Non-linear micrascopy of biophysical processes in living cells

光学显微镜是能够非侵入性地研究活细胞和生物组织的最强大的工具之一。所提出的新的非线性光学显微镜的特点是成形单元，能够操纵激光束的光谱和空间分布，以及调整飞秒脉冲的不同光谱分量的时间定相和偏振。定制形状的飞秒脉冲将用于生物样品中荧光团的相干控制激发。优化的激光脉冲将提高荧光团激发的效率并减少光漂白。飞秒脉冲啁啾调制将能够对细胞内的pH分布进行成像。相干控制显微镜将建立在现有的最先进的多模态非线性激光扫描显微镜的基础上，能够同时记录三次和二次谐波产生，以及多光子激发荧光图像。这一独特的仪器将用于研究肌细胞收缩过程中线粒体的生物能量学。该显微镜还将用于阐明单个分离叶绿体和原位叶绿体中光捕获的调节机制。新的显微镜将作为一个平台，为非侵入性生物研究创建新的非线性成像模式。

Optical microscopy is among the most powerful tools that enable to investigate non-invasively living cells and biological tissue. The proposed new non-linear optical microscope features the shaping unit that is able to manipulate spectral and spatial profiles of the laser beam, as well as adjust the temporal phasing and polarization of different spectral components of the femtosecond pulse. The custom shaped femtosecond pulses will be used for coherent control excitation of fluorophores in the biological samples. The optimized laser pulses will enhance the efficiency of fluorophore excitation and reduce photobleaching. The femtosecond pulse chirp modulation will enable to image the pH distribution inside the cells. The coherent control microscope will be built on the basis of already existing state-of-the-art multimodal non-linear laser scanning microscope capable of simultaneously recording third and second harmonic generation, and multiphoton excitation fluorescence images. The unique instrument will be used to investigate bioenergetics of mitochondria during contraction of myocytes. The microscope will also be applied to elucidate regulation mechanisms of light harvesting in single isolated and in situ chloroplasts. The new microscope will serve as a platform for creation of new non-linear imaging modalities for non-invasive biological investigations.