Three kinds of 3D human skin models (MEL300 series) and growth medium (EPI-100 containing KGF113) were purchased from the Bio-Medical Department of Kurabo Industries Ltd. from MatTek Corp. Hydrogen peroxide (Wako Pure Chemical Industries, Inc.) was used to stimulate the skin models. A CO2 incubator was used to maintain the skin models at 37 °C under a 5% CO2 atmosphere. Five pieces of the Asian skin model were subjected to H2O2 stimulation in which 10 µL of 2 mM H2O2 was added to the 5 mL of medium containing each piece of the Asian skin model. Another
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piece was used as the control, with no H2O2 stimulation. This procedure was repeated for both the Black and Caucasian skin models. Stimulation was applied once for each piece of a skin model.
A CCD-equipped Raman spectrometer (RS in Fig. 1b) was used with a microscope attachment. Laser light tuned to 785 nm (L785) was reflected with an edge filter (EF). The laser power was averaged to 130 mW. The light was focused onto the target material with an objective lens (L1: 20×, NA 0.4). The scattered light of the target material reached the RS through L1, EF, a notch filter (NF), an imaging lens (L2), and a slit (S: 100 µm in width). For sufficient filtration of the light, the NF filter was placed perpendicular to the path. To establish the time course of the stimulation, as shown in Fig. 1c, the skin model samples were taken out of the incubator piece by piece to make the Raman measurements. We obtained Raman spectra at seven random locations, focusing on the surface of each skin sample avoiding apparent pigmentation spots. The focal point was chosen so that the Raman signal was maximum. The time for the measurement of a spectrum was 600 s (exposure 10 s, with 60 scans).
The Raman spectrum, which we called in this paper the ‘pure’ spectrum, was calculated as y(ν) = [yexp(ν) – a(ν)]/b(ν), where yexp(ν), a(ν), and b(ν) are the spectra obtained experimentally with the following procedures. (i) The spectrum yexp(ν) was obtained by irradiating the target material on an aluminum foil support with 785 nm light. (ii) The reference spectrum a(ν) was measured by irradiating the aluminum foil with 785 nm light with no target material (the aluminum foil was placed above the focal point of the light). (iii) Another reference spectrum, b(ν), was obtained without laser irradiation by placing a white light source at the focal point. A tungsten lump was used as the white light source, and a pin hole was used so that the white light passing through
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it became a spot (0.9 mm in diameter). The attachment of a variable resistor connected to a battery allowed the light intensity of the light source to be adjusted.
The baselines of the pure Raman spectra were then corrected. The baseline-corrected Raman spectrum y2(ν) was calculated as y2(ν) = y(ν) – ypoly(ν), in which ypoly(ν) is a fitted polynomial curve constructed with the following procedures. (i) For a spectrum truncated between the minimum Raman shift position νmin and the maximum position νmax, the degree of the function d was selected to fit the baseline using a polynomial function (this time d = 8). (ii) Using the least squares method, the polynomial function ypoly was first fitted to the Raman spectrum y. (iii) The Raman spectrum y was divided into upper and lower parts, relative to the fitted baseline ypoly. (iv) The number of data points on the upper side of y was designated NA, and the number on the lower side of y was designated NB. If NA < NB, the upper part of y was removed from the whole of y, and the Raman spectrum y was replaced with the lower part of the spectrum. Then, procedure (ii) was repeated. When NA e NB, the baseline was considered the best fit and optimal.
3. Results and discussion
As explained in Introduction, human skin is sensitive to sunlight, especially to UV light, which generates ROS in dermal cells. In this research, one of ROS, hydrogen peroxide, was used as a substitute of UV light. Here, we confirmed that the pigmentation of the black type skin model was reproduced by the stimulation of H2O2. Fig. 2a shows the skin models containing Black type melanocytes without and with the stimulation of the H2O2 (10 µL of 10 mM H2O2 was added to the 5 mL of medium containing a single piece of the Black type skin model. The black type was here chosen to exemplify
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pigmentation of the skin models since the existence of melanin was the most readily recognizable). The image of the skin model at a week after the stimulation gave several clear spots (almost black in color) of melanin above the supporting layer (collagen) while that without the stimulation (the control) provided less spots of melanin. Also, we checked that the absorbance value at 405 nm of the skin model after the H2O2 stimulation was clearly increased compared to that of the control (Fig. 2b).
