The electron diffractometer: a dedicated user-friendly device for electron crystallography experiments

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.

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Enhanced information from biological materials through technological improvements in cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124343 ◽

1992 ◽

Vol 50 (1) ◽

pp. 788-789

Author(s):

Marc J.C. de Jong ◽

Wim M. Busing ◽

Max T. Otten

Keyword(s):

Electron Microscopy ◽

Electron Beam ◽

Biological Materials ◽

Electron Crystallography ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

The Many ◽

Technological Improvements ◽

Beam Damage

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.

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Direct phase determination in electron crystallography: small organic molecules

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100130468 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1166-1167

Author(s):

Douglas L. Dorset

Keyword(s):

Organic Molecules ◽

Data Sets ◽

Temperature Structure ◽

3D Analysis ◽

Intensity Data ◽

Electron Crystallography ◽

Phase Determination ◽

Measured Intensity ◽

3D Data

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.

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Defocus ramp correction in spot-scan imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100121053 ◽

1992 ◽

Vol 50 (1) ◽

pp. 130-131

Author(s):

Kenneth H. Downing

Keyword(s):

Computer Control ◽

Three Dimensional ◽

Sufficient Accuracy ◽

Objective Lens ◽

Electron Crystallography ◽

Contrast Transfer Function ◽

Tilt Axis ◽

Specimen Height ◽

The Difference ◽

Envelope Functions

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.

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The Added Value of Graphical Input and Display for Electron Lens Design

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179701 ◽

1990 ◽

Vol 48 (1) ◽

pp. 190-191

Author(s):

B. Lencova ◽

G. Wisselink

Keyword(s):

Flux Density ◽

Numerical Data ◽

Added Value ◽

Lens Design ◽

Step Size ◽

Magnetic Lens ◽

Flux Lines ◽

Fine Mesh ◽

Electron Lens ◽

User Friendly

Recent progress in computer technology enables the calculation of lens fields and focal properties on commonly available computers such as IBM ATs. If we add to this the use of graphics, we greatly increase the applicability of design programs for electron lenses. Most programs for field computation are based on the finite element method (FEM). They are written in Fortran 77, so that they are easily transferred from PCs to larger machines.The design process has recently been made significantly more user friendly by adding input programs written in Turbo Pascal, which allows a flexible implementation of computer graphics. The input programs have not only menu driven input and modification of numerical data, but also graphics editing of the data. The input programs create files which are subsequently read by the Fortran programs. From the main menu of our magnetic lens design program, further options are chosen by using function keys or numbers. Some options (lens initialization and setting, fine mesh, current densities, etc.) open other menus where computation parameters can be set or numerical data can be entered with the help of a simple line editor. The "draw lens" option enables graphical editing of the mesh - see fig. I. The geometry of the electron lens is specified in terms of coordinates and indices of a coarse quadrilateral mesh. In this mesh, the fine mesh with smoothly changing step size is calculated by an automeshing procedure. The options shown in fig. 1 allow modification of the number of coarse mesh lines, change of coordinates of mesh points or lines, and specification of lens parts. Interactive and graphical modification of the fine mesh can be called from the fine mesh menu. Finally, the lens computation can be called. Our FEM program allows up to 8000 mesh points on an AT computer. Another menu allows the display of computed results stored in output files and graphical display of axial flux density, flux density in magnetic parts, and the flux lines in magnetic lenses - see fig. 2. A series of several lens excitations with user specified or default magnetization curves can be calculated and displayed in one session.

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Factors in Photographic Emulsions for Low Dose Electron Crystallography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s143192760000101x ◽

1980 ◽

Vol 38 ◽

pp. 230-231

Author(s):

T.W. Jeng ◽

W. Chiu

Keyword(s):

Radiation Damage ◽

Low Dose ◽

Damage Effect ◽

Photographic Emulsion ◽

Electron Crystallography ◽

Electron Microscopic ◽

X Ray ◽

Signal Contrast ◽

Diffraction Patterns ◽

Photographic Emulsions

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).

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A Review of 21 iPad Applications for Augmentative and Alternative Communication Purposes

Perspectives on Augmentative and Alternative Communication ◽

10.1044/aac21.2.60 ◽

2012 ◽

Vol 21 (2) ◽

pp. 60-71 ◽

Cited By ~ 24

Author(s):

Ashley Alliano ◽

Kimberly Herriger ◽

Anthony D. Koutsoftas ◽

Theresa E. Bartolotta

Keyword(s):

Augmentative And Alternative Communication ◽

Cost Effective ◽

Alternative Form ◽

Alternative Communication ◽

Text To Speech ◽

Reference Guide ◽

Expressive Communication ◽

Communication Needs ◽

User Friendly ◽

Ipad Applications

Abstract Using the iPad tablet for Augmentative and Alternative Communication (AAC) purposes can facilitate many communicative needs, is cost-effective, and is socially acceptable. Many individuals with communication difficulties can use iPad applications (apps) to augment communication, provide an alternative form of communication, or target receptive and expressive language goals. In this paper, we will review a collection of iPad apps that can be used to address a variety of receptive and expressive communication needs. Based on recommendations from Gosnell, Costello, and Shane (2011), we describe the features of 21 apps that can serve as a reference guide for speech-language pathologists. We systematically identified 21 apps that use symbols only, symbols and text-to-speech, and text-to-speech only. We provide descriptions of the purpose of each app, along with the following feature descriptions: speech settings, representation, display, feedback features, rate enhancement, access, motor competencies, and cost. In this review, we describe these apps and how individuals with complex communication needs can use them for a variety of communication purposes and to target a variety of treatment goals. We present information in a user-friendly table format that clinicians can use as a reference guide.

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