A spot auto-focus method with high definition ACF and applications to electron tomography

The technique of spot auto-focus (spot AF) is normal in the field of optical camera and microscope. However, in the field of electron microscope it is not normal. In electron tomography the spot AF is necessary to focus on the view field of a specimen on the tilt-axis. The automated wobbler method (tilt beam induced displacement method) and the ‘diffractogram’ method are not suitable for the spot AF. We devised a new spot AF method with a high definition auto correlation function (HD-ACF) of spot image area. A profile of the peak region in the center of the ACF describes the sharpness of image contrast and the ACF can be obtained even from relatively small area. The ACF of an image is generally computed with FFTs. However, the fineness of the profile, especially in the center region, is not enough to analyze in detail. In general, the peak profile rapidly decreases. Our HD-ACF resolves finely the distribution of the peak profile because the profile is calculated with sub-pixel precision by slightly changing the phase of Fourier coefficients [1]. Therefore, the gradient of the peak profile except the noise peak is measured with high precision, which is directly related to the sharpness of the image contrast. We have developed a spot AF for a transmission electron microscope (TEM) with the HD-ACF and preliminary have applied to an acquisition procedure of a specimen tilt series (0 to ± 60deg). (The specimen was a yeast cell thin section and the equipment was Hitachi HT-7700 linked to an external PC controller.) As a result, a successful tilt series well focused on the tilt-axis was obtained, which resulted in a tomography reconstruction with a desired high resolution [2]. © 2016, Oxford University Press. All rights reserved.