Calibration methods for quantitative image processing in electron microscopy

Lamvik, M. K.; Davilla, S. D.

doi:10.1002/jemt.1060110202

Cited by 4 publications

(2 citation statements)

References 21 publications

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“…Subsequently, Lamvik and Davilla (1989) showed that path length through standard particles, such as polystyrene spheres of known constant density, could be used to confirm directly the accuracy of the mass density measurements in images of particles recorded on film. By integrating intensities in the digitized film along different path lengths through the spheres, the mass could be determined, as long as the film density was within the linear intensity response region.…”

Section: Introductionmentioning

confidence: 99%

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

Costello

Johnsen

Metlapally

et al. 2010

Experimental Eye Research

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The goal of this project was to determine the relative refractive index (RI) of the interior of multilamellar bodies (MLBs) compared to the adjacent cytoplasm within human nuclear fiber cells. MLBs have been characterized previously as 1-4 μm diameter spherical particles covered by multiple lipid bilayers surrounding a cytoplasmic core of variable density. Age-related nuclear cataracts have more MLBs than transparent donor lenses and were predicted to have high forward scattering according to Mie scattering theory, assuming different RIs for the MLB and cytoplasm. In this study quantitative values of relative RI were determined from specific MLBs in electron micrographs of thin sections and used to calculate new Mie scattering plots. Fresh lenses were Vibratome sectioned, immersion fixed and en bloc stained with osmium tetroxide and uranyl acetate, or uranyl acetate alone, prior to dehydration and embedding in epoxy or acrylic resins. Thin sections 70 nm thick were cut on a diamond knife and imaged without grid stains at 60 kV using a CCD camera on a transmission electron microscope (TEM). Integrated intensities in digital electron micrographs were related directly to protein density, which is linearly related to RI for a given substance. The RI of the MLB interior was calculated assuming an RI value of 1.42 for the cytoplasm from the literature. Calculated RI values for MLBs ranged from 1.35 to 1.53. Thus, some MLBs appeared to have interior protein densities similar to or less than the adjacent cytoplasm whereas others had significantly higher densities. The higher density MLBs occurred preferentially in older and more advanced cataracts suggesting a maturation process. The bilayer coats were more often observed in MLBs from transparent donors and early stage cataracts indicating that bilayer loss was part of the MLB maturation, producing large low-density spaces around dense MLB cores. These spaces were frequently observed in advanced cataracts from India as large low-density crescents and annular rings. Predicted scattering from Mie plots using particles with dense cores and low-density rims was higher than reported previously, although the most important factor was the relative RI , not the MLB coat or lack thereof. In conclusion, the Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript measurements confirm the high protein density and RI of some MLB interiors compared to adjacent cytoplasm. This high RI ratio used in the Mie calculations suggests that for 2 000 MLBs/ mm 3...

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Section: Introductionmentioning

confidence: 99%

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

Costello

Johnsen

Metlapally

et al. 2010

Experimental Eye Research

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“…The pixel values were either reversed (black to white and vice versa) or adjusted for maximum clarity and a smooth filtering function applied. These and other image processing algorithms and functions are very valuable means of removing noise and dirt ithat usually find their way into images (Fermin et aL, 1992;Hawkes, 1988;Lamvik and Davilla, 1989). The processed image was "dumped directly from the video screen to the Polaroid video recorder and printed on color paper.…”

Section: Image Processingmentioning

confidence: 99%

High resolution and image processing of otoconia matrix

Fermin

1993

Microscopy Res & Technique

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This study was designed to investigate patterns of fibrils organization in histochemically stained otoconia. Transmission electron microscope and video imaging were used. These data indicate that otoconia of the chick (Gallus domesticus) inner ear may have central cores in vivo. The data also show that the ultrastructural organization of fibrils fixed with aldehydes and histochemical stains follows trajectories that conform to the hexagonal shape of otoconia. These changes in direction may contribute to the formation of a central core. The existence of central cores is important for the in vivo buoyancy of otoconia. Packing of fibrils is tighter after phosphotungstic acid (PTA) stained otoconia than with other histochemical stains, which usually produce looser packing of fibrils and seemingly larger central core. TEM of tilted and untilted material showed that turning of fibrils occurs at the points where the face angles of otoconia form and where central cores exist. Video image processing of the images allowed reconstructing a template which, if assumed to repeat and change trajectories, would fit the pattern of fibrils seen in fixed otoconia. Since it is highly unlikely that aldehyde primary fixation or PTA stain caused such drastic change in the direction of fibrils, the template derived from these results may closely approximate patterns of otoconia fibrils packing in vivo. However, if the above is correct, the perfect crystallographic diffraction pattern of unfixed otoconia do not correspond to patterns of fixed fibrils.

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Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography

Shi

Hsiung

Pace

et al. 1995

Biophysical Journal

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Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals.

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Calibration methods for quantitative image processing in electron microscopy

Cited by 4 publications

References 21 publications

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

Multilamellar spherical particles as potential sources of excessive light scattering in human age-related nuclear cataracts

High resolution and image processing of otoconia matrix

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography

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