Figure 3a shows all the pure Raman spectra produced in the present study. The offset values of the spectra differed greatly. When the background value of a spectrum was low, the corresponding baseline was close to a straight line. If a baseline shifted upward, it was varied to a more concave-down structure, and accompanying this change, the Raman peaks became more planar. In Fig. 3b, the spectra of each model were averaged over the time course. The offset values, wavenumber averaged, were
~0.24, ~0.16, and ~0.11 for the Black, Asian, and Caucasian models, respectively.
These values are probably influenced by the eumelanin bio-polymers of human skin, which gives two broad peaks of Raman bands at 1580 and 1380 cm-1 whose full widths of half heights are around 100 cm-1 and whose positions are not influenced by the wavelengths of excitation lights (457.9, 514.5, 632.8, and 785nm).20
Figure 4a shows the baseline-corrected spectra. The major bands at 1007 cm–1, 1455 cm–1, and 1662 cm–1 are assigned to the ring breath mode of phenylalanine, the CH2 bending mode, and the amide I mode of proteins, respectively.13, 21 Fig. 4b shows the time-dependent intensity profiles for these specific bands. For the Caucasian model, almost all the Raman bands underwent large intensity changes within 4–5 days of stimulation and the bands became stable thereafter. For the Black model, the Raman bands remained almost unchanged throughout the observation period. For the Asian
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model, the spectral changes were intermediate compared with those of the Caucasian and Black models. The Raman responses of the Caucasian and Asian models occurred within 4–5 days of H2O2 stimulation (Fig. 4b) are both interesting and important because the phenomenon is probably relevant to the early-stage sensing and diagnosis of pigmentation disorders of the skin (macroscopically, pigmentation was recognized only at 10–14 days after stimulation).
For clarifying whether the spectral changes observed in the above (Fig. 4b) are specific for effects of reactive oxygen species, the time-dependent intensity changes of the remarkable Raman signals for the Caucasian skin type after the H2O2 stimulation (Fig. 4b) were compared to those without the H2O2 stimulation (the control). We confirmed that the drastic changes in the Raman signals within 4-5 days for the stimulated Caucasian type were not observed in the control.
Let us here discuss the information obtained using the Raman confocal microscope to the live tissue analysis. The intensities of Raman bands were measured in the fixed focal volume given by the Raman confocal microscope, and the information obtained there is affected by many factors (superposition of many chemical reactions).
Even in the situation it is possible to estimate changes in the concentrations for Raman active chemical species since the intensity of a Raman band is changed in a proportional manner to concentrations. In Fig. 4b, concerning to the Caucasian skin models, the remarkable Raman bands at 1455, 1662, and 1007 cm-1 gave intensity changes with synchronicity as function of time, whose bands correspond to the vibrations of the CH2, amide, and phenylalanine portions, respectively. The observation of the intensity changes with synchronicity suggests that the concentrations of the Raman active molecules in the fixed focal volume were changed in the same manner. It is probable
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that the observed dermal cells were changed to different states after the H2O2 stimulation to give the synchronous changes in the Raman bands.
