Koster A J, Chen H, Sedat J W, Agard D A
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Ultramicroscopy. 1992 Oct;46(1-4):207-27. doi: 10.1016/0304-3991(92)90016-d.
Instrumentation and methodology for the automatic collection of tomographic tilt series data for the three-dimensional reconstruction of single particles is described. The system consists of a Philips EM 430 TEM, with a Gatan 673 cooled slow-scan CCD camera and a Philips C400 microscope computer control unit attached. The procedure for data collection includes direct digital recording of the images on the CCD camera and the automatic measurement and correction of (a) image shifts resulting from tilting the specimen, (b) variation of defocus and (c) the eucentric height position of the specimen. Experiments are described illustrating the possibilities and limitations of automatic data collection. Data collection at a magnification of 30k shows that the exposure time of the specimen to the beam is reduced by a factor of 10-100 compared to manual operation of the TEM.
本文描述了用于单颗粒三维重建的断层倾斜系列数据自动采集的仪器设备和方法。该系统由一台飞利浦EM 430透射电子显微镜、一台加坦673冷却慢扫描电荷耦合器件相机以及一台连接的飞利浦C400显微镜计算机控制单元组成。数据采集过程包括在电荷耦合器件相机上直接进行图像的数字记录，以及对（a）因样品倾斜导致的图像偏移、（b）散焦变化和（c）样品的同心高度位置进行自动测量和校正。文中描述了实验，阐述了自动数据采集的可能性和局限性。在30k放大倍数下的数据采集表明，与透射电子显微镜的手动操作相比，样品对电子束的曝光时间减少了10到100倍。
