Performance of a 2k CCD camera designed for electron crystallography at 400 kV

Abstract

We discuss the performance of a charge-coupled device (CCD) camera that has been designed for use in electron crystallographic studies of proteins. There have been many previous publications describing the characteristics and performance of CCD-based cameras in electron microscopy; here we focus on characteristics relevant to protein studies at 400 kV. The low exposure that must be used in such studies produces a very poor signal-to-noise ratio, so any loss of signal-to-noise ratio in the recording process must be avoided. Images must contain a sufficient number of molecules to allow identification of the reciprocal lattice, thus requiring a large image format. Electron diffraction patterns may contain some spots with intensity around 10 −7 times that of the central beam, so the largest possible dynamic range is helpful. Some of the characteristics we discuss are most easily measured with crystals, but the conclusions also apply for other work such as single-particle analyses. The camera has been optimized for work at 400 kV with a P43 scintillator fiber-optically coupled to a CCD with 24 μm pixels. The scintillator in this camera is thicker than generally used at lower voltages, which provides an adequate signal level but slightly degrades the resolution. Operation at 400 kV leads to a point spread function that is broader than the CCD pixel size. Images are thus binned by a factor of two to double the effective pixel size, with the resulting loss of a factor of two in the size of areas that can be recorded in a single frame. A large CCD with a 2048×2048 pixel array is used to compensate for this loss and provide a sufficient signal for the crystallographic image processing used in this work. Images and electron diffraction patterns recorded on the CCD are compared with data recorded on photographic film. While the quality of the images recorded on the CCD at the low exposures required in protein studies is not quite as good as that on film, electron diffraction data recorded on the CCD are superior to that on film.