In Fig. 4b, the 3D human skin models containing melanocytes exhibited different responses to H2O2. It is of interest that Caucasian keratinocytes commonly used in all the three skin-type models, the majority of the dermal cells, were influenced by the different types of the melanocytes present (the skin models are fabricated by spreading dermal cells on the collagen sheet with the approximate ratio of one melanocyte to twenty keratinocytes; we can model three types of the skin models by choosing Caucasian, Asian, and Black type melanocytes; within melanosomes of a melanocyte, melanin is synthesized and transformed into surrounding keratinocytes through dendrites of the melanocyte; the melanocytes were remained in the basal layer while keratinocytes were proliferated upward in order to form the three dimensionally developed skin models with the basal layer, prickle cell layer, granular cell layer, and keratin layer.) Melanin is a pigment with a complex chemical structure. Two types of melanin coexist in almost all human beings, and their ratio determines an individual’s hair and skin color.22, 23 One is eumelanin, which is brown to black in color, and the other is pheomelanin, yellow to red in color. These are probably the main compounds that cause differences in dermal cells in terms of their sensitivity to H2O2. Melanin is related to the photoprotection of the skin through its ability to absorb and scatter light.22,
23 Another function of melanin is in the reduction or generation of ROS.22, 23 While ROS reduction is induced by eumelanin, ROS production is done by pheomelanin.22, 23
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4. Conclusion
With H2O2 stimulation, the time-dependent Raman spectra of three kinds of skin models exhibited different responses. Changes in the Raman signals were observed at the early stage, compared with the macroscopic pigmentation after stimulation. The properties of the skin models were characterized using the Raman technique. Raman bands for the Caucasian skin model showed large intensity changes within 4–5 days of stimulation, compared to those for the Asian and Black skin model. Eventually, eumelanin and pheomelanin are considered to be the main compounds that differentiate skin properties in terms of their sensitivity to H2O2. The findings are important since the early stage Raman detections of the skin identities may allow us to classify disorders or diseases such as stains, freckles, and cancers of the skin in a newly started manner. Our research group is at present interested in measuring the Raman spectra of single melanocytes or keratinocytes, distinguishing the locations in the target cells to study more specific chemical reactions.
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Christensen, D. H.; Hercogova, J.; Rossen, K.; Thomsen, H. K.; Gniadecki, R.;
Hansen, L. K.; Wulf, H. C.; J. Invest. Dermatol. 2004, 122, 443-449.
3. Caspers, P. J.; Lucassen, G. W.; Puppels, G. J.; Biophys. J. 2003, 85, 572-580.
4. Ferraro, J. R.; Nakamoto, K.; Brown, C. W.; Introductory Raman spectroscopy, Amsterdam, Elsevier, 2003.
5. Yu, N. T.; Jo, B. H.; Chang, R. C.; Huber, J. D.; Arch. Biochem. Biophys. 1974, 160, 614-622.
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17. Katagiri, T.; Yamamoto, Y. S.; Ozaki, Y.; Matsuura, Y.; Sato, H.; Appl. Spectrosc.
2009, 63, 103-107.
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23. Takeuchi, S.; Zhang, W.; Wakamatsu, K.; Ito, S.; Hearing, V. J.; Kraemer, K. H.;
Brash, D. E.; Proc. Natl. Acad. Sci. U S A. 2004, 101, 15076-15081.
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Figure 1. (a) An example of 3D human skin models containing melanocytes (the hematoxylin and eosin (HE) stained tissue image). SC, stratum corneum; BL, basal layer; SL, supporting layer; M, melanin. At the right side, a magnified schematic representation around the BL. KC, keratinocyte; MC, melanocyte. (b) The Raman spectroscopic system. (c) A schematic representation of the Raman measurements relative to the time course after stimulation. The rectangle represents the six-well-plate used to hold the cells; the open circle represents a piece of the skin model; the asterisk indicates the operation of picking a piece of the model skin out of the incubator. The
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label ‘(0)’ on the vertical axis indicates when the H2O2 stimulations were applied to the three types of skin models.
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Figure 2. The skin models containing Black type melanocytes (a) without and with the stimulation of the hydrogen peroxide (the hematoxylin and eosin (HE)-stained tissue images). A single piece of the Black type skin model was stimulated by 10 µl of H2O2
(10 mM). (b) the corresponding absorbance values at 405 nm. The error bars correspond to the standard deviation values. The thicknesses of the skin models are approximately 250 to 300 µm.
