Stroboscopic image capture: reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin

Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser [Ultramicroscopy 16 (1985) 139-150], which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range Charge-coupled device (CCD) camera for image recording and a liquid N2-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F(image)(g)/F(image)(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.