scholarly journals Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

2016 ◽  
Vol 22 (6) ◽  
pp. 1316-1328 ◽  
Author(s):  
Michael Marko ◽  
Chyongere Hsieh ◽  
Eric Leith ◽  
David Mastronarde ◽  
Sohei Motoki
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Practical Experience ◽  
Phase Plate ◽  
Native State ◽  
Automated Data Collection ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Phase Plates ◽  
Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

Phase Contrast Enhancement with Phase Plates in Biological Electron Microscopy

2010 ◽  
Vol 18 (4) ◽  
pp. 10-13 ◽  
Author(s):  
Kuniaki Nagayama ◽  
Radostin Danev ◽  
Hideki Shigematsu ◽  
Naoki Hosogi ◽  
Yoshiyuki Fukuda ◽  
...  
Keyword(s):  
Thin Film ◽  
Electron Microscopy ◽  
Phase Contrast ◽  
Phase Plate ◽  
Electron Loss ◽  
Whole Cells ◽  
Transmission Electron ◽  
Different Types ◽  
Transparent Objects ◽  
Phase Plates

Theoretically, transmission electron microscopy (TEM) is compatible with three different types of phase plate: thin-film, electrostatic, and magnetic. However, designing functional phase plates has been an arduous process that has suffered from unavoidable technical obstacles such as phase-plate charging and difficulties associated with micro-fabrication of electrostatic and magnetic phase plates. This review discusses phase-contrast schemes that allow visualization of transparent objects with high contrast. Next it deals with recent studies on biological applications ranging from proteins and viruses to whole cells. Finally, future prospects for overcoming the problem of phase-plate charging and for designing the next generation of phase-plates to solve the problem of electron loss inherent in thin-film phase plates are discussed.

scholarly journals High-resolution transmission electron microscopy with an electrostatic Zach phase plate

2016 ◽  
Vol 18 (5) ◽  
pp. 053005 ◽  
Author(s):  
S Hettler ◽  
M Dries ◽  
J Zeelen ◽  
M Oster ◽  
R R Schröder ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Phase Plate ◽  
Transmission Electron

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

High Resolution Cryo-Electron Microscopy Sheds A New Light Upon Biomembrane Architecture

1990 ◽  
Vol 48 (1) ◽  
pp. 494-495
Author(s):  
Kume P. Nahoaki ◽  
Yuba D. Akiko ◽  
Yoshizawa C. Akiyasu ◽  
Sato B. Satoshi ◽  
Fujiyoshi Yoshinori
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Photosynthetic Bacterium ◽  
Chromatium Vinosum ◽  
Irradiation Damage ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Cooling Mechanism ◽  
Chicken Erythrocytes

Cryo-electron microscopy, together with the rapid cryo-fixation technique, has made it possible to observe chemically unfixed unstained specimens under a transmission electron microscope. Aoki et al. constructed a new cryo-stage in order to reduce irradiation damage upon biological specimens further by cooling them with liquid helium. It was also designed to eliminate shaking of specimens as much as possible by the sophisticated mounting and the refined cooling mechanism of the stage. The stage has proved to achieve a higher contrast and higher resolution of images than a commonly-used cryo-stage cooled solely by liquid nitrogen does. This high resolution cryo-electron microscope (HiRCEM) was originally developed aiming at the improvement of resolution in structural analysis of proteins or nucleic acids, but it has turned out to be quite useful for detecting the architecture of biomembranes as well. This is due to the existence of phosphorus atoms of phospholipids which are heavier than C, H, O, N atoms abundant within biomembranes, and also due to the great optical depth of the membrane where it is aligned parallel to the electron beams.We have examined several kinds of biomembranes as well as the synthesized liposomes under HiRCEM. Among them were the plasma membrane of human erythrocytes (ghosts), the nuclear membrane of chicken erythrocytes, the endoplasmic reticular membrane of rat nerve cells, the chromatophore membrane of a photosynthetic bacterium Chromatium vinosum , the thylakoid membrane of spinach chloroplasts and the envelope of influenza type A virus (PR8 & X31). Liposomes we employed were synthesized from the 2:1 mixture of phosphatidylcholine and cholesterol.

scholarly journals Near-Atomic Resolution Structure Determination in Over-Focus with Volta Phase Plate by Cs-corrected Cryo-EM

2017 ◽  
Author(s):  
Xiao Fan ◽  
Lingyun Zhao ◽  
Chuan Liu ◽  
Jin-Can Zhang ◽  
Kelong Fan ◽  
...  
Keyword(s):  
Optical Properties ◽  
High Resolution ◽  
Spherical Aberration ◽  
Atomic Resolution ◽  
Objective Lens ◽  
Phase Plate ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Imaging Strategy ◽  
Imaging Conditions

SummaryVolta phase plate (VPP) is a recently developed transmission electron microscope (TEM) apparatus that can significantly enhance the image contrast of biological samples in cryo-electron microscopy (cryo-EM) therefore impose the possibility to solve structures of relatively small macromolecules at high resolution. In this work, we performed theoretical analysis and found that using phase plate on objective lens spherical aberration (Cs)-corrected TEM may gain some interesting optical properties, including the over-focus imaging of macromolecules. We subsequently evaluated the imaging strategy of frozen-hydrated apo-ferritin with VPP on a Cs-corrected TEM and obtained the structure of apo-ferritin at near atomic resolution from both under- and over-focused dataset, illustrating the feasibility and new potential of combining VPP with Cs-corrected TEM for high resolution cryo-EM.HighlightsThe successful combination of volta phase plate and Cs-corrector in single particle cryo-EM.Near-atomic structure determined from over-focused images by cryo-EM. VPP-Cs-corrector coupled EM provides interesting optical properties.In BriefWe took the unique advantage of the optical system by combining the volta phase plate and Cs-corrector in a modern TEM to collect high resolution micrographs of frozen-hydrated apo-ferritin in over-focus imaging conditions and determined the structure of apo-ferritin at 3.0 Angstrom resolution.