0.00 0.05 0.10 0.15
Absorbance at 405 nm
Control 10 mM
H2O2 10 mM H2O2 Control
BL SL
-M M
(b) (a)
~100µm
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Figure 3. (a) The complete Raman data used in the present research (126 spectra). (b) Time-averaged spectra for the Black, Asian, and Caucasian skin types.
(a) (b)
Int ens it y Int ens it y
Raman shift, ν (cm
-1) Raman shift, ν (cm
-1) Black
Asian
Caucasian
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Figure 4. (a) Raman spectra after baseline corrections for the Black, Asian, and Caucasian models, respectively. (b) Time-dependent intensity profiles for the Black, Asian, and Caucasian skin types. For the data points, solid circles, asterisks, and open circles are used to represent the intensity values at 1007, 1662, and 1455 cm–1, respectively.
(a) (b)
1455cm-1 1662cm-1 1007cm-1
Raman shift, ν(cm-1) Intensity(arb.unit) Black
Asian
Caucasian
0 2 4 6 8 10121416 0
0
0
Intensity(arb.unit)
Day after stimulation Black
Asian
Caucasian
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This chapter was referenced from S. Morita, S. Takanezawa, A. Date, S. Watanabe, and Y. Sako, 'Raman Spectroscopic Analysis of H2O2-Stimulated Three-Dimensional Human Skin Models Containing Asian, Black, and Caucasian Melanocytes', J.
Spectrosc. (2013) ID: 903450.
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Acknowledgements
During the course of the study, I have received much favor, help and encouragement from many peoples. I would like to express great gratitude to Chief scientist Yasushi Sako from RIKEN, for his really sincere discussion, helpful advices, continuous instructions, and encouragements in this study. Especially, his fruitful discussion about biology and physics is foundation for constructing the study. I would like to great appreciate to Professor Yukihiro Ozaki, my supervisor, for precious discussion, exact advices, cordial encouragement, and consistent instructions. Obviously, his great discussion about vibrational spectroscopy, chemistry, and chemometrics is fundamental for constructing this study. They always showed me the really enthusiastic attitude for research. Without these discussions, advices support, and encouragements, it is impossible to construct present study.
I wish express my heartfelt gratitude to Associate Professor Shin-ichi Morita from Tohoku University, for his valuable discussion, and really enthusiastic encouragement and support. He has guided me a lot about my research and life from undergraduate students. He showed me the way of life and research, and expected me to think more deeply for research. I must thank to Professor Hidetoshi Sato, for his precious discussion and for giving me a chance to learn the biological Raman studies.
He showed me frontier research of Raman spectroscopy in biology and medicine.
I also thank to important two researchers, Dr. Michio Hiroshima and Dr.
Toshiaki Suzuki. They give me important suggestions, discussions and insights, including techniques about instruments, methods, and analyses in biology and optics. I would like to appreciate to Dr. Norimichi Kawashima and Dr. Takurou N. Murakami.
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They showed me the delight, passion and patient in science study when I was undergraduate student in Toin University of Yokohama. I wish appreciate to Dr. Daitaro Ishikawa and Dr. Nicolas Spegazzini for their great kindness and encouragements while they were post doctor in Ozaki laboratory. I also express many thanks to all members in Cellular Informatics Laboratory in RIKEN, and members in Ozaki Laboratory. They really helped and encouraged me in research and life. All of them are really nice peoples.
I would like to thank Junior Associate Research fellowship in RIKEN supporting on my study and living. Finally, I cordially and deeply thank to my family for their supports, encouragements, considerations and great confidences.
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Publication list
Original paper
(1) S. Takanezawa, S. Morita, Y. Ozaki, and Y. Sako, ‘Raman Spectral Dynamics of Single Cells in the Early Stages of Growth Factor Stimulation’, Biophys. J. 2015.
