scholarly journals Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy

2020 ◽  
Author(s):  
Ryan Lane ◽  
Yoram Vos ◽  
Anouk H. G. Wolters ◽  
Luc van Kessel ◽  
Ben N.G. Giepmans ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Image Quality ◽  
Large Scale ◽  
Signal To Noise Ratio ◽  
Negative Bias ◽  
Acquisition Rate ◽  
Bias Potential ◽  
Signal Collection ◽  
Block Face ◽  
Imaging Conditions

AbstractLarge-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 hours. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.

scholarly journals Plasma Cleaning Improves the Image Quality of Serial Block-face Scanning Electron Microscopy (SBFSEM) Volumetric Data Sets

2017 ◽  
Vol 23 (S1) ◽  
pp. 1266-1267 ◽  
Author(s):  
Barbara Armbruster ◽  
Christopher Booth ◽  
Stuart Searle ◽  
Michael Cable ◽  
Ronald Vane
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Image Quality ◽  
Data Sets ◽  
Plasma Cleaning ◽  
Volumetric Data ◽  
Face Scanning ◽  
Block Face ◽  
Scanning Electron

“Resolution” in atomic-resolution Electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 498-499
Author(s):  
Michael A. O'Keefe ◽  
John C.H. Spence
Keyword(s):  
Electron Microscopy ◽  
Signal To Noise Ratio ◽  
Dark Field ◽  
Lattice Imaging ◽  
Rayleigh Criterion ◽  
Transmission Electron ◽  
Resolution Electron ◽  
Incoherent Imaging ◽  
General Relations ◽  
Imaging Conditions

The Rayleigh resolution criterion was developed for incoherent imaging conditions and cannot, in general, be applied to coherent high-resolution transmission electron microscopy (HRTEM). In fact, the Rayleigh criterion may lead to paradoxical results since it considers only two scatterers and does not account for the signal-to-noise ratio. It may, however, be used for dark-field HAAD STEM imaging. For the case of strong multiple scattering in HRTEM lattice imaging, the only general relations that can be assumed between the image of a specimen and its projected crystal potential are those imposed by symmetry, and the “local” HRTEM column approximation. The effects of limited resolution may produce an image of lower symmetry than that of the object. Also, misalignment along symmetry axes may preserve some symmetry elements. Under coherent conditions the ability to distinguish atoms in an image depends on their scattering phases, which are a property of the sample.

scholarly journals Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy

2021 ◽  
Vol 5 ◽  
pp. 100046
Author(s):  
Ryan Lane ◽  
Yoram Vos ◽  
Anouk H.G. Wolters ◽  
Luc van Kessel ◽  
S. Elisa Chen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Large Scale ◽  
Bias Potential

scholarly journals A petascale automated imaging pipeline for mapping neuronal circuits with high-throughput transmission electron microscopy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenjing Yin ◽  
Derrick Brittain ◽  
Jay Borseth ◽  
Marie E. Scott ◽  
Derric Williams ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Parallel Imaging ◽  
Large Scale ◽  
Network Connectivity ◽  
Brain Regions ◽  
Acquisition Rate ◽  
Tissue Sections ◽  
Transmission Electron ◽  
Electron Microscopes ◽  
The Brain

Abstract Electron microscopy (EM) is widely used for studying cellular structure and network connectivity in the brain. We have built a parallel imaging pipeline using transmission electron microscopes that scales this technology, implements 24/7 continuous autonomous imaging, and enables the acquisition of petascale datasets. The suitability of this architecture for large-scale imaging was demonstrated by acquiring a volume of more than 1 mm3 of mouse neocortex, spanning four different visual areas at synaptic resolution, in less than 6 months. Over 26,500 ultrathin tissue sections from the same block were imaged, yielding a dataset of more than 2 petabytes. The combined burst acquisition rate of the pipeline is 3 Gpixel per sec and the net rate is 600 Mpixel per sec with six microscopes running in parallel. This work demonstrates the feasibility of acquiring EM datasets at the scale of cortical microcircuits in multiple brain regions and species.

An Environmental Wet Cell For High Voltage Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 18-19
Author(s):  
N.J. Tighe ◽  
H.M. Flower ◽  
P.R. Swann
Keyword(s):  
Electron Microscopy ◽  
High Temperature ◽  
Image Quality ◽  
Inelastic Scattering ◽  
High Voltage ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Environmental Cell ◽  
Water Saturated ◽  
High Temperature Gas

A differentially pumped environmental cell has been developed for use in the AEI EM7 million volt microscope. In the initial version the column of gas traversed by the beam was 5.5mm. This permited inclusion of a tilting hot stage in the cell for investigating high temperature gas-specimen reactions. In order to examine specimens in the wet state it was found that a pressure of approximately 400 torr of water saturated helium was needed around the specimen to prevent dehydration. Inelastic scattering by the water resulted in a sharp loss of image quality. Therefore a modified cell with an ‘airgap’ of only 1.5mm has been constructed. The shorter electron path through the gas permits examination of specimens at the necessary pressure of moist helium; the specimen can still be tilted about the side entry rod axis by ±7°C to obtain stereopairs.

