Three-dimensional reconstruction of large subcellular structures: A method for combining axial tilt tomography with serial reconstruction of thick sections

1992 ◽  
Vol 50 (1) ◽  
pp. 904-905
Author(s):  
Gabriel E. Soto ◽  
Maryann E. Martone ◽  
Stephan Lamont ◽  
Bridget O. Carragher ◽  
Thomas J. Deerinck ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
3 Dimensional ◽  
Voltage Electron ◽  
Large Numbers ◽  
Dimensional Reconstruction ◽  
Axial Tilt

The study of subcellular structures requires the resolution afforded by the electron microscope. However, cellular organelle systems can extend for tens of microns and therefore cannot be encompassed in a single thin section required for conventional electron microscopic observation. Even with the use of high voltage electron microscopy, section thickness is limited to no more than a few microns. Visualization of 3-dimensional cellular structure in large volumes of tissue can be achieved by using 3-dimensional reconstructions based on serial sections. This approach is often tedious, requiring an extremely large series of thin sections in order to encompass the structure of interest. This method also suffers from technical difficulties in obtaining, processing and maintaining adequate registration over large numbers of sections. We have been exploring a method in which the number of sections is reduced by employing a series of thick sections in which the structures of interest are selectively stained. Three-dimensional information is extracted from each section using axial tilt tomography. The resulting serial volumes are then aligned and linked to form a single volume which is displayed using volume rendering techniques.

Three-Dimensional Analysis of Tranverse Tubules in Normal and Failing Heart: A Combined Confocal and High Voltage Electron Microscope Study

1997 ◽  
Vol 3 (S2) ◽  
pp. 231-232
Author(s):  
M. E. Martone ◽  
V. M. Edelman ◽  
A. Thor ◽  
S. J. Young ◽  
S. P. Lamont ◽  
...  
Keyword(s):  
High Voltage ◽  
Three Dimensional ◽  
Cardiac Cells ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Spontaneously Hypertensive ◽  
3 Dimensional ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
T Tubules

Early electron microscopic studies documented that significant changes in the membrane systems of cardiac cells occur in both ischemic and non-ischemic heart failure. These studies relied on analysis of two-dimensional sections and although quantitative changes were observed, the overall organization of the tranverse tubules (T-tubules) and the sarcoplasmic reticulum could not be assessed. In a 3-dimensional study using high voltage electron microscopy (EM) of the T-tubules in spontaneously hypertensive rats, Nakamura and Hama (1991) observed that concomitant with an increase in surface area, the T-tubule system becomes progressively more disorganized and exhibits structural irregularities such as increased numbers of longitudinal tubules, numerous short dead end branches and complex tubular aggregates. These authors suggested that this disorganization may interfere with synchronous contraction over the entire cell.In the present study, we examined the 3-dimensional organization of T-tubules in the left ventricle of explanted human hearts using confocal microscopy and EM tomography.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

High-voltage electron microscopic studies of inflammatory cell attachment during blood-brain barrier inflammation

1990 ◽  
Vol 48 (3) ◽  
pp. 276-277
Author(s):  
A.S. Lossinsky ◽  
M.J. Song
Keyword(s):  
High Voltage ◽  
Inflammatory Cell ◽  
Cell Attachment ◽  
Electron Microscopic ◽  
Vascular Perfusion ◽  
Tissue Samples ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron

Previous studies have suggested the usefulness of high-voltage electron microscopy (HVEM) for investigating blood-bram barrier (BBB) injury and the mechanism of inflammatory-cell (IC) attachment. These studies indicated that, in evaluating standard conventional thin sections, one might miss cellular attachment sites of ICs in their process of attaching to the luminal endothelial cell (EC) surface of cerebral blood vessels. Our current studies in animals subjected to autoimmune disease suggest that HVEM may be useful in localizing precise receptor sites involved in early IC attachment.Experimental autoimmune encephalomyelitis (EAE) was induced in mice and rats according to standard procedures. Tissue samples from cerebellum, thalamus or spinal cords were embedded in plastic following vascular perfusion with buffered aldehyde. Thick (0.5-0.7 μm) sections were cut on glass knives and collected on Formvar-coated slot grids stained with uranylacetate and lead citrate and examined with the AEI EM7 1.2 MV HVEM in Albany, NY at 1000 kV.

scholarly journals The chondriome of selected trypanosomatids. A three-dimensional study based on serial thick sections and high voltage electron microscopy.

1975 ◽  
Vol 66 (2) ◽  
pp. 404-413 ◽  
Author(s):  
J J Paulin
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Flagellar Pocket ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
Trypomastigote Form ◽  
High Voltage Electron ◽  
Three Dimensional Models

The unitary nature of the chondriome of two species of trypanosomatids, Blastocrithidia culicis and Trypanosoma cruzi, has been demonstrated by utilizing serial thick-sectioning techniques combined with high voltage electron microscopy. Profiles of mitochondrial elements seen in thin sections and suspected to be parts of a continuum were confirmed by serial thick sectioning (0.25-0.50 mum thick) and stereopair analysis to be parts of the same mitochondrion. Three-dimensional models obtained from tracings of mitochondrial profiles on cellulose acetate reveal the mitochondrion of B. culicis to consist of a posterior mass with six tubular extensions extending upward and terminating in the anterior apex. The kinetoplast was found suspended between two of the tubular extensions, or less frequently, protuding as a nodule from one of the extensions. A bifurcation of one of the extensions was found in some specimens. The mitochondrion of T. cruzi consists of a triangular-shaped convoluted tubule, the base being the kinetoplast portion while the apex is directed posteriorly. The mitochondrion bifurcates behind the flagellar pocket, lateral to the kinetoplast, sending two entwined extensions into the tenuous anterior apex. Whether the mitochondrion of T. cruzi is unitary in the trypomastigote form was not determined in this study, since only epimastigote forms were used.

