Improved structural preservation of high-pressure frozen cartilage

1993 ◽  
Vol 51 ◽  
pp. 108-109
Author(s):  
Daniel Studer ◽  
Jeannine Wagner ◽  
Ernst B. Hunziker
Keyword(s):  
Extracellular Matrix ◽  
High Pressure ◽  
High Resolution ◽  
Growth Plate ◽  
High Resolution Imaging ◽  
Chemical Fixation ◽  
Growth Plate Cartilage ◽  
Rat Growth ◽  
Resolution Imaging ◽  
Structural Preservation

Adult cartilage is a unique tissue in that it is avascular and lacks innervation. The chondrocytes are embedded in an extracellular matrix which in most cases occupies 60 to 90% of the tissue volume. The ultrastructure and composition of rat growth plate cartilage has been investigated (1). It was found that during conventional chemical fixation of me tissue (2), about 60% of the extracellular matrix proteoglycans were lost (3). This artefact can be prevented if proteoglycans are precipitated by cationic dyes (eg. ruthenium hexaamine trichloride) during fixation (4) which forms relatively large precipitates by rendering high resolution imaging impossible. High pressure frozen extracellular matrix revealed a fine meshwork which was attributed to proteoglycan distribution (5). A meshwork due to segregation during freezing was not seen in recent experiments where adaequately frozen cartilage was investigated.200μm thick sections of rat growth plate cartilage were excised in a bath of hexadecene (6). The sections were again processed under hexadecene, firstly punched (diameter 1.7mm) and then transferred to an aluminum sandwhich (corresponding in size to the sample).

scholarly journals Quantitative high-resolution imaging of live microbial cells at high hydrostatic pressure

2019 ◽  
Author(s):  
A. C. Bourges ◽  
A. Lazarev ◽  
N. Declerck ◽  
K. L. Rogers ◽  
C. A. Royer
Keyword(s):  
High Pressure ◽  
High Resolution ◽  
Microbial Biomass ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Great Promise ◽  
Physical Parameters ◽  
High Resolution Imaging ◽  
Microbial Cells ◽  
Resolution Imaging

ABSTRACTThe majority of the Earth’s microbial biomass exists in the Deep Biosphere, in the deep ocean and within the Earth’s crust. While other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high-pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold pressure on the Earth’s surface. The mechanisms of pressure adaptation are also important to in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here we describe implementation of a high-pressure sample chamber with a 2-photon scanning microscope system allowing for the first time, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We adapted this setup for Fluorescence Lifetime Imaging Microscopy with Phasor analysis (FLIM/Phasor) and investigated metabolic responses to pressure of live cells from mesophilic yeast and bacterial strains, as well as the piezophilic archaeon, Archaeoglobus fulgidus. We also monitored by fluorescence intensity fluctuation-based methods (scanning Number and Brightness (sN&B) and Raster scanning Imaging Correlation Spectroscopy (RICS)) the effect of pressure on the chromosome-associated protein HU and on the ParB partition protein in E. coli, revealing partially reversible dissociation of ParB foci and concomitant nucleoid condensation.SIGNIFICANCEThe majority of the Earth’s microbial biomass exists in high-pressure environments where pressures can reach over 100 MPa. The molecular mechanisms that allow microbes to flourish under such extreme conditions remain to be discovered. The high pressure, high resolution imaging system presented here revealed pressure dependent changes in metabolism and protein interactions in live microbial cells, demonstrating great promise for understanding deep life.

ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix

2017 ◽  
Vol 23 (7) ◽  
pp. 890-898 ◽  
Author(s):  
Alejandro E Mayorca-Guiliani ◽  
Chris D Madsen ◽  
Thomas R Cox ◽  
Edward R Horton ◽  
Freja A Venning ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
High Resolution ◽  
Proteomic Analysis ◽  
High Resolution Imaging ◽  
Resolution Imaging

scholarly journals Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing

2021 ◽  
Author(s):  
Felix J.B. Baeuerlein ◽  
Jose C. Pastor-Pareja ◽  
Ruben Fernandez-Busnadiego
Keyword(s):  
High Pressure ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
High Resolution Imaging ◽  
Molecular Architecture ◽  
High Pressure Freezing ◽  
Cryo Electron Tomography ◽  
Resolution Imaging

Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to tissues, as they usually require high-pressure freezing for vitrification. Here we show that dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of native tissues.

scholarly journals Freeze-substitution staining of rat growth plate cartilage with alcian blue for electron microscopic study of proteoglycans.

1989 ◽  
Vol 37 (3) ◽  
pp. 383-387 ◽  
Author(s):  
M E Maitland ◽  
A L Arsenault
Keyword(s):  
Extracellular Matrix ◽  
Growth Plate ◽  
Alcian Blue ◽  
Freeze Substitution ◽  
Electron Microscopic Examination ◽  
Visual Interpretation ◽  
Electron Microscopic ◽  
Growth Plate Cartilage ◽  
Rat Growth ◽  
Cell Processes

To selectively stain polyanionic macromolecules of growth plate cartilage and to prevent artifacts induced by aqueous fixation, proximal tibial growth plates were excised from rats, slam-frozen, and freeze-substituted in 100% methanol containing the cationic dye Alcian blue. Electron microscopic examination showed the tissue stained with Alcian blue to be comparable in ultrastructural preservation to tissues slam-frozen and freeze-substituted in the absence of Alcian blue. The extracellular matrix exhibited a characteristic staining pattern when stained by this method. The pericellular rim was identified as a band of varying width encircling the chondrocyte and its cell processes. Peripheral to the pericellular rim the heterogeneity of staining within the extracellular matrix increased, taking the form of polymorphic densities. X-ray microanalysis showed that the visual interpretation of electron density was related to the concentration of copper present, and that the concentration of sulfur was variable in the pericellular rim and in the interterritorial matrix. The difficulties associated with aqueous fixation and staining procedures are discussed in contrast to the improved preservation achieved by cryogenic methods.