Accepted.
(2) S. Morita*, S. Takanezawa*, M. Hiroshima, T. Mitsui, Y. Ozaki, and Y. Sako,
‘Raman and Autofluorescence Spectrum Dynamics along the HRG-induced Differentiation Pathway of MCF-7 Cells’, Biophys. J. 107 (2014) 2221-2229.
*These authors contributed equally.
(3) S. Takanezawa, A. Baba, Y. Sako, Y. Ozaki, A. Date, K. Toyama, and S. Morita,
‘Image enhancement of optical images for binary system of melanocytes and keratinocytes’, Proc. SPIE (2013) ID: 887901.
(4) S. Takanezawa, Y. Ozaki, Y. Sako, and S. Morita,’ Mechanism of sequential order determination in bio-Raman correlation analysis’, Proc. SPIE (2013) ID: 887902.
(5) S. Morita, S. Takanezawa, A. Date, S. Watanabe, and Y. Sako, 'Raman Spectroscopic Analysis of H2O2-Stimulated Three-Dimensional Human Skin Models Containing Asian, Black, and Caucasian Melanocytes', J. Spectrosc. (2013) ID: 903450.
(6) S. Takanezawa, S. Morita, A. Maruyama, T. N. Murakami, N. Kawashima, H. Endo, K. Iijima, T. Asakura, T. Shimosegawa, and H. Sato, ‘Two-Dimensional Raman Correlation Analysis of Diseased Esophagus in a Rat’, APEX, 3 (2010)
077001-077003.
Invited Oral Presentation
高根沢 聡太、「成長因子刺激初期における単一細胞のラマンスペクトルダイナ
143
ミクス」、研究会「理論と実験」2014、広島大学、2014年10月
Oral Presentations (International)
(1) ËS. Takanezawa, Y. Sako, Y. Ozaki, and S. Morita, Sequential Order Determination of Chemical Species in Perturbed Living Cellular System. The 7th International Symposium on Two-dimensional Correlation Spectroscopy. Seoul, Korea, Aug.
2013.
(2) ËS. Takanezawa, S. Morita, Y. Ozaki, and Y. Sako, Determination of Sequential Order using Bio-Raman Correlation Spectroscopy. SPIE 2013 Nano-bio sensing, Imaging & Spectroscopy, ICC Jeju Jeju-do, Korea, Feb. 2013.
(3) ËS. Takanezawa, S. Morita, M. Hiroshima, T. N. Murakami, N. Kawashima, T.
Mitsui, and Y. Sako ,Raman Spectroscopy and Correlation Analysis on Live Cell Dynamics, Sixth International Meeting on 2-Dimensional Correlation Spectroscopy, Doubletree Hotel Sonoma Wine Country, America, Jun. 2011.
(4) ËS. Takanezawa, S. Morita, A. Maruyama, T. N. Murakami, N. Kawashima, T.
Asakura, T. Shimosegawa and H. Sato, Two-Dimensional Raman Correlation Analysis of Diseased Esophagus in a Rat, TOIN International Symposium on Biomedical Engineering, Toin University of Yokohama, Japan, Nov. 2010.
(5) ËS. Morita, S. Takanezawa, M. Hiroshima, T. Mitui, and Y. Sako. Non-Invasive and Non-Laveling Raman Analysis of Single Cell Dynamics ,TOIN International Symposium on Biomedical Engineering, Toin University of Yokohama, Japan, Nov.
2010.
144
Poster Presentations (International)
(1) S. Takanezawa, S. Morita, Y. Sako, and Y. Ozaki, Raman Analysis on Early Stage of Cell Differentiation, 7th International Conference on Advanced Vibrational Spectroscopy, Kobe, Japan, Aug. 2013.
(2) S. Takanezawa, A. Date, K. Toyama, Y. Sako, Y. Ozaki, and S. Morita, Raman Study on Human Skin Models, 7th International Conference on Advanced Vibrational Spectroscopy, Kobe, Japan, Aug. 2013.
