Future Directions of Electron Crystallography

Author(s):  
Yoshinori Fujiyoshi
Author(s):  
Benjamin F. Trump ◽  
Irene K. Berezesky ◽  
Raymond T. Jones

The role of electron microscopy and associated techniques is assured in diagnostic pathology. At the present time, most of the progress has been made on tissues examined by transmission electron microscopy (TEM) and correlated with light microscopy (LM) and by cytochemistry using both plastic and paraffin-embedded materials. As mentioned elsewhere in this symposium, this has revolutionized many fields of pathology including diagnostic, anatomic and clinical pathology. It began with the kidney; however, it has now been extended to most other organ systems and to tumor diagnosis in general. The results of the past few years tend to indicate the future directions and needs of this expanding field. Now, in addition to routine EM, pathologists have access to the many newly developed methods and instruments mentioned below which should aid considerably not only in diagnostic pathology but in investigative pathology as well.


Author(s):  
A. Engel ◽  
A. Holzenburg ◽  
K. Stauffer ◽  
J. Rosenbusch ◽  
U. Aebi

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.


Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.


Author(s):  
Douglas L. Dorset

The quantitative use of electron diffraction intensity data for the determination of crystal structures represents the pioneering achievement in the electron crystallography of organic molecules, an effort largely begun by B. K. Vainshtein and his co-workers. However, despite numerous representative structure analyses yielding results consistent with X-ray determination, this entire effort was viewed with considerable mistrust by many crystallographers. This was no doubt due to the rather high crystallographic R-factors reported for some structures and, more importantly, the failure to convince many skeptics that the measured intensity data were adequate for ab initio structure determinations.We have recently demonstrated the utility of these data sets for structure analyses by direct phase determination based on the probabilistic estimate of three- and four-phase structure invariant sums. Examples include the structure of diketopiperazine using Vainshtein's 3D data, a similar 3D analysis of the room temperature structure of thiourea, and a zonal determination of the urea structure, the latter also based on data collected by the Moscow group.


Author(s):  
Kenneth H. Downing

Three-dimensional structures of a number of samples have been determined by electron crystallography. The procedures used in this work include recording images of fairly large areas of a specimen at high tilt angles. There is then a large defocus ramp across the image, and parts of the image are far out of focus. In the regions where the defocus is large, the contrast transfer function (CTF) varies rapidly across the image, especially at high resolution. Not only is the CTF then difficult to determine with sufficient accuracy to correct properly, but the image contrast is reduced by envelope functions which tend toward a low value at high defocus.We have combined computer control of the electron microscope with spot-scan imaging in order to eliminate most of the defocus ramp and its effects in the images of tilted specimens. In recording the spot-scan image, the beam is scanned along rows that are parallel to the tilt axis, so that along each row of spots the focus is constant. Between scan rows, the objective lens current is changed to correct for the difference in specimen height from one scan to the next.


Author(s):  
T.W. Jeng ◽  
W. Chiu

With the advances in preparing biological materials in a thin and highly ordered form, and in maintaining them hydrated under vacuum, electron crystallography has become an important tool for biological structure investigation at high resolution (1,2). However, the electron radiation damage would limit the capability of recording reflections with low intensities in an electron diffraction pattern. It has been demonstrated that the use of a low temperature stage can reduce the radiation damage effect and that one can expose the specimen with a higher dose in order to increase the signal contrast (3). A further improvement can be made by selecting a proper photographic emulsion. The primary factors in evaluating the suitability of photographic emulsion for recording low dose diffraction patterns are speed, fog level, electron response at low electron exposure, linearity, and usable range of exposure. We have compared these factors with three photographic emulsions including Kodak electron microscopic plate (EMP), Industrex AA x-ray film (AA x-ray) and Kodak nuclear track film (NTB3).


2019 ◽  
Vol 28 (4) ◽  
pp. 877-894
Author(s):  
Nur Azyani Amri ◽  
Tian Kar Quar ◽  
Foong Yen Chong

Purpose This study examined the current pediatric amplification practice with an emphasis on hearing aid verification using probe microphone measurement (PMM), among audiologists in Klang Valley, Malaysia. Frequency of practice, access to PMM system, practiced protocols, barriers, and perception toward the benefits of PMM were identified through a survey. Method A questionnaire was distributed to and filled in by the audiologists who provided pediatric amplification service in Klang Valley, Malaysia. One hundred eight ( N = 108) audiologists, composed of 90.3% women and 9.7% men (age range: 23–48 years), participated in the survey. Results PMM was not a clinical routine practiced by a majority of the audiologists, despite its recognition as the best clinical practice that should be incorporated into protocols for fitting hearing aids in children. Variations in practice existed warranting further steps to improve the current practice for children with hearing impairment. The lack of access to PMM equipment was 1 major barrier for the audiologists to practice real-ear verification. Practitioners' characteristics such as time constraints, low confidence, and knowledge levels were also identified as barriers that impede the uptake of the evidence-based practice. Conclusions The implementation of PMM in clinical practice remains a challenge to the audiology profession. A knowledge-transfer approach that takes into consideration the barriers and involves effective collaboration or engagement between the knowledge providers and potential stakeholders is required to promote the clinical application of evidence-based best practice.


2008 ◽  
Vol 18 (1) ◽  
pp. 31-40 ◽  
Author(s):  
David J. Zajac

Abstract The purpose of this opinion article is to review the impact of the principles and technology of speech science on clinical practice in the area of craniofacial disorders. Current practice relative to (a) speech aerodynamic assessment, (b) computer-assisted single-word speech intelligibility testing, and (c) behavioral management of hypernasal resonance are reviewed. Future directions and/or refinement of each area are also identified. It is suggested that both challenging and rewarding times are in store for clinical researchers in craniofacial disorders.


VASA ◽  
2013 ◽  
Vol 42 (4) ◽  
pp. 239-251
Author(s):  
Kai Mortensen ◽  
Klaas Franzen ◽  
Michael Reppel ◽  
Joachim Weil

The aim of this review is to give a contemporary update on renal denervation therapy focusing particularly on the scientific background and present literature as well as on different technical approaches and potential future directions.


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