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.

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.

scholarly journals Using AlphaFold to predict the impact of single mutations on protein stability and function

2021 ◽  
Author(s):  
Marina A Pak ◽  
Karina A Markhieva ◽  
Mariia S Novikova ◽  
Dmitry S Petrov ◽  
Ilya S Vorobyev ◽  
...  
Keyword(s):  
Protein Folding ◽  
Protein Stability ◽  
Structure Prediction ◽  
Large Scale ◽  
3D Structure ◽  
Three Dimensional ◽  
Single Mutation ◽  
Before And After ◽  
And Function ◽  
The Impact

AlphaFold changed the field of structural biology by achieving three-dimensional (3D) structure prediction from protein sequence at experimental quality. The astounding success even led to claims that the protein folding problem is "solved". However, protein folding problem is more than just structure prediction from sequence. Presently, it is unknown if the AlphaFold-triggered revolution could help to solve other problems related to protein folding. Here we assay the ability of AlphaFold to predict the impact of single mutations on protein stability (ΔΔG) and function. To study the question we extracted metrics from AlphaFold predictions before and after single mutation in a protein and correlated the predicted change with the experimentally known ΔΔG values. Additionally, we correlated the AlphaFold predictions on the impact of a single mutation on structure with a large scale dataset of single mutations in GFP with the experimentally assayed levels of fluorescence. We found a very weak or no correlation between AlphaFold output metrics and change of protein stability or fluorescence. Our results imply that AlphaFold cannot be immediately applied to other problems or applications in protein folding.

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.

An Integrated Biological Imaging Facility: Capabilities of the Biological Microscopy and Image Reconstruction Resource

1997 ◽  
Vol 3 (S2) ◽  
pp. 271-272
Author(s):  
J. Frank ◽  
C.A. Mannella ◽  
C. Rieder
Keyword(s):  
Electron Microscopy ◽  
Image Reconstruction ◽  
3D Structure ◽  
Biological Imaging ◽  
Cell Manipulation ◽  
Projection Data ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Current Area

The Biological Microscopy and Image Reconstruction Resource (BMIRR) is operated by the Wadsworth Center as a national biotechnology resource, with funding through the NIH Center for Research Resources and from NSF. This biological imaging resource has evolved continuously over the past two decades. Early development focussed on correlative, same-cell light and electron microscopic techniques, combining the capabilities of video-enhanced light microscopy and high-voltage electron microscopy. A current area of development is electron microscopic tomography, whereby the full 3D capabilities of higher voltage (400-1200 KV) electron microscopy is brought to bear on biological problems. In particular, the recent development of techniques for merging projection data from two mutually perpendicular tilt series has permitted significantly improved resolution, reducing the missing wedge of information to a missing pyramid. Attention is now turning to optimization of conditions for applying tomography to frozen-hydrated specimens, using automated data collection on our cryo-IVEM. Combined with parallel advances in same-cell manipulation and viewing, the BMIRR provides biologists with a unique combination of imaging and computational tools for research into the 3D structure and dynamics that underly cellular processes (see figures and refs. 2-4).

Three-Dimensional Visualization of the T-System of Frog Muscle Using High Voltage Electron Microscopy and a Lanthanum Stain

1977 ◽  
Vol 35 ◽  
pp. 570-571 ◽  
Author(s):  
Lee D. Peachey ◽  
Clara Franzini-Armstrong
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Biological Tissues ◽  
High Voltage Electron Microscopy ◽  
Transverse Tubular System ◽  
Peroxidase Reaction ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
T System

The effective study of biological tissues in thick slices of embedded material by high voltage electron microscopy (HVEM) requires highly selective staining of those structures to be visualized so that they are not hidden or obscured by other structures in the image. A tilt pair of micrographs with subsequent stereoscopic viewing can be an important aid in three-dimensional visualization of these images, once an appropriate stain has been found. The peroxidase reaction has been used for this purpose in visualizing the T-system (transverse tubular system) of frog skeletal muscle by HVEM (1). We have found infiltration with lanthanum hydroxide to be particularly useful for three-dimensional visualization of certain aspects of the structure of the T- system in skeletal muscles of the frog. Specifically, lanthanum more completely fills the lumen of the tubules and is denser than the peroxidase reaction product.

