Sample Shrinkage and Radiation Damage of Plastic Sections

Luther, Pradeep K.

doi:10.1007/978-0-387-69008-7_2

Cited by 31 publications

(26 citation statements)

References 63 publications

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“…Another technique for cryo-sample preparation is cryo-sectioning in which the high pressure frozen thick specimen is trimmed using a diamond knife. Unfortunately, previous studies have shown that there is considerable variation in section thickness, especially for thin sections (Luther, 2006). Focused ion beam (FIB) milling has gained considerable acceptance in recent years as a precision section preparation method.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer–Lambert law

Yan

Edwards

Pankratz

et al. 2015

Journal of Structural Biology

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Cryo-electron tomography (cryo-ET) is an emerging technique that can elucidate the architecture of macromolecular complexes and cellular ultrastructure in a near-native state. Some important sample parameters, such as thickness and tilt, are needed for 3-D reconstruction. However, these parameters can currently only be determined using trial 3-D reconstructions. Accurate electron mean free path plays a significant role in modeling image formation process essential for simulation of electron microscopy images and model-based iterative 3-D reconstruction methods; however, their values are voltage and sample dependent and have only been experimentally measured for a limited number of sample conditions. Here, we report a computational method, tomoThickness, based on the Beer-Lambert law, to simultaneously determine the sample thickness, tilt and electron inelastic mean free path by solving an overdetermined nonlinear least square optimization problem utilizing the strong constraints of tilt relationships. The method has been extensively tested with both stained and cryo datasets. The fitted electron mean free paths are consistent with reported experimental measurements. The accurate thickness estimation eliminates the need for a generous assignment of Z-dimension size of the tomogram. Interestingly, we have also found that nearly all samples are a few degrees tilted relative to the electron beam. Compensation of the intrinsic sample tilt can result in horizontal structure and reduced Z-dimension of tomograms. Our fast, pre-reconstruction method can thus provide important sample parameters that can help improve performance of tomographic reconstruction of a wide range of samples.

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Section: Introductionmentioning

confidence: 99%

Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer–Lambert law

Yan

Edwards

Pankratz

et al. 2015

Journal of Structural Biology

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“…We tried to restore the original (un-shrunken) dimensions of the section in the tomogram for morphometric accuracy by estimating the amount of shrinkage by means of two independent methods (see Section 2). Both methods indicated that the sections shrank in thickness to 63-64% of the original thickness in the area exposed to the beam, and the resin shrinkage during beam irradiation has been demonstrated to occur predominantly along the beam axis, that is in the thickness of the section (Luther, 2006). Indeed, there was no significant lateral displacement within the sections.…”

Section: Depth Compensated Et Reconstructionmentioning

confidence: 94%

Helical arrangement of filaments in microvillar actin bundles

Ohta

Higashi

Sawaguchi

et al. 2012

Journal of Structural Biology

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“…High-quality reconstruction results from ET are hampered by several major obstacles: First, tilt angles in ET owing to technical and material characteristics are generally limited to a range within ±70° due to scattering of the primary electron beam. The resulting limitation in tilt and inability to collect high angle projections is often referred to as the missing wedge problem (Frank, 2008); Second, reconstructions are affected by the distortions that result from the imperfect optics of the electron microscope and the sample warping due to radiation exposure damage (Luther, 2006). Additionally, electron micrographs are quite noisy and low contrast, especially under low dose collection conditions or when employing higher electron beam voltage.…”

Section: Introductionmentioning

confidence: 99%

Electron tomography simulator with realistic 3D phantom for evaluation of acquisition, alignment and reconstruction methods

Wan

Katchalski

Churas

et al. 2017

Journal of Structural Biology

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Because of the significance of electron microscope tomography in the investigation of biological structure at nanometer scales, ongoing improvement efforts have been continuous over recent years. This is particularly true in the case of software developments. Nevertheless, verification of improvements delivered by new algorithms and software remains difficult. Current analysis tools do not provide adaptable and consistent methods for quality assessment. This is particularly true with images of biological samples, due to image complexity, variability, low contrast and noise. We report an electron tomography (ET) simulator with accurate ray optics modeling of image formation that includes curvilinear trajectories through the sample, warping of the sample and noise. As a demonstration of the utility of our approach, we have concentrated on providing verification of the class of reconstruction methods applicable to wide field images of stained plastic-embedded samples. Accordingly, we have also constructed digital phantoms derived from serial block face scanning electron microscope images. These phantoms are also easily modified to include alignment features to test alignment algorithms. The combination of more realistic phantoms with more faithful simulations facilitates objective comparison of acquisition parameters, alignment and reconstruction algorithms and their range of applicability. With proper phantoms, this approach can also be modified to include more complex optical models, including distance-dependent blurring and phase contrast functions, such as may occur in cryotomography.

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Sample Shrinkage and Radiation Damage of Plastic Sections

Cited by 31 publications

References 63 publications

Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer–Lambert law

Simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on Beer–Lambert law

Helical arrangement of filaments in microvillar actin bundles

Electron tomography simulator with realistic 3D phantom for evaluation of acquisition, alignment and reconstruction methods

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