Development of staining-free molecular imaging microscope for biological molecules and its application for quality control of cultured cells

生物分子免染色分子成像显微镜的研制及其在培养细胞质量控制中的应用

• 批准号: 17200032
• 负责人: ARAKI Tsutomu
• 金额: $ 31.28万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17200032/
• 关键词: molecular imaging; SHG microscope; orientation of collagen molecule; cultured cell; CARS microscope; nanosecond fluorescence lifetime; collagen gel; skin; 光老化; カルシウムイオン

Aims of this research project are to develop new microscopes by which molecular imaging of the biological specimen can be obtained without unwanted staining treatment, and to apply these microscopes for quality control of the cultured cells. The results are written as follows.(1) We have developed a high-speed SHG microscope and measured in situ tomographic images of collagen molecules in human dermis through the skin. The measurement time to obtain one image was shortened to one second that value is about 1/60 of the conventional equipment.(2) We have developed another SHG microscope with a controlled polarization pattern to detect 3D / molecular orientation. Using this microscope, we have demonstrated the 3D collagen profiles in Achilles tendon.(3) Deformation and remodeling of collagen gel due to endotherial cells have been observed by SHG microscopy in order to apply that microscopy for quality control of the cell culture.(4) We have developed a high-speed CARS microscopy system. Using this system, a real-time imaging of liposomes has been demonstrated.(5) We have measured calcium contents in various vascular walls and analyzed the heterogeneity of the contents based on biomechanical effect.(6) A new quantification technique for calcium ion concentration has been proposed based on fluorescence lifetime measurement. The waveform distortion caused by the frequency band width limitation of the instruments has been compensated by a newly proposed algorithm.

本研究项目的目的是开发新的显微镜，通过它可以在不进行不必要的染色处理的情况下获得生物标本的分子成像，并将这些显微镜应用于培养细胞的质量控制。研究结果如下：(1)研制了一台高速倍频显微镜，并通过皮肤对真皮中的胶原分子进行了原位断层图像测量。获得一幅图像的测量时间缩短到1秒，约为传统设备的1/60。(2)我们研制了另一台具有可控偏振图案的倍频显微镜，用于检测3D/分子取向。利用这台显微镜，我们展示了跟腱的三维胶原轮廓。(3)利用倍频显微镜观察了内皮细胞引起的胶原凝胶的变形和重塑，以便将其应用于细胞培养的质量控制。(4)我们研制了一套高速CARS显微镜系统。利用该系统实现了脂质体的实时成像。(5)测量了不同血管壁的钙含量，并基于生物力学效应分析了其含量的不均一性。(6)提出了一种新的基于荧光寿命测量的钙离子浓度定量技术。提出了一种新的算法来补偿由于仪器频带限制而引起的波形失真。

项目成果

Second-haromic-generation microscopy using excitation beam with controlled polarization pattern to determine three-dimensional molecular orientation

使用具有受控偏振模式的激发光束来确定三维分子取向的第二哈罗代显微镜

• 发表时间: 2005
• 期刊: Jap.J.Appl.Phys. 巻44(34)
• 作者: K.;Yoshiki;K.Yoshiki
• 通讯作者: K.Yoshiki

Relationship between arterial calcification and hemodynamics

动脉钙化与血流动力学的关系

• 发表时间: 2005
• 期刊: Proc.2nd Jap.-Switz.Workshop on Biomech.
• 作者: K.;Yoshiki;K.Yoshiki;R.Nakayama;S.Fukushima
• 通讯作者: S.Fukushima

Age-related changes of elements in human corpus callosum and relationships among these elements

人体胼胝体各成分随年龄的变化及各成分之间的关​​系

• 发表时间: 2008
• 期刊: Biol. Trace Element Res. 121
• 作者: 垂水信二;倉爪亮;橋爪;S. Tohno
• 通讯作者: S. Tohno

SHG(第2高調波発生光)顕微鏡を用いた真皮コラーゲン線維分布の観察

使用SHG（二次谐波发生光）显微镜观察真皮胶原纤维分布

• 发表时间: 2007
• 期刊: 光学 36
• 作者: 保中志元;橋爪 誠;光石 衛;et al.;伊藤誠啓
• 通讯作者: 伊藤誠啓

Moderate Accumulation of Calcium and Phosphorus in the Splenic Artery with Aging and Low Accumulation of Those in the Pulmonary Artery with Aging

• DOI: 10.1007/s12011-007-0052-6
• 发表时间: 2007-05
• 期刊: Biological Trace Element Research
• 影响因子: 3.9
• 作者: Iria Maria Prieto Payo;Nutcharin Ongkana;S. Tohno;C. Azuma;T. Minami;Y. Moriwake;Y. Tohno