Monte Carlo calculations of (a) and (b) electron trajectories in a thick phosphor scintillator mounted on fiber optics and covered with a metallization and protective layer. These layers are indicated by the dashed lines. Because of the lower density and atomic number of the fiber optics, the range of the electrons is much larger than in the scintillator. (c) Predicted width of the point spread function estimated from simulations at various accelerating voltages.
(a) Block diagram of the CCD decelerator system. (b) Cross section through the system, showing arrangement of the decelerator stack, CCD and electronics in the HV tank, and the electron beam path.
(a) Images of a grating replica taken with the microscope set at and the decelerator at (left to right): 0, 100, and . There is an increase in magnification of about 20% with the potential, as predicted, and some distortions are detectable by sighting along the grating lines. Contrast has been adjusted for uniform appearance. (b) Magnification of image on CCD as a function of the net incident electron energy. Curves are shown for four series of measurements, starting from 200, 230, 250, and , and decelerating the beam by up to . The “net energy” is corrected for the calibrations of the power supplies (see text). (c) System gain (arbitrary scale) as a function of incident voltage for the decelerator camera with a thin (triangles) or thick (circles) phosphor. The gain is corrected for changes in magnification, and the net energy is corrected for the voltage calibration errors.
(a) Line scans across an image of the edge of the electron microscope’s beam stop, with the accelerating voltage set to 300 or to and the beam decelerated as indicated. (b) MTF curves derived from the beam stop images.
Fourier transforms of carbon film images recorded with the microscope at and the decelerator set to (a) and (b) . The magnification at the CCD was , and the Nyquist frequency corresponds to . The nominal microscope magnification was changed from 25 to to account for the increase in magnification as the deceleration voltage increases, and to maintain the same exposure at the level of the specimen or detector. The images were gain normalized with reference images recorded at the corresponding voltages. The improvement in clarity of the Thon rings, which arise from the focus-dependent phase contrast transfer function, is an illustration of the improvement in DQE, or SNR. Curves were obtained from rotationally averaged transforms.
DQE measurements with the decelerator-CCD system. (a) A series of measurements of DQE vs spatial frequency with various microscope and decelerator voltage combinations. The corrected net voltage difference is given. (b) A summary of the DQE dependence on voltage, for two spatial frequencies; the upper curves (each curve representing a different microscope voltage) are for low frequencies, around 0.05 Nyquist, while the lower curves are for half-Nyquist frequency (0.25 on the graph at the left). The essential point is that the DQE improves from 0.08 at to 0.4—i.e., by a factor of 5—at the midfrequencies, and a factor of 2 at the low frequencies.
Characterization of distortions. (a) Array of illumination spots made by stepping the beam in small increments through a two-dimensional lattice. The microscope was set to , the decelerator to . (b) Representation of displacements of the spots from ideal lattice positions as determined by program QUADSERCH (Ref. 19). Line segments correspond to ten times the actual displacement. (c) Image after correction for displacements using program CCUNBEND. (d) Fourier transform of the original image; diffraction spots are spread over many pixels, even at low spatial frequencies. (e) Fourier transform after correction; diffraction spots are concentrated essentially to a single pixel and extend to the edge of the transform.
Representation of the SNR for diffraction spots in the Fourier transform of an image of bR, a two-dimensional protein crystal. The image was taken with the microscope set to and the decelerator to , with a nominal magnification of . The edge of the transform corresponds to a resolution of . The size of each circle reflects the IQ (Ref. 19) for each spot. The IQ is a measure of the signal-to-noise ratio calculated as where A is the spot amplitude and B the local background. An IQ of 1 indicates a peak-to-background ratio greater than 7, while a ratio between 1 and 1.33 gives an IQ of 8. Circle sizes are shown at the lower left. Excellent SNR is obtained essentially out to the edge of the transform.
Images of a typical plastic-embedded and stained cell section. The sample was decorated with diameter gold beads, which would be used as fiducial markers in aligning a tomographic series of images. Each image shows an area comprising out of the original image. The microscope was set at , the decelerator at [(a)–(c)] or [(d)–(f)] . Nominal microscope magnification was [(a)–(c)] or [(d)–(f)] , giving a final magnification of for both conditions. Exposures at the CCD were [(a) and (d)] [(b) and (e)] , and [(c) and (f)] and . The insets in each image show the same set of gold beads. Contrast has been adjusted for uniform appearance of the images.
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