Development of simultaneous measurement of micro/macro-molecular kinetics in a living cell by laser speckle microscopy

研制了一种用于活体细胞观察的激光散斑显微成像系统。该系统由光学显微镜、激光光源和摄像机组成。该系统的优点在于，细胞条件可以通过观察散斑运动来即时确定，而无需任何特殊的准备，如染色和遗传操作。在实验中，将活的和固定的HeLa细胞的斑点波动与细胞周围培养基的温度变化进行了比较，并对其视频图像进行了定量分析。利用散斑起伏的频谱分析了这些细胞上的激光散斑起伏。在该频率分析中，在活细胞中观察到几Hz附近的特定峰。在这项研究中还发现，通过使用飞秒激光照明，可以在这种激光散斑显微系统中同时观察生物分子的自发荧光和活细胞中的激光散斑。通过应用该系统，不仅可以估计细胞活性，而且可以估计包括细胞质在内的细胞间物质的实时运动，而不需要复杂的标记过程。

A laser speckle microscopy imaging system for observation of living cells was developed. This system consists of an optical microscope, a laser source, and a video camera. The system has an advantage in that cellular conditions could be instantaneously determined by observing speckle movements without any special preparation such as staining and genetic manipulation. In the experiments, the speckle fluctuations of living and fixed HeLa cells were compared with the change in the temperature of the culture medium around the cells, and their video images were quantitatively analyzed. Laser speckle fluctuations on these cells were also analyzed by using frequency spectra of speckle fluctuations. In this frequency analysis, particular peak around a few Hz was observed in a living cell. It was also found in this study that by using a femto second laser illumination, simultaneous observation of auto-fluorescence from bio-molecules and laser speckle in a living cell could be possible in this laser speckle microscopic system. By applying this system, it is possible to estimate not only cellular activity but also real-time movements of intercellular materials including the cytoplasm without requiring complicated labeling processes.