Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300 kV

Booth, Christopher R.; Jakana, Joanita; Chiu, Wah

doi:10.1016/j.jsb.2006.08.019

Cited by 30 publications

(38 citation statements)

References 18 publications

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“…The camera used in this study is the same as that characterized previously with carbon film data (Booth et al, 2006). The MTF and the DQE are 0.07 and 0.03 at 0.67 × Nyquist frequency (P. Mooney, unpublished data), respectively, using methods described previously (Mooney, 2007).…”

Section: Discussionmentioning

confidence: 99%

“…In the case of the CCD camera, there is no chemical fog but there is electronic noise. Given the same total specimen dose, the SNR of the CCD image, calculated as a function of spatial frequency, is not affected by the imaging magnification (Booth et al, 2006). Therefore, it is reasonable to use the same dose as constrained by the radiation damage to record the images at a higher magnification on the CCD as long as it is within the linear range of detector response.…”

Section: Discussionmentioning

confidence: 99%

“…Their advantages over film are numerous (see reviews: (Faruqi and Subramaniam, 2000;Mooney, 2007)): first, the CCD camera makes the recorded images available for instant processing and analysis to provide a rapid feedback for specimen and image quality; second, it has a larger dynamic range and higher linearity relative to film well suited for electron diffraction (Brink and Chiu, 1994); third, it can provide slightly higher signal to noise ratio (SNR) at low resolution than photographic film (Booth et al, 2006;Booth et al, 2004;Sander et al, 2005); and lastly, it can facilitate automatic and high-throughput data collection (Dierksen et al, 1995;Koster et al, 1992;Stagg et al, 2006;Zhang et al, 2003). Due to these advantages, CCD cameras have been widely used in structural studies from cells to macromolecules at a broad range of resolutions.…”

Section: Introductionmentioning

confidence: 99%

“…Recent characterization of a Gatan Ultrascan 4k × 4k CCD (model US4000, 15 μm/pixel) with amorphous carbon film images suggested that this latest generation of the Gatan CCD camera has a SNR comparable to photographic film on a 300-kV electron microscope for data out to at least 0.5 × Nyquist frequency (Booth et al, 2006). Here we report the use of trehaloseembedded two-dimensional (2D) crystalline bacteriorhodopsin (bR) and ice-embedded ε 15 bacteriophage as biological test specimens to further evaluate the performance of the same Gatan Ultrascan 4k × 4k CCD in a 300-kV (JEM3000SFF) electron cryomicroscope.…”

Section: Introductionmentioning

confidence: 99%

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Achievable resolution from images of biological specimens acquired from a 4k×4k CCD camera in a 300-kV electron cryomicroscope

Chen

Jakana

Liu

et al. 2008

Journal of Structural Biology

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Bacteriorhodopsin and ε 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k × 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000 × effective magnification averaged 15.7° out to 5.8-Å resolution relative to Henderson's published values. Using a single-particle reconstruction technique, we obtained an 8.2-Å icosahedral structure of ε 15 bacteriophage with the CCD images collected at an effective magnification of 56,000 ×. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Achievable resolution from images of biological specimens acquired from a 4k×4k CCD camera in a 300-kV electron cryomicroscope

Chen

Jakana

Liu

et al. 2008

Journal of Structural Biology

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“…Results presented by Sherman et al 12 are among the most favorable for IVEM applications, but still show a strong loss of both signal and signal-to-noise ratio ͑SNR͒ at high resolution, compared to film. Measurements by Booth et al 13 produced a favorable comparison of a CCD and film under certain conditions. Our own experience with a CCD optimized for working at 400 kV included quantitative comparisons of images of purple membrane and other specimens on film and on the CCD.…”

Section: Introductionmentioning

confidence: 99%

A charge coupled device camera with electron decelerator for intermediate voltage electron microscopy

Downing

Mooney²

2008

Review of Scientific Instruments

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Electron microscopists are increasingly turning to intermediate voltage electron microscopes ͑IVEMs͒ operating at 300-400 kV for a wide range of studies. They are also increasingly taking advantage of slow-scan charge coupled device ͑CCD͒ cameras, which have become widely used on electron microscopes. Under some conditions, CCDs provide an improvement in data quality over photographic film, as well as the many advantages of direct digital readout. However, CCD performance is seriously degraded on IVEMs compared to the more conventional 100 kV microscopes. In order to increase the efficiency and quality of data recording on IVEMs, we have developed a CCD camera system in which the electrons are decelerated to below 100 kV before impacting the camera, resulting in greatly improved performance in both signal quality and resolution compared to other CCDs used in electron microscopy. These improvements will allow high-quality image and diffraction data to be collected directly with the CCD, enabling improvements in data collection for applications including high-resolution electron crystallography, single particle reconstruction of protein structures, tomographic studies of cell ultrastructure, and remote microscope operation. This approach will enable us to use even larger format CCD chips that are being developed with smaller pixels.

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Electron Cryomicroscopy of Molecular Nanomachines and Cells

2010

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Electron cryomicroscopy (cryo‐EM) is an emerging biophysical tool that can be used to determine the structures of molecular nanomachines in fully solvated conformations at subnanometer resolutions (<1 nm). Such cryo‐EM maps can reveal long α‐helices and large β‐sheets. In the highest resolution cryo‐EM density maps, it is possible to see bulky side chains and trace the Cα backbone of molecular subunits within a multicomponent nanomachine. Electron cryotomography (cryo‐ET) is equally powerful because of the unique cellular context in which it can capture and reveal cellular nanomachines. Despite reaching only 4–10 nm resolution, cryo‐ET reconstructions are capable of imaging whole cells and distinguishing their molecular components. Both of these methods are complementary to conventional methods of structure determination, including X‐ray crystallography and NMR spectroscopy. Hybrid methods that combine these structural techniques with cryo‐EM and cryo‐ET result in a complete view of nanomachines from atomic detail to their spatial and temporal location within a cell. This chapter will describe the experimental and computational pipeline in cryo‐EM and cryo‐ET and illustrate their effectiveness with examples from an ion channel, spherical virus, cellular motor and the survey of a complete cell.

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Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300 kV

Cited by 30 publications

References 18 publications

Achievable resolution from images of biological specimens acquired from a 4k×4k CCD camera in a 300-kV electron cryomicroscope

Achievable resolution from images of biological specimens acquired from a 4k×4k CCD camera in a 300-kV electron cryomicroscope

A charge coupled device camera with electron decelerator for intermediate voltage electron microscopy

Electron Cryomicroscopy of Molecular Nanomachines and Cells

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