): 1314-1319.
[62] P. Calzavara-Pinton, C. Longo, M. Venturini, et al. Reflectance confocal microscopy for in vivo skin imaging. Photochemistry and photobiology, 2008, 84(6): 1421-1430.