(3) S. Takanezawa, Y. Sako, Y. Ozaki, and S. Morita, Bio-Raman Correlation Analysis and the Sequential Order Determination, The 7th International Symposium on Two-dimensional Correlation Spectroscopy. Seoul, Korea, Aug. 2013.
(4) S. Takanezawa, S. Morita, A. Date, S. Watanabe, T. N. Murakami, N. Kawashima, H.
SATO, and Y. Sako, Raman Study on H2O2-Stimulated Three-Dimensional Human Skin Models, Sixth International Conference on Advanced Vibrational Spectroscopy, Doubletree Hotel Sonoma Wine Country, America, Jun. 2011.
(5) S. Takanezawa, T. Yanagida, S. Morita, T. N. Murakami, N. Kawashima, H. Sato, and Y. Sako, Study on Penetration Depth of External Reflection Infrared Spectroscopy at the Air/Water Interface, Sixth international conference on molecular electronics and bioelectronics, Sendai International Center, Mar. 2011.
(6) S. Takanezawa, S. Morita, H. Shinzawa, T. N. Murakami, N. Kawashima, T.
Asakura, T. Shimosegawa, and H. Sato, Two-Dimensional Correlation Raman Analysis of Diseased Esophagus of a Rat, The 5th International Symposium on Two-Dimensional Correlation Spectroscopy, Wroclaw, Poland, Aug. 2009.
(7) T. Sugawara, S. Takanezawa, N. Kawashima, and T. N. Murakami, Kinetic Study on Degradation of Nafion by Fenton Reaction, 216th ECS Meeting (2009), Vienna,
145
Austria, Oct. 2009.
(8) S. Takanezawa, S. Morita, A. Maruyama, T. N. Murakami, N. Kawashima, T.
Asakura, T. Shimosegawa, and H. Sato, Raman Analysis of Diseased Esophagus of a Rat,TOIN International Symposium on Biomedical Engineering, Toin University of Yokohama, Japan, October 2009.
(9) T. Sugawara, S. Takanezawa, N. Kawashima, and T. N. Murakami, Kinetic Study on Degradation of Nafion by Fenton Reaction (2), TOIN International Symposium on Biomedical Engineering, Toin University of Yokohama, Japan, October 2009.
(10) S. Takanezawa, S. Morita, H. Sato, T. N. Murakamami, and N. Kawashima, Raman Spectroscopy of a Single Living Cell in Photodynamic Therapy, TOIN International Symposium on Biomedical Engineering, Toin University of Yokohama, Japan, October 2008.
(11) T. Sugawara, S. Takanezawa, N. Sakai, T. N. Murakami, and N. Kawashima, Kinetic Study of the Degradative Oxidation of Nafion by Reactive Oxygen Species, TOIN International Symposium on Biomedical Engineering, Toin University of Yokohama, Japan, October 2008.