The application of three-dimensional tomographic reconstruction methods to high-voltage electron microscopy

1987 ◽  
Vol 45 ◽  
pp. 570-573
Author(s):  
B. F. McEwen ◽  
C. L. Rieder ◽  
M. Radermacher ◽  
R. A. Grassucci ◽  
J. N. Turner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Depth Of Field ◽  
Specimen Thickness ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Reconstruction Methods

High-voltage electron microscopy (HVEM) has considerably increased the thickness limit of biological specimens that can be visualized at high resolution. Because of its increased penetration power, HVEM is potentially the most powerful tool available for obtaining three-dimensional (3D) information concerning the structure of cells. In the past, such information was primarily obtained from serial thin sections or techniques based on surface shadowing, but these methods have severe problems and limitations which can only be overcome by imaging greater depths in the samples (see refs. 1 and 2). HVEM has yet to realize its potential for 3D structural determination because of the confusion arising from the overlap of features at different depths in the sample. Due to the relatively large depth of field, which exceeds the specimen thickness, HVEM (like all electron microscopy) produces an image that is essentially a projection of the sample.

Ultrastructural three-dimensional reconstruction of a T lymphocyte

1992 ◽  
Vol 50 (1) ◽  
pp. 592-593
Author(s):  
Karen K. Bovard ◽  
Joseph N. Marcus
Keyword(s):  
Electron Microscopy ◽  
T Lymphocyte ◽  
High Voltage ◽  
Three Dimensional ◽  
High Voltage Electron Microscopy ◽  
Analysis Techniques ◽  
Nucleolar Organizing Regions ◽  
Voltage Electron ◽  
Dimensional Reconstruction ◽  
Limited Depth

There have been many electron microscopy studies of normal and neoplastic lymphocyte microanatomy. However, only a few use three-dimensional analysis techniques to visualize organelle relationships, such as nuclear shape and chromatin distribution, nucleolar organizing regions, and, by high voltage electron microscopy, the microtubular networks over a limited depth though the cell. To date, there has been no three-dimensional serial reconstuction of a T Lymphocyte and its organelles.

Three-dimensional imaging and physiology of live neurons and glia: Confocal light and correlative high-voltage Electron Microscopy

1993 ◽  
Vol 51 ◽  
pp. 162-163
Author(s):  
J.N. Turner ◽  
D.H. Szarowski ◽  
W. Shain ◽  
M. Davis-Cox ◽  
D.O. Carpenter ◽  
...  
Keyword(s):  
Large Scale ◽  
Culture Media ◽  
Shape Change ◽  
Complex Function ◽  
3D Structure ◽  
Three Dimensional ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
And Function

Correlating physiologic measures with three-dimensional (3D) imaging at the light and electron microscopic levels is a powerful combination of methods for studying the structure and function of biological systems. Neurobiology is an ideal field for the application of these methods because neurons and glia have complex and extensive 3D structure, and their physiology is under intense study. Neurons, such as those studied here from Aplysia, can be more than 100 μm in diameter, and glia undergo large scale 3D shape change as a function of a number of physiologic parameters. The ability to accurately quantitate the 3D structure, volume and surface area of live neurons and glia is important to our understanding of the complex function of these cells.Neurons were isolated from the major ganglia of juvenile Aplysia Californica and glia were obtained from long term cultures of LRM 55 cells or as primary isolates from rats. Cultures were exposed to Dil dissolved in DMSO with or without 20% Pluronic F-127 and added to the culture media. The imaging instrument was an Olympus IMT-2 and a Bio-Rad MRC-600.

Tomographic methods for detailed examination of large structures in the nervous system

1993 ◽  
Vol 51 ◽  
pp. 96-97
Author(s):  
Gabriel E. Soto ◽  
Stephen J. Young ◽  
Maryann E. Martone ◽  
Thomas J. Deerinck ◽  
Stephan Lamont ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Detailed Examination ◽  
Thick Section ◽  
Section Thickness ◽  
Serial Sectioning ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Large Structures ◽  
Electron Microscopes ◽  
Axial Tilt

One of the limitations of electron microscopy has been the requirement for very thin samples to allow penetration of the electron beam. It is often the case that structures of interest are not contained within a single thin section. In these cases, serial sectioning techniques are required to reconstruct the object in its entirety. The use of higher voltage electron microscopes has allowed researchers to examine specimens up to fifty times thicker than those suitable for a conventional TEM. However, images from thick sections are often difficult to interpret as the electron micrograph is essentially a projection of the overlapping material within the section. The method of computerized axial tilt electron microscopic tomography offers the potential to visualize and analyze information contained in a thick section by deriving a three dimensional volume from a series of projections acquired by collecting images of the specimen at successive tilt increments about the Y axis. Unfortunately there are practical limitations to the resolution that can be obtained using this technique with very thick sections. Resolution of the tomogram increases with finer tilt sampling and an increased range of tilts but decreases with section thickness.

Sign in / Sign up

Export Citation Format

Share Document