Sanderson M J
Department of Physiology, University of Massachusetts Medical School, Worcester 01655, USA.
Methods. 2000 Aug;21(4):325-34. doi: 10.1006/meth.2000.1022.
High-speed imaging is an ideal technique to accurately resolve the temporal and spatial characteristics of rapid events at either the molecular or cellular level. In this article, the digital imaging techniques used to simultaneously acquire transillumination phase-contrast images, at 240 images s(-1) (high-speed), to characterize ciliary beat frequency, and fluorescence images, at 30 images s(-1) (fast), to measure intracellular calcium concentration ([Ca2+]i), are described. With this technique, a precise correlation between the changes in ciliary beat frequency with changes in [Ca2+]i can be made. Simultaneous imaging is achieved by using different wavelengths of light to form the phase-contrast and fluorescent images and selectively directing these light wavelengths to different cameras with dichroic mirrors and bandpass filters. High-speed images compatible with standard video recording equipment are obtained by prematurely resetting the raster scan of a CCD camera with additional vertical synchronization pulses. The fast [Ca2+]i images are determined using the ratiometric dye fura-2 and a recording technique that monitors rapid changes in fluorescence at a single wavelength and uses intermittent reference images for calibration.
高速成像是一种理想的技术，能够在分子或细胞水平上精确解析快速事件的时间和空间特征。本文描述了用于同时获取透射相衬图像（以240幅/秒的速度，即高速，用于表征纤毛搏动频率）和荧光图像（以30幅/秒的速度，即快速，用于测量细胞内钙浓度[Ca2+]i）的数字成像技术。通过该技术，可以建立纤毛搏动频率变化与[Ca2+]i变化之间的精确关联。利用不同波长的光形成相衬图像和荧光图像，并通过二向色镜和带通滤波器将这些光波长选择性地导向不同的相机，从而实现同时成像。通过使用额外的垂直同步脉冲提前重置CCD相机的光栅扫描，可获得与标准视频记录设备兼容的高速图像。快速的[Ca2+]i图像使用比率染料fura-2和一种记录技术来确定，该技术在单一波长下监测荧光的快速变化，并使用间歇参考图像进行校准。