Oral presentations (Domestic)
(1) 高根沢聡太、盛田伸一、尾崎幸洋、佐甲靖志、「細胞運命決定過程における 細胞内状態変動のラマン分光分析」、第 3 回日本生物物理学会関東支部研究 会、明治大学中野キャンパス、2014年3月
(2) S. Takanezawa, S. Morita, Y. Ozaki, and Y. Sako, 「Measurements of Single Cell Dynamics upon Stimulation with a Differentiation Factor Using Raman Micro-Spectroscopy」、第50回生物物理学会年会、名古屋大学、2012年9月
146
(3) 高根沢聡太、盛田伸一、伊達朗、渡辺慎、村上拓郎、川島德道、佐藤英俊、
佐甲靖志、「ラマン分光法によるヒト皮膚三次元モデルの色素沈着過程の研 究」、第58回応用物理学関係連合講演会、神奈川工科大学、2011年3月 (4) 盛田伸一、高根沢聡太、廣島道夫、三井敏之、佐甲靖志、「ラマン分光によ
る生体システムおよび生細胞の非侵襲的計測および分析」、第48回日本生物 物理学会年会、東北大学(川内キャンパス)、2010年9月
(5) 高根沢聡太、盛田伸一、丸山篤史、村上拓郎、川島德道、遠藤博之、飯島克 則、朝倉徹、下瀬川徹、佐藤英俊、「生体組織病変の二次元相関解析」、第57 回応用物理学会学術講演会、東海大(湘南キャンパス)、2010年3月
(6) 盛田伸一，高根沢聡太，伊達朗，稲益直子，渡辺慎，村上拓郎，川島徳道，
佐藤英俊、「生体組織に由来するラマン信号の解析」、第 57 回応用物理学関 係連合講演会、東海大(湘南キャンパス)、2010年3月
(7) 菅原智子、高根沢聡太、川島德道、村上拓郎、｢PEFC 内 Nafion®フィルムの 状態予測を目的とした酸化反応(3)｣、日本化学会第90回関東支部大会、近畿 大学(本部キャンパス) 、2010年3月
(8) 高根沢聡太、盛田伸一、丸山篤史、村上拓郎、川島德道、遠藤博之、飯島克 則、朝倉徹、下瀬川徹、佐藤英俊、「ラット食道病変のラマンプロファイル 解析」、第70回応用物理学会学術講演会、富山大(五福キャンパス)、2009年 9月
(9) 高根沢聡太、Ë盛田伸一、伊達朗、稲益直子、渡辺慎、村上拓郎、川島德道、
佐藤英俊、「ヒト皮膚の振動分光による分析」、第 70 回応用物理学会学術講 演会、富山大(五福キャンパス)、2009年9月
(10) 菅原智子、高根沢聡太、川島德道、村上拓郎、｢PEFC 内 Nafion®フィル
ムの状態予測を目的とした酸化反応｣、電気化学会第76回大会、京都大学(吉
147
田キャンパス)、2009年3月
Poster presentations (Domestic)
(1) S. Takanezawa, S. Morita, Y. Ozaki, and Y. Sako, 「Dynamics of Cellular Chemical State upon Stimulation with Growth Factors Analyzed by Raman Micro-spectroscopy」、第５２回生物物理学会年会、札幌コンベンションセン ター、2014年9月
(2) S. Takanezawa, S. Morita, Y. Sako, Y. Ozaki, 「Detection of Cellular Responses to a Differentiation Factor Using Raman Spectroscopy」、第５１回日本生物物理学会 年会、国立京都国際会館、2013年10月
(3) 高根沢聡太、盛田伸一、伊達朗、渡辺慎、稲益直子、村上拓郎、川島德道、
佐藤英俊、佐甲靖志、「ヒト皮膚三次元モデルにおける色素沈着の顕微ラマ ン分光分析」、第 48 回日本生物物理学会年会、東北大学(川内キャンパス)、
2010年9月
(4) 盛田伸一、高根沢聡太、廣島道夫、三井敏之、佐甲靖志、「ラマン分光によ る生体システムおよび生細胞の非侵襲的計測および分析」、第48回日本生物 物理学会年会、東北大学(川内キャンパス)、2010年9月
(5) 高根沢聡太、盛田伸一、伊達朗、渡辺慎、稲益直子、村上拓郎、川島德道、
佐藤英俊、佐甲靖志、「ラマン分光法による三次元皮膚モデルの研究」、日本 化学会第4回関東支部大会、筑波大学(筑波大学第一キャンパスエリア)、2010 年8月
(6) 高根沢聡太、盛田伸一、丸山篤史、村上拓郎、川島德道、遠藤博之、飯島克 則、朝倉徹、下瀬川徹、佐藤英俊、「ラット食道病変のラマンプロファイル 分析」、第9回国際バイオEXPO、東京ビックサイト、2010年7月