Fast auto-acquisition tomography tilt series by using HD video camera in ultra-high voltage electron microscope

The ultra-high voltage electron microscope (UHVEM) H-3000 with the world highest acceleration voltage of 3 MV can observe remarkable three dimensional microstructures of microns-thick samples[1]. Acquiring a tilt series of electron tomography is laborious work and thus an automatic technique is highly desired. We proposed the Auto-Focus system using image Sharpness (AFS)[2,3] for UHVEM tomography tilt series acquisition. In the method, five images with different defocus values are firstly acquired and the image sharpness are calculated. The sharpness are then fitted to a quasi-Gaussian function to decide the best focus value[3]. Defocused images acquired by the slow scan CCD (SS-CCD) camera (Hitachi F486BK) are of high quality but one minute is taken for acquisition of five defocused images.In this study, we introduce a high-definition video camera (HD video camera; Hamamatsu Photonics K. K. C9721S) for fast acquisition of images[4]. It is an analog camera but the camera image is captured by a PC and the effective image resolution is 1280×1023 pixels. This resolution is lower than that of the SS-CCD camera of 4096×4096 pixels. However, the HD video camera captures one image for only 1/30 second. In exchange for the faster acquisition the S/N of images are low. To improve the S/N, 22 captured frames are integrated so that each image sharpness is enough to become lower fitting error. As countermeasure against low resolution, we selected a large defocus step, which is typically five times of the manual defocus step, to discriminate different defocused images.By using HD video camera for autofocus process, the time consumption for each autofocus procedure was reduced to about six seconds. It took one second for correction of an image position and the total correction time was seven seconds, which was shorter by one order than that using SS-CCD camera. When we used SS-CCD camera for final image capture, it took 30 seconds to record one tilt image. We can obtain a tilt series of 61 images within 30 minutes. Accuracy and repeatability were good enough to practical use (Figure 1). We successfully reduced the total acquisition time of a tomography tilt series in half than before.jmicro;63/suppl_1/i25/DFU066F1F1DFU066F1Fig. 1.Objective lens current change with a tilt angle during acquisition of tomography series (Sample: a rat hepatocyte, thickness: 2 m, magnification: 4k, acc. voltage: 2 MV). Tilt angle range is ±60 degree with 2 degree step angle. Two series were acquired in the same area. Both data were almost same and the deviation was smaller than the minimum step by manual, so auto-focus worked well. We also developed a computer-aided three dimensional (3D) visualization and analysis software for electron tomography "HawkC" which can sectionalize the 3D data semi-automatically[5,6]. If this auto-acquisition system is used with IMOD reconstruction software[7] and HawkC software, we will be able to do on-line UHVEM tomography. The system would help pathology examination in the future.This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, under a Grant-in-Aid for Scientific Research (Grant No. 23560024, 23560786), and SENTAN, Japan Science and Technology Agency, Japan.