Black-white contrast of dislocation loops

1978 ◽  
Vol 36 (1) ◽  
pp. 328-329
Author(s):  
J. J. Hren ◽  
W. D. Cooper ◽  
L. J. Sykes
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Dislocation Loops ◽  
Major Premise ◽  
Burgers Vector ◽  
Transmission Electron ◽  
Primary Means ◽  
Contrast Analysis ◽  
Dot Product ◽  
Imaging Conditions

Small dislocation loops observed by transmission electron microscopy exhibit a characteristic black-white strain contrast when observed under dynamical imaging conditions. In many cases, the topography and orientation of the image may be used to determine the nature of the loop crystallography. Two distinct but somewhat overlapping procedures have been developed for the contrast analysis and identification of small dislocation loops. One group of investigators has emphasized the use of the topography of the image as the principle tool for analysis. The major premise of this method is that the characteristic details of the image topography are dependent only on the magnitude of the dot product between the loop Burgers vector and the diffracting vector. This technique is commonly referred to as the (g•b) analysis. A second group of investigators has emphasized the use of the orientation of the direction of black-white contrast as the primary means of analysis.

Image quality vs data obtainment in biological electron microscopy

1986 ◽  
Vol 44 ◽  
pp. 42-43
Author(s):  
J. E. Johnson
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Image Quality ◽  
High Speed ◽  
Early Period ◽  
Early Years ◽  
Fluorescent Screen ◽  
Embedding Medium

In the early years of biological electron microscopy, scientists had their hands full attempting to describe the cellular microcosm that was suddenly before them on the fluorescent screen. Mitochondria, Golgi, endoplasmic reticulum, and other myriad organelles were being examined, micrographed, and documented in the literature. A major problem of that early period was the development of methods to cut sections thin enough to study under the electron beam. A microtome designed in 1943 moved the specimen toward a rotary “Cyclone” knife revolving at 12,500 RPM, or 1000 times as fast as an ordinary microtome. It was claimed that no embedding medium was necessary or that soft embedding media could be used. Collecting the sections thus cut sounded a little precarious: “The 0.1 micron sections cut with the high speed knife fly out at a tangent and are dispersed in the air. They may be collected... on... screens held near the knife“.

Progress in high-resolution CRYO-SEM imaging of viral particles

1996 ◽  
Vol 54 ◽  
pp. 818-819
Author(s):  
Ya Chen ◽  
Geoffrey Letchworth ◽  
John White
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Structural Information ◽  
Signal To Noise Ratio ◽  
Flexible Structures ◽  
Simian Virus ◽  
Topographic Features ◽  
Viral Particles ◽  
Scanning Electron

Low-temperature high-resolution scanning electron microscopy (cryo-HRSEM) has been successfully utilized to image biological macromolecular complexes at nanometer resolution. Recently, imaging of individual viral particles such as reovirus using cryo-HRSEM or simian virus (SIV) using HRSEM, HV-STEM and AFM have been reported. Although conventional electron microscopy (e.g., negative staining, replica, embedding and section), or cryo-TEM technique are widely used in studying of the architectures of viral particles, scanning electron microscopy presents two major advantages. First, secondary electron signal of SEM represents mostly surface topographic features. The topographic details of a biological assembly can be viewed directly and will not be obscured by signals from the opposite surface or from internal structures. Second, SEM may produce high contrast and signal-to-noise ratio images. As a result of this important feature, it is capable of visualizing not only individual virus particles, but also asymmetric or flexible structures. The 2-3 nm resolution obtained using high resolution cryo-SEM made it possible to provide useful surface structural information of macromolecule complexes within cells and tissues. In this study, cryo-HRSEM is utilized to visualize the distribution of glycoproteins of a herpesvirus.

Image Quality Enhancing by Efficient Histogram Equalization

2014 ◽  
Vol 2 (2) ◽  
pp. 47-58
Author(s):  
Ismail Sh. Baqer
Keyword(s):  
Image Quality ◽  
Contrast Enhancement ◽  
Mean Squared Error ◽  
Signal To Noise Ratio ◽  
Histogram Equalization ◽  
Gray Level ◽  
Signal To Noise ◽  
Squared Error ◽  
Noise Ratio

A two Level Image Quality enhancement is proposed in this paper. In the first level, Dualistic Sub-Image Histogram Equalization DSIHE method decomposes the original image into two sub-images based on median of original images. The second level deals with spikes shaped noise that may appear in the image after processing. We presents three methods of image enhancement GHE, LHE and proposed DSIHE that improve the visual quality of images. A comparative calculations is being carried out on above mentioned techniques to examine objective and subjective image quality parameters e.g. Peak Signal-to-Noise Ratio PSNR values, entropy H and mean squared error MSE to measure the quality of gray scale enhanced images. For handling gray-level images, convenient Histogram Equalization methods e.g. GHE and LHE tend to change the mean brightness of an image to middle level of the gray-level range limiting their appropriateness for contrast enhancement in consumer electronics such as TV monitors. The DSIHE methods seem to overcome this disadvantage as they tend to preserve both, the brightness and contrast enhancement. Experimental results show that the proposed technique gives better results in terms of Discrete Entropy, Signal to Noise ratio and Mean Squared Error values than the Global and Local histogram-based equalization methods

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