Three-dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ

Locadiff, an innovative imaging technique based on diffuse waves, has recently been developed in order to image mechanical changes in heterogeneous, geological, or man-made materials. This manuscript reports the on-site application of Locadiff to locate several pre-existing cracks on an aeronautical wind tunnel made of pre-stressed concrete. Using 32 transducers working at ultrasonic frequencies (80–220 kHz) where multiple scattering occurs, we monitor during 15 min an area of 2.5 m×2.5 m of a 35-cm-thick wall. With the wind tunnel in its routine operation, structural changes around the cracks are detected, thanks to their closing or opening due to slight pressure changes. By mapping the density of such microstructure changes in the bulk of the material, locating three pre-existing cracks is properly performed in three dimensions.

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Development of synchrotron X-ray micro-tomography under extreme conditions of pressure and temperature

Journal of Synchrotron Radiation ◽

10.1107/s1600577516016623 ◽

2017 ◽

Vol 24 (1) ◽

pp. 240-247 ◽

Cited By ~ 8

Author(s):

M. Álvarez-Murga ◽

J. P. Perrillat ◽

Y. Le Godec ◽

F. Bergame ◽

J. Philippe ◽

...

Keyword(s):

High Pressure ◽

Structural Changes ◽

Three Dimensional ◽

Crystallographic Structure ◽

Three Dimensional Imaging ◽

X Ray ◽

Wide Range ◽

Micro Tomography ◽

Non Destructive

X-ray tomography is a non-destructive three-dimensional imaging/microanalysis technique selective to a wide range of properties such as density, chemical composition, chemical states and crystallographic structure with extremely high sensitivity and spatial resolution. Here the development of in situ high-pressure high-temperature micro-tomography using a rotating module for the Paris–Edinburgh cell combined with synchrotron radiation is described. By rotating the sample chamber by 360°, the limited angular aperture of ordinary high-pressure cells is surmounted. Such a non-destructive high-resolution probe provides three-dimensional insight on the morphological and structural evolution of crystalline as well as amorphous phases during high pressure and temperature treatment. To demonstrate the potentials of this new experimental technique the compression behavior of a basalt glass is investigated by X-ray absorption tomography, and diffraction/scattering tomography imaging of the structural changes during the polymerization of C60 molecules under pressure is performed. Small size and weight of the loading frame and rotating module means that this apparatus is portable, and can be readily installed on most synchrotron facilities to take advantage of the diversity of three-dimensional imaging techniques available at beamlines. This experimental breakthrough should open new ways for in situ imaging of materials under extreme pressure–temperature–stress conditions, impacting diverse areas in physics, chemistry, geology or materials sciences.

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Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005161x ◽

1974 ◽

Vol 32 ◽

pp. 320-321

Author(s):

J. P. Revel

Keyword(s):

Developmental Stages ◽

Three Dimensional ◽

Cell Movement ◽

Cellular Interactions ◽

Serial Sections ◽

Cell Sheets ◽

Freeze Etching ◽

Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

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In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100083035 ◽

1978 ◽

Vol 36 (2) ◽

pp. 434-435

Author(s):

D. Reis ◽

B. Vian ◽

J. C. Roland

Keyword(s):

Cell Wall ◽

Self Assembly ◽

Plant Cell Wall ◽

Higher Plants ◽

Three Dimensional ◽

Cytochemical Study ◽

Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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In situ investigation of the primary recrystallization of alpha titanium in HVEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100110313 ◽

1978 ◽

Vol 36 (1) ◽

pp. 634-635

Author(s):

S. Naka ◽

R. Penelle ◽

R. Valle

Keyword(s):

