scholarly journals Three dimensional multi-molecule tracking in thick samples with extended depth-of-field

2015 ◽  
Vol 23 (2) ◽  
pp. 787 ◽  
Author(s):  
Heng Li ◽  
Danni Chen ◽  
Gaixia Xu ◽  
Bin Yu ◽  
Hanben Niu
1999 ◽  
Vol 24 (4) ◽  
pp. 253 ◽  
Author(s):  
Daniel L. Marks ◽  
Ronald A. Stack ◽  
David J. Brady ◽  
Joseph van der Gracht

2018 ◽  
Vol 42 (3) ◽  
pp. 377-384 ◽  
Author(s):  
S. N. Koreshev ◽  
D. S. Smorodinov ◽  
O. V. Nikanorov ◽  
M. A. Frolova

A quantitative evaluation of the depth of field of optical systems is given. Results of the calculation of the distribution of the complex amplitude and intensity in a three-dimensional scattering pattern formed by the optical system for an on-axis point object are presented. The work was carried out as part of developing optical systems with an extended depth of field for a synthesized hologram of a point object located on a perpendicular constructed to the hologram center.


2012 ◽  
Vol 95 (11) ◽  
pp. 14-27 ◽  
Author(s):  
Keiichiro Kagawa ◽  
Kenji Yamada ◽  
Eiji Tanaka ◽  
Jun Tanida

Author(s):  
A.M. Jones ◽  
A. Max Fiskin

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.


Author(s):  
Jaap Brink ◽  
Wah Chiu

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.


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