Alternative approaches to ultra-high resolution imaging

1991 ◽  
Vol 49 ◽  
pp. 650-651
Author(s):  
J.M. Cowley
Keyword(s):  
High Resolution ◽  
High Voltage ◽  
High Resolution Electron Microscopy ◽  
Crystal Defects ◽  
High Resolution Imaging ◽  
General Applicability ◽  
Resolution Electron ◽  
Radiation Resistant ◽  
Resolution Imaging ◽  
Alternative Approaches

By extrapolation of past experience, it would seem that the future of ultra-high resolution electron microscopy rests with the advances of electron optical engineering that are improving the instrumental stability of high voltage microscopes to achieve the theoretical resolutions of 1Å or better at 1MeV or higher energies. While these high voltage instruments will undoubtedly produce valuable results on chosen specimens, their general applicability has been questioned on the basis of the excessive radiation damage effects which may significantly modify the detailed structures of crystal defects within even the most radiation resistant materials in a period of a few seconds. Other considerations such as those of cost and convenience of use add to the inducement to consider seriously the possibilities for alternative approaches to the achievement of comparable resolutions.

Principle and practice of high-resolution imaging with a field-emission TEM

1992 ◽  
Vol 50 (1) ◽  
pp. 138-139
Author(s):  
Max T. Otten ◽  
Wim M.J. Coene
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Incidence Angle ◽  
Temporal Coherence ◽  
Energy Spread ◽  
High Resolution Imaging ◽  
Major Improvement ◽  
High Resolution Images ◽  
Resolution Imaging

High-resolution imaging with a LaB6 instrument is limited by the spatial and temporal coherence, with little contrast remaining beyond the point resolution. A Field Emission Gun (FEG) reduces the incidence angle by a factor 5 to 10 and the energy spread by 2 to 3. Since the incidence angle is the dominant limitation for LaB6 the FEG provides a major improvement in contrast transfer, reducing the information limit to roughly one half of the point resolution. The strong improvement, predicted from high-resolution theory, can be seen readily in diffractograms (Fig. 1) and high-resolution images (Fig. 2). Even if the information in the image is limited deliberately to the point resolution by using an objective aperture, the improved contrast transfer close to the point resolution (Fig. 1) is already worthwhile.

Imaging of ferroelectric domain walls by electron holography

1992 ◽  
Vol 50 (1) ◽  
pp. 246-247
Author(s):  
Xiao Zhang
Keyword(s):  
Magnetic Field ◽  
High Resolution ◽  
Domain Walls ◽  
Ferroelectric Domain ◽  
Object Wave ◽  
Electron Holography ◽  
High Resolution Imaging ◽  
Quantitative Measurements ◽  
Resolution Imaging ◽  
Electron Microscopes

Electron holography has recently been available to modern electron microscopy labs with the development of field emission electron microscopes. The unique advantage of recording both amplitude and phase of the object wave makes electron holography a effective tool to study electron optical phase objects. The visibility of the phase shifts of the object wave makes it possible to directly image the distributions of an electric or a magnetic field at high resolution. This work presents preliminary results of first high resolution imaging of ferroelectric domain walls by electron holography in BaTiO3 and quantitative measurements of electrostatic field distribution across domain walls.

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

Nano-probe x-ray analysis and high-resolution imaging on planar defects in high-pressure synthesized infinite-layer superconductor

1994 ◽  
Vol 52 ◽  
pp. 728-729
Author(s):  
Y. Y. Wang ◽  
H. Zhang ◽  
V. P. Dravid ◽  
H. Zhang ◽  
L. D. Marks ◽  
...  
Keyword(s):  
High Pressure ◽  
High Resolution ◽  
Atomic Structure ◽  
Emission Spectra ◽  
High Resolution Electron Microscopy ◽  
Planar Defects ◽  
X Ray ◽  
Infinite Layer ◽  
Resolution Electron ◽  
Resolution Imaging

Azuma et al. observed planar defects in a high pressure synthesized infinitelayer compound (i.e. ACuO2 (A=cation)), which exhibits superconductivity at ~110 K. It was proposed that the defects are cation deficient and that the superconductivity in this material is related to the planar defects. In this report, we present quantitative analysis of the planar defects utilizing nanometer probe xray microanalysis, high resolution electron microscopy, and image simulation to determine the chemical composition and atomic structure of the planar defects. We propose an atomic structure model for the planar defects.Infinite-layer samples with the nominal chemical formula, (Sr1-xCax)yCuO2 (x=0.3; y=0.9,1.0,1.1), were prepared using solid state synthesized low pressure forms of (Sr1-xCax)CuO2 with additions of CuO or (Sr1-xCax)2CuO3, followed by a high pressure treatment.Quantitative x-ray microanalysis, with a 1 nm probe, was performed using a cold field emission gun TEM (Hitachi HF-2000) equipped with an Oxford Pentafet thin-window x-ray detector. The probe was positioned on the planar defects, which has a 0.74 nm width, and x-ray emission spectra from the defects were compared with those obtained from vicinity regions.

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