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

本研究项目的目的是开发新的显微镜，通过这种显微镜可以获得生物标本的分子成像，而无需进行不必要的染色处理，并将这些显微镜用于培养细胞的质量控制。结果写如下。(1)我们已经开发了一种高速SHG显微镜和测量在原位断层图像的胶原分子在人体真皮通过皮肤。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)我们已经开发了另一种具有受控偏振模式的SHG显微镜来检测3D /分子取向。使用这种显微镜，我们已经证明了跟腱中的3D胶原蛋白分布。(3)由于内皮细胞的胶原凝胶的变形和重塑已经通过SHG显微镜观察，以便将该显微镜用于细胞培养的质量控制。(4)我们已经开发了一个高速汽车显微系统。使用该系统，脂质体的实时成像已被证明。(5)我们测量了不同血管壁的钙含量，并根据生物力学效应分析了钙含量的异质性。(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。针对仪器频带宽度限制引起的波形失真，提出了一种新的补偿算法。

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.