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Title: Fully Mechanically Controlled Automated Electron Microscopic Tomography

Abstract

Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins' functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000-160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.

Authors:
 [1];  [2];  [3];  [3];  [3];  [3];  [3];  [4];  [5];  [6];  [7];  [8];  [3]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry; Xi'an Jiaotong Univ. (China). State Key Lab. for Manufacturing System Engineering
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry; Xi'an Jiaotong Univ. (China). School of Electrical Engineering
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Foundry
  4. Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.
  5. Pfizer BioThermapeutics Pharmaceutical Sciences, Pear River, NY (United States)
  6. Pfizer BioTherapeutics Pharmaceutical Sciences, St. Louis, MO (United States)
  7. Xi'an Jiaotong Univ. (China). School of Electrical Engineering
  8. Xi'an Jiaotong Univ. (China). State Key Lab. for Manufacturing System Engineering
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1379510
Grant/Contract Number:
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 6; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; electron microscopy; electronic structure of atoms and molecules; molecular imaging; single-molecule biophysics

Citation Formats

Liu, Jinxin, Li, Hongchang, Zhang, Lei, Rames, Matthew, Zhang, Meng, Yu, Yadong, Peng, Bo, Celis, César Díaz, Xu, April, Zou, Qin, Yang, Xu, Chen, Xuefeng, and Ren, Gang. Fully Mechanically Controlled Automated Electron Microscopic Tomography. United States: N. p., 2016. Web. doi:10.1038/srep29231.
Liu, Jinxin, Li, Hongchang, Zhang, Lei, Rames, Matthew, Zhang, Meng, Yu, Yadong, Peng, Bo, Celis, César Díaz, Xu, April, Zou, Qin, Yang, Xu, Chen, Xuefeng, & Ren, Gang. Fully Mechanically Controlled Automated Electron Microscopic Tomography. United States. doi:10.1038/srep29231.
Liu, Jinxin, Li, Hongchang, Zhang, Lei, Rames, Matthew, Zhang, Meng, Yu, Yadong, Peng, Bo, Celis, César Díaz, Xu, April, Zou, Qin, Yang, Xu, Chen, Xuefeng, and Ren, Gang. 2016. "Fully Mechanically Controlled Automated Electron Microscopic Tomography". United States. doi:10.1038/srep29231. https://www.osti.gov/servlets/purl/1379510.
@article{osti_1379510,
title = {Fully Mechanically Controlled Automated Electron Microscopic Tomography},
author = {Liu, Jinxin and Li, Hongchang and Zhang, Lei and Rames, Matthew and Zhang, Meng and Yu, Yadong and Peng, Bo and Celis, César Díaz and Xu, April and Zou, Qin and Yang, Xu and Chen, Xuefeng and Ren, Gang},
abstractNote = {Knowledge of three-dimensional (3D) structures of each individual particles of asymmetric and flexible proteins is essential in understanding those proteins' functions; but their structures are difficult to determine. Electron tomography (ET) provides a tool for imaging a single and unique biological object from a series of tilted angles, but it is challenging to image a single protein for three-dimensional (3D) reconstruction due to the imperfect mechanical control capability of the specimen goniometer under both a medium to high magnification (approximately 50,000-160,000×) and an optimized beam coherence condition. Here, we report a fully mechanical control method for automating ET data acquisition without using beam tilt/shift processes. This method could reduce the accumulation of beam tilt/shift that used to compensate the error from the mechanical control, but downgraded the beam coherence. Our method was developed by minimizing the error of the target object center during the tilting process through a closed-loop proportional-integral (PI) control algorithm. The validations by both negative staining (NS) and cryo-electron microscopy (cryo-EM) suggest that this method has a comparable capability to other ET methods in tracking target proteins while maintaining optimized beam coherence conditions for imaging.},
doi = {10.1038/srep29231},
journal = {Scientific Reports},
number = 1,
volume = 6,
place = {United States},
year = 2016,
month = 7
}