Dimensional Analysis ◽

Texture Evolution ◽

Cold Work ◽

Three Dimensional ◽

Primary Recrystallization ◽

Thin Foils ◽

In Situ Investigation ◽

Grain Growth Model ◽

Alpha Titanium

The in situ experimentation technique in HVEM seems to be particularly suitable to clarify the processes involved in recrystallization. The material under investigation was unidirectionally cold-rolled titanium of commercial purity. The problem was approached in two different ways. The three-dimensional analysis of textures was used to describe the texture evolution during the primary recrystallization. Observations of bulk-annealed specimens or thin foils annealed in the microscope were also made in order to provide information concerning the mechanisms involved in the formation of new grains. In contrast to the already published work on titanium, this investigation takes into consideration different values of the cold-work ratio, the temperature and the annealing time.Two different models are commonly used to explain the recrystallization textures i.e. the selective grain growth model (Beck) or the oriented nucleation model (Burgers). The three-dimensional analysis of both the rolling and recrystallization textures was performed to identify the mechanismsl involved in the recrystallization of titanium.

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Detection and mapping of interactions between chromatin and the nuclear envelope in drosophila embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140191 ◽

1995 ◽

Vol 53 ◽

pp. 764-765

Author(s):

W.F. Marshall ◽

A.F. Dernburg ◽

B. Harmon ◽

J.W. Sedat

Keyword(s):

Nuclear Envelope ◽

Chromatin Condensation ◽

Three Dimensional ◽

Physiological Role ◽

Nuclear Surface ◽

Intact Cells ◽

Molecular Determinants ◽

Surface Harmonic ◽

Drosophila Embryos

Interactions between chromatin and nuclear envelope (NE) have been implicated in chromatin condensation, gene regulation, nuclear reassembly, and organization of chromosomes within the nucleus. To further investigate the physiological role played by such interactions, it will be necessary to determine which loci specifically interact with the nuclear envelope. This will not only facilitate identification of the molecular determinants of this interaction, but will also allow manipulation of the pattern of chromatin-NE interactions to probe possible functions. We have developed a microscopic approach to detect and map chromatin-NE interactions inside intact cells.Fluorescence in situ hybridization (FISH) is used to localize specific chromosomal regions within the nucleus of Drosophila embryos and anti-lamin immunofluorescence is used to detect the nuclear envelope. Widefield deconvolution microscopy is then used to obtain a three-dimensional image of the sample (Fig. 1). The nuclear surface is represented by a surface-harmonic expansion (Fig 2). A statistical test for association of the FISH spot with the surface is then performed.

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Application of stereological analysis of cell volume to isolated myocytes in culture with and without adrenergic innervation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010015962x ◽

1990 ◽

Vol 48 (3) ◽

pp. 416-417

Author(s):

Jane K. Rosenthal ◽

Dianne L. Atkins ◽

William J. Marvin ◽

Penny A. Krumm

Keyword(s):

Cardiac Myocytes ◽

Structural Changes ◽

Three Dimensional ◽

Adrenergic Innervation ◽

Culture Cell ◽

Serial Sections ◽

Newborn Rats ◽

Primary Culture Cell ◽

Biological Studies ◽

Cell Volumes

To comprehend structural changes in cardiac myocytes accompanying adrenergic innervation, it is essential that a three dimensional analysis be performed. To date, biological studies which utilize stereological methods have been limited to cells in tissue and in organs. Our laboratory has utilized current stereological techniques for measuring absolute volumes of individual myocytes in primary culture. Cell volumes are calculated for two distinct groups of cells at 96 hours in culture: isolated myocytes and myocytes innervated with adrenergic neurons (Figure 1).Cardiac myocytes are cultured from the ventricular apices of newborn rats. Cells are plated directly onto tissue culture dishes with or without preplated explants from the paravertebral thoracolumbar sympathetic chain. On day four cultures are photographed and marked for one-to-one cell location. Following conventional fixation and embeddment in eponate-12, the cells are relocated and mounted for microtomy. The cells are completely sectioned at 120nm in their parallel orientation to the surface of the dish (Figure 2). Serial sections are collected on formvar coated slotted grids and are recorded in sequence.