High-voltage electron microscopy of maxillary gland of spirochete-infected brine shrimp: lesion of efferent tubule

1988 ◽  
Vol 46 ◽  
pp. 362-363
Author(s):  
G. E. Tyson ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Brine Shrimp ◽  
Natural Populations ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Infected Adult

Natural populations of the brine shrimp, Artemia, may possess spirochete- infected animals in low numbers. The ultrastructure of Artemia's spirochete has been described by conventional transmission electron microscopy. In infected shrimp, spirochetal cells were abundant in the blood and also occurred intra- and extracellularly in the three organs examined, i.e. the maxillary gland (segmental excretory organ), the integument, and certain muscles The efferent-tubule region of the maxillary gland possessed a distinctive lesion comprised of a group of spirochetes, together with numerous small vesicles, situated in a cave-like indentation of the base of the tubule epithelium. in some instances the basal lamina at a lesion site was clearly discontinuous. High-voltage electron microscopy has now been used to study lesions of the efferent tubule, with the aim of understanding better their three-dimensional structure.Tissue from one maxillary gland of an infected, adult, female brine shrimp was used for HVEM study.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

Scanning electron microscopic study of collagen fibrillogenesis and extracellular compartmentalization in the chick embryo tendon

1986 ◽  
Vol 44 ◽  
pp. 208-209
Author(s):  
Grace C.H. Yang
Keyword(s):  
Chick Embryo ◽  
Extracellular Space ◽  
Fibroblast Cell ◽  
Collagen Fibrils ◽  
Electron Microscopic ◽  
Voltage Electron ◽  
Scanning Electron Microscopic Study ◽  
Microscopic Studies ◽  
And Function

The size and organization of collagen fibrils in the extracellular matrix is an important determinant of tissue structure and function. The synthesis and deposition of collagen involves multiple steps which begin within the cell and continue in the extracellular space. High-voltage electron microscopic studies of the chick embryo cornea and tendon suggested that the extracellular space is compartmentalized by the fibroblasts for the regulation of collagen fibril, bundle, and tissue specific macroaggregate formation. The purpose of this study is to gather direct evidence regarding the association of the fibroblast cell surface with newly formed collagen fibrils, and to define the role of the fibroblast in the control and the precise positioning of collagen fibrils, bundles, and macroaggregates during chick tendon development.

High-voltage cytochemistry for three-dimensional observation of nuclei and calculation of total numbers of nuclear pores in retinal Mueller glia

1990 ◽  
Vol 48 (3) ◽  
pp. 956-957
Author(s):  
H. Ishigooka ◽  
S. Ueno ◽  
L.M. Hjelmeland ◽  
M.B. Landers ◽  
K. Ogawa
Keyword(s):  
Nuclear Envelope ◽  
High Voltage ◽  
Three Dimensional ◽  
Reaction Medium ◽  
Nuclear Pores ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
G6pase Activity ◽  
Mueller Glia

Introduction: We have demonstrated that Glucose-6-phosphatase (G6Pase) activity is localized to the endoplasmic reticulum and nuclear envelope of Mueller glia in the normal and pathological guinea pig retina. Using a combination of this cytochemical technique and high voltage electron microscopy, the distribution of nuclear pores could be clearly observed on the nuclear envelope of Mueller glia because of their anatomical lack of reaction products. This technique was developed to study the three-dimensional structure of nuclei and to calculate total numbers of nuclear pores utilizing a computer graphic analysis system in the normal and pathological retina.Materials and methods: Normal and photocoagulated retina of pigmented adult guinea pigs were perfused with a cold mixture of 0.25% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, and the enucleated globes were hemisected and immersed in the same fixative for 30 min. After sectioning and incubation in the reaction medium for the detection of G6Pase activity by the method of Wachstein-Meisel, the sections were postfixed, dehydrated and embedded in Spurr’s epoxy resin. Serial thick sections (1.0um) were prepared for the observation by a Hitachi high voltage electron microscope (H 1250-M) with an accelerating voltage of 1000 Kv. and pictures were analyzed and three-dimensionally reconstructed by TRI (RATOC Co., Ltd.).

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