Performance of a slow-scan CCD camera for macromolecular imaging in a 400 kV electron cryomicroscope

The feasibility and limitations of a 1024 x 1024 slow-scan charge-coupled device (CCD) camera were evaluated for imaging in a 400kV electron cryomicroscope. Catalase crystals and amorphous carbon film were used as test specimens. Using catalase crystals, it was found that the finite (24 microns) pixel size of the slow-scan CCD camera governs the ultimate resolution in the acquired images. For instance, spot-scan images of ice-embedded catalase crystals showed resolutions of 8 A and 4 A at effective magnifications of 67,000 x and 132,000 x, respectively. Using an amorphous carbon film, the damping effect of the modulation transfer function (MTF) of the slow-scan CCD camera on the specimen's Fourier spectrum relative to that of the photographic film was evaluated. The MTF of the slow-scan CCD camera fell off more rapidly compared to that of the photographic film and reached the value of 0.2 at the Nyquist frequency. Despite this attenuation, the signal-to-noise ratio of the CCD data, as determined from reflections of negatively-stained catalase crystals, was found to decrease to approximately 50% of that of photographic film data. The phases computed from images of the same negatively-stained catalase crystals recorded consecutively on both the slow-scan CCD camera and photographic film were found to be comparable to each other within 12 degrees. Ways of minimizing the effect of the MTF of the slow-scan CCD camera on the acquired images are also presented.