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Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163903 ◽

1996 ◽

Vol 54 ◽

pp. 288-289

Author(s):

Greg V. Martin ◽

Ann L. Hubbard

Keyword(s):

Three Dimensional ◽

Integral Membrane Proteins ◽

Polarized Secretion ◽

Microscope Images ◽

Computer Aided ◽

Intracellular Organelles ◽

Cytoskeletal Elements ◽

Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

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Going Beyond 3-D Imaging: Automated 3-D Montaged image Analysis of Cytological Specimens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100163873 ◽

1996 ◽

Vol 54 ◽

pp. 282-283

Author(s):

Badrinath Roysam ◽

Hakan Ancin ◽

Douglas E. Becker ◽

Robert W. Mackin ◽

Matthew M. Chestnut ◽

...

Keyword(s):

Image Analysis ◽

Structural Changes ◽

Sampling Methods ◽

Spatial Clustering ◽

Three Dimensional ◽

Field Of View ◽

Cell Counting ◽

Depth Of Field ◽

Tissue Cell ◽

Tissue Organization

This paper summarizes recent advances made by this group in the automated three-dimensional (3-D) image analysis of cytological specimens that are much thicker than the depth of field, and much wider than the field of view of the microscope. The imaging of thick samples is motivated by the need to sample large volumes of tissue rapidly, make more accurate measurements than possible with 2-D sampling, and also to perform analysis in a manner that preserves the relative locations and 3-D structures of the cells. The motivation to study specimens much wider than the field of view arises when measurements and insights at the tissue, rather than the cell level are needed.The term “analysis” indicates a activities ranging from cell counting, neuron tracing, cell morphometry, measurement of tracers, through characterization of large populations of cells with regard to higher-level tissue organization by detecting patterns such as 3-D spatial clustering, the presence of subpopulations, and their relationships to each other. Of even more interest are changes in these parameters as a function of development, and as a reaction to external stimuli. There is a widespread need to measure structural changes in tissue caused by toxins, physiologic states, biochemicals, aging, development, and electrochemical or physical stimuli. These agents could affect the number of cells per unit volume of tissue, cell volume and shape, and cause structural changes in individual cells, inter-connections, or subtle changes in higher-level tissue architecture. It is important to process large intact volumes of tissue to achieve adequate sampling and sensitivity to subtle changes. It is desirable to perform such studies rapidly, with utmost automation, and at minimal cost. Automated 3-D image analysis methods offer unique advantages and opportunities, without making simplifying assumptions of tissue uniformity, unlike random sampling methods such as stereology.12 Although stereological methods are known to be statistically unbiased, they may not be statistically efficient. Another disadvantage of sampling methods is the lack of full visual confirmation - an attractive feature of image analysis based methods.

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Conformational characterization of nucleosome structure by Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146813 ◽

1993 ◽

Vol 51 ◽

pp. 194-195

Author(s):

Gregory J. Czarnota

Keyword(s):

Electron Microscopy ◽

Structural Changes ◽

Physiological State ◽

Gene Activation ◽

Three Dimensional ◽

Macromolecular Complex ◽

Ultrastructural Studies ◽

Ionic Environment ◽

Nucleosome Structure ◽

Dimensional Reconstruction

Chromatin structure at the fundamental level of the nucleosome is important in vital cellular processes. Recent biochemical and genetic analyses show that nucleosome structure and structural changes are very active participants in gene expression, facilitating or inhibiting transcription and reflecting the physiological state of the cell. Structural states and transitions for this macromolecular complex, composed of DNA wound about a heterotypic octamer of variously modified histone proteins, have been measured by physico-chemical techniques and by enzyme-accessibility and are recognized to occur with various post-translational modifications, gene activation, transformation and with ionic-environment. In spite of studies which indicate various forms of nucleosome structure, all current x-ray and neutron diffraction studies have consistently resulted in only one structure, suggestive of a static conformation. In contrast, two-dimensional electron microscopy studies and three-dimensional reconstruction techniques have yielded different structures. These fundamental differences between EM and other ultrastructural studies have created a long standing quandary, which I have addressed and resolved using spectroscopic electron microscopy and statistical analyses of nucleosome images in a study of nucleosome structure with ionic environment.

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