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首页> 外文期刊>Brain cell biology >Development of microscopic systems for high-speed dual-excitation ratiometric Ca2+ imaging.
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Development of microscopic systems for high-speed dual-excitation ratiometric Ca2+ imaging.

机译：高速双激发比例式Ca2 +成像显微系统的开发。

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摘要

For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems. (1) A system in which two laser beams are alternated on every scanning line, allowing us to obtain confocal images using dual-excitation ratiometric dyes. This system increases the rate at which ratio measurements can be made to 200 Hz and provides confocal images at 1-10 Hz depending on the image size. (2) A truly simultaneous dual-excitation ratiometry system that used linearly polarized excitation light and polarization detection, allowing us to obtain ratiometric images without any time lag. This system, however, is based on statistical features of the fluorescence polarization and is limited to samples that contain a large number of fluorophores. In addition, this method requires complicated calculations. (3) An efficient, nearly simultaneous dual-excitation ratiometry system that allows us to rapidly switch between two synchronized excitation-detection components by employing two high-power light-emitting diodes (LEDs) and two high-speed liquid crystal shutters. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity.
机译：对于Ca（2+）浓度（[Ca（2+）]）的定量测​​量，比例染料是优选的，因为使用这种染料可以校正细胞内染料的不均匀负载或分配以及细胞中的变化。厚度。尽管用于测量[Ca（2+）]的双激发比例染料，如Fura-2，Fura-Red和Peratiometric-pericam，已广泛用于各种应用，但很难将它们用于监测快速Ca（2+）动态或Ca（2+）变化在高度运动的细胞中。为了克服这个问题，我们开发了三个新的双励磁比率测量系统。 （1）一种系统，其中在每条扫描线上交替出现两个激光束，这使我们能够使用双激发比例染料获得共焦图像。该系统将比率测量的速率提高到200 Hz，并根据图像尺寸提供1-10 Hz的共聚焦图像。 （2）使用线性偏振激发光和偏振检测的真正同时双激发比率测量系统，使我们能够获得比率图像而没有任何时间延迟。但是，该系统基于荧光偏振的统计特征，并且仅限于包含大量荧光团的样品。另外，该方法需要复杂的计算。 （3）一个高效的，几乎同时存在的双激励比率测量系统，该系统允许我们通过使用两个大功率发光二极管（LED）和两个高速液晶快门在两个同步的激励检测组件之间快速切换。两个百叶窗的打开/关闭操作与两个LED的开/关切换同步。该系统将比率测量的速率提高到1 kHz，并根据信号强度提供10-100 Hz的比率图像。

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