Monitoring SEM Performance

2001 ◽  
Vol 7 (S2) ◽  
pp. 822-823
Author(s):  
Stephen K. Chapman
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Resolution ◽  
Scanning Electron Microscope ◽  
Standard Procedure ◽  
Contamination Rate ◽  
Transmission Electron ◽  
Set Up ◽  
Scanning Electron

I was trained as a transmission electron microscope engineer in the mid 1960s. I took resolution tests at least once each year and calibrated all of the microscopes that I attended, it was considered a standard procedure for those maintaining an instrument. Moving into the scanning electron microscope field in the mid 1970s it was natural to carry this practice over to that instrument, but in those days this was considered to be extreme. Now, as a consultant in electron microscopy, I routinely carry out SEM resolution, magnification calibration and contamination rate tests on the instruments that I use. I train operators in the role of preventative maintenance and encourage them to know as much as possible about their instruments as this increases their ability to fault find and maintain their own instruments.Resolution - in many laboratories most tungsten hairpin instruments are set up for extended filament life rater than for high resolution.

In-Focus Electrostatic Zach Phase Plate Imaging for Transmission Electron Microscopy with Tunable Phase Contrast of Frozen Hydrated Biological Samples

2014 ◽  
Vol 20 (1) ◽  
pp. 175-183 ◽  
Author(s):  
Nicole Frindt ◽  
Marco Oster ◽  
Simon Hettler ◽  
Björn Gamm ◽  
Levin Dieterle ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Phase Contrast ◽  
Structural Information ◽  
Signal To Noise Ratio ◽  
Phase Plate ◽  
Spatial Frequencies ◽  
Transmission Electron ◽  
Sample Orientation ◽  
Phase Plates

AbstractTransmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.

scholarly journals High-Resolution Transmission Electron Microscopy With Zach Phase Plate

2015 ◽  
Vol 21 (S3) ◽  
pp. 1581-1582
Author(s):  
Simon Hettler ◽  
Manuel Dries ◽  
Tina Schulze ◽  
Marco Oster ◽  
Christian Wacker ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Resolution ◽  
Phase Plate ◽  
Transmission Electron

scholarly journals Investigation of hole-free phase plate performance in transmission electron microscopy under different operation conditions by experiments and simulations

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Rebecca Pretzsch ◽  
Manuel Dries ◽  
Simon Hettler ◽  
Martin Spiecker ◽  
Martin Obermair ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Phase Shift ◽  
Zero Order ◽  
Phase Shifting ◽  
Negative Phase ◽  
Phase Plate ◽  
Microscopy Imaging ◽  
Transmission Electron ◽  
Phase Plates

Abstract Hole-free phase plates (HFPPs), also known as Volta phase plates, were already demonstrated to be well suited for in-focus transmission electron microscopy imaging of organic objects. However, the underlying physical processes have not been fully understood yet. To further elucidate the imaging properties of HFPPs, phase shift measurements were carried out under different experimental conditions. Both positive and negative phase shifts occur depending on the diameter of the zero-order electron beam and the HFPP film temperature. The analysis of Thon ring patterns of an amorphous carbon test sample reveals that the phase-shifting patch can be significantly larger than the size of the zero-order beam on the HFPP film. An HFPP was used for in-focus phase contrast imaging of carbon nanotube (CNT) bundles under positive and negative phase-shifting conditions. The comparison of experimental and simulated images of CNT bundles gives detailed information on the phase shift profile, which depends on the spatial frequency in the vicinity of the zero-order beam. The shape of the phase shift profile also explains halo-like image artifacts that surround the imaged objects.

scholarly journals Biological Applications at the Cutting Edge of Cryo-Electron Microscopy

2018 ◽  
Vol 24 (4) ◽  
pp. 406-419 ◽  
Author(s):  
Rebecca S. Dillard ◽  
Cheri M. Hampton ◽  
Joshua D. Strauss ◽  
Zunlong Ke ◽  
Deanna Altomara ◽  
...  
Keyword(s):  
Biological Sciences ◽  
Electron Microscopy ◽  
Sample Preparation ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Biological Applications ◽  
Preparation Methods ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Phase Plates

AbstractCryo-electron microscopy (cryo-EM) is a powerful tool for macromolecular to near-atomic resolution structure determination in the biological sciences. The specimen is maintained in a near-native environment within a thin film of vitreous ice and imaged in a transmission electron microscope. The images can then be processed by a number of computational methods to produce three-dimensional information. Recent advances in sample preparation, imaging, and data processing have led to tremendous growth in the field of cryo-EM by providing higher resolution structures and the ability to investigate macromolecules within the context of the cell. Here, we review developments in sample preparation methods and substrates, detectors, phase plates, and cryo-correlative light and electron microscopy that have contributed to this expansion. We also have included specific biological applications.

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