Journal Article:
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  • A closed-loop proportional-integral (PI) control software is provided for fully mechanically controlled automated electron microscopic tomography. The software is developed based on Gatan DigitalMicrograph, and is compatible with Zeiss LIBRA 120 transmission electron microscope. However, it can be expanded to other TEM instrument with modification. The software consists of a graphical user interface, a digital PI controller, an image analyzing unit, and other drive units (i.e.: image acquire unit and goniometer drive unit). During a tomography data collection process, the image analyzing unit analyzes both the accumulated shift and defocus value of the latest acquired image, and provides the resultsmore » to the digital PI controller. The digital PI control compares the results with the preset values and determines the optimum adjustments of the goniometer. The goniometer drive unit adjusts the spatial position of the specimen according to the instructions given by the digital PI controller for the next tilt angle and image acquisition. The goniometer drive unit achieves high precision positioning by using a backlash elimination method. The major benefits of the software are: 1) the goniometer drive unit keeps pre-aligned/optimized beam conditions unchanged and achieves position tracking solely through mechanical control; 2) the image analyzing unit relies on only historical data and therefore does not require additional images/exposures; 3) the PI controller enables the system to dynamically track the imaging target with extremely low system error.« less
  • Purpose: Thousands of cone-beam computed tomography (CBCT) scanners for vascular, maxillofacial, neurological, and body imaging are in clinical use today, but there is no consensus on uniform acceptance and constancy testing for image quality (IQ) and dose yet. The authors developed a quality assurance (QA) framework for fully automated and time-efficient performance evaluation of these systems. In addition, the dependence of objective Fourier-based IQ metrics on direction and position in 3D volumes was investigated for CBCT. Methods: The authors designed a dedicated QA phantom 10 cm in length consisting of five compartments, each with a diameter of 10 cm, andmore » an optional extension ring 16 cm in diameter. A homogeneous section of water-equivalent material allows measuring CT value accuracy, image noise and uniformity, and multidimensional global and local noise power spectra (NPS). For the quantitative determination of 3D high-contrast spatial resolution, the modulation transfer function (MTF) of centrally and peripherally positioned aluminum spheres was computed from edge profiles. Additional in-plane and axial resolution patterns were used to assess resolution qualitatively. The characterization of low-contrast detectability as well as CT value linearity and artifact behavior was tested by utilizing sections with soft-tissue-equivalent and metallic inserts. For an automated QA procedure, a phantom detection algorithm was implemented. All tests used in the dedicated QA program were initially verified in simulation studies and experimentally confirmed on a clinical dental CBCT system. Results: The automated IQ evaluation of volume data sets of the dental CBCT system was achieved with the proposed phantom requiring only one scan for the determination of all desired parameters. Typically, less than 5 min were needed for phantom set-up, scanning, and data analysis. Quantitative evaluation of system performance over time by comparison to previous examinations was also verified. The maximum percentage interscan variation of repeated measurements was less than 4% and 1.7% on average for all investigated quality criteria. The NPS-based image noise differed by less than 5% from the conventional standard deviation approach and spatially selective 10% MTF values were well comparable to subjective results obtained with 3D resolution pattern. Determining only transverse spatial resolution and global noise behavior in the central field of measurement turned out to be insufficient. Conclusions: The proposed framework transfers QA routines employed in conventional CT in an advanced version to CBCT for fully automated and time-efficient evaluation of technical equipment. With the modular phantom design, a routine as well as an expert version for assessing IQ is provided. The QA program can be used for arbitrary CT units to evaluate 3D imaging characteristics automatically.« less
  • A full field transmission x-ray microscope (TXM) has been developed and commissioned at the National Synchrotron Light Source at Brookhaven National Laboratory. The capabilities we developed in auto-tomography, local tomography, and spectroscopic imaging that overcome many of the limitations and difficulties in existing transmission x-ray microscopes are described and experimentally demonstrated. Sub-50 nm resolution in 3-dimension (3D) with markerless automated tomography has been achieved. These capabilities open up scientific opportunities in many research fields.
  • A full field transmission x-ray microscope (TXM) has been developed and commissioned at the National Synchrotron Light Source at Brookhaven National Laboratory. The capabilities we developed in auto-tomography, local tomography, and spectroscopic imaging that overcome many of the limitations and difficulties in existing transmission x-ray microscopes are described and experimentally demonstrated. Sub-50 nm resolution in 3-dimension (3D) with markerless automated tomography has been achieved. These capabilities open up scientific opportunities in many research fields.