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Molecular Biology of the Cell Feb 2014Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important...
Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.
Topics: Animals; Cell Biology; Cells; Fixatives; Humans; Microscopy, Electron, Transmission; Microtomy; Osmium Tetroxide; Specimen Handling; Staining and Labeling
PubMed: 24482357
DOI: 10.1091/mbc.E12-12-0863 -
Journal of Radiation Research Mar 1992A variety of techniques have been developed to detect single-base changes for the two different purposes. One is the detection of mutational events without phenotypic... (Review)
Review
A variety of techniques have been developed to detect single-base changes for the two different purposes. One is the detection of mutational events without phenotypic selection, and another is the rapid and conventional identification of mutations such as the specific base changes related to activation of oncogene, genetic diseases, etc. In this study, the utility of the two methods, single strand conformation polymorphism (SSCP) and chemical cleavage, was explored using 13 E. coli lacI- mutations cloned onto M13 phase. The 167 base region encompassing mutations was amplified by PCR as dsDNA. Following denaturation, these PCR products were analyzed by non-denaturing polyacrylamide gel electrophoresis (SSCP) and the separation of the ssDNA fragment carrying the altered sequence from the original sequence was found to be dependent on the location and type of the change. Hetero duplexes of changed/original sequences were also prepared by hybridization of the above PCR products. Mismatched C and T bases were modified by hydroxylamine and osmium tetroxide, respectively, and subsequently treated with piperidine to analyze the cleaved DNA fragments on a polyacrylamide gel (Chemical Cleavage). The cleavage efficiency was also found to be influenced by the type of mismatch and its surrounding sequence. Such observed characteristics should contribute to a better appreciation for these types of mutational systems, which in turn should lead to insight into the mechanisms of mutagenesis.
Topics: Base Sequence; DNA, Bacterial; Escherichia coli; Molecular Sequence Data; Mutation; Polymorphism, Genetic
PubMed: 1507183
DOI: 10.1269/jrr.33.supplement_95 -
Annals of Medicine and Surgery (2012) Oct 2021Electron microscopy is a powerful tool to study biological samples at higher magnification. The higher magnifications achieved by the electron microscopes are helpful to...
Electron microscopy is a powerful tool to study biological samples at higher magnification. The higher magnifications achieved by the electron microscopes are helpful to the researchers to study surface morphology as well as cellular morphology of the samples. The blood sample surface morphology can be visualized at higher magnification by scanning electron microscope (SEM). For the examination of the blood cells at the cellular level, transmission electron microscopes (TEM) are used. In this article, we have described the step-by-step standard protocol for the preparation of blood samples for electron microscopy. The prepared blood samples can be visualized under SEM and TEM. The obtained electron micrographs of blood cells can be used for differential diagnosis of various diseases at the cellular level.
PubMed: 34691432
DOI: 10.1016/j.amsu.2021.102895 -
The Journal of Comparative Neurology Aug 2012Synaptic function depends on interactions among sets of proteins that assemble into complex supramolecular machines. Molecular biology, electrophysiology, and live-cell...
Synaptic function depends on interactions among sets of proteins that assemble into complex supramolecular machines. Molecular biology, electrophysiology, and live-cell imaging studies have provided tantalizing glimpses into the inner workings of the synapse, but fundamental questions remain regarding the functional organization of these "nano-machines." Electron tomography reveals the internal structure of synapses in three dimensions with exceptional spatial resolution. Here we report results from an electron tomographic study of axospinous synapses in neocortex and hippocampus of the adult rat, based on aldehyde-fixed material stabilized with tannic acid in lieu of postfixation with osmium tetroxide. Our results provide a new window into the structural basis of excitatory synaptic processing in the mammalian brain.
Topics: Animals; Artifacts; Dendritic Spines; Electron Microscope Tomography; Male; Osmium Tetroxide; Post-Synaptic Density; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Synapses
PubMed: 22684938
DOI: 10.1002/cne.23067 -
Ultramicroscopy Nov 2022Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde...
Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde and osmium tetroxide are widely used for transmission electron microscopy (TEM) and lead to adequate preservation of muscle ultrastructure, but do not preserve the molecular features of samples. Methacarn is suggested to be a preferable chemical fixative for light microscopy because it maintains immunohistological features of samples. However, the efficacy of methacarn to preserve ultrastructural features as a primary chemical fixative for TEM is currently unclear. Additionally, cryo-preservation of samples for TEM analysis involves freezing processes such as plunge freezing, slam freezing, or high pressure freezing. High pressure freezing is the considered the gold standard but requires costly equipment and may not be a viable option for many labs collecting tissue samples from remote locations. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant that may allow for better structural preservation of samples by impairing ice damage that occurs during plunge/snap freezing. We aimed to assess the effectiveness of methacarn as a primary chemical fixative and determine the effect of pre-coating samples with DMSO before plunge/snap freezing tissues to be prepared for TEM. The micrographs of the methcarn-fixed samples indicate a loss of Z-disk integrity, intermyofibrillar space, mitochondria structure, and lipids. Ultimately, methacarn is not a viable primary fixative for tissue sample preparation for TEM. Similarly, liquid nitrogen freezing of samples wrapped in aluminum foil produced non-uniform Z-disk alignments that appeared smeared with swollen mitochondria. DMSO coating before freezing appears to lessen the alterations to contractile and mitochondrial morphological structures. DMSO appears to be useful for preserving the ultrastructure of sarcomeres if samples are covered before freezing.
Topics: Acetic Acid; Aluminum; Chloroform; Cryopreservation; Dimethyl Sulfoxide; Fixatives; Glutaral; Ice; Methanol; Microscopy, Electron, Transmission; Muscles; Osmium Tetroxide
PubMed: 35988477
DOI: 10.1016/j.ultramic.2022.113600 -
Journal of Anatomy May 2012The high spatial resolution of micro-computed tomography (micro-CT) is ideal for 3D imaging of coronary arteries in intact mouse heart specimens. Previously, micro-CT of...
The high spatial resolution of micro-computed tomography (micro-CT) is ideal for 3D imaging of coronary arteries in intact mouse heart specimens. Previously, micro-CT of mouse heart specimens utilized intravascular contrast agents that hardened within the vessel lumen and allowed a vascular cast to be made. However, for mouse coronary artery disease models, it is highly desirable to image coronary artery walls and highlight plaques. For this purpose, we describe an ex vivo contrast-enhanced micro-CT imaging technique based on tissue staining with osmium tetroxide (OsO(4) ) solution. As a tissue-staining contrast agent, OsO(4) is retained in the vessel wall and surrounding tissue during the fixation process and cleared from the vessel lumens. Its high X-ray attenuation makes the artery wall visible in CT. Additionally, since OsO(4) preferentially binds to lipids, it highlights lipid deposition in the artery wall. We performed micro-CT of heart specimens of 5- to 25-week-old C57BL/6 wild-type mice and 5- to 13-week-old apolipoprotein E knockout (apoE(-/-) ) mice at 10 μm resolution. The results show that walls of coronary arteries as small as 45 μm in diameter are visible using a table-top micro-CT scanner. Similar image clarity was achieved with 1/2000th the scan time using a synchrotron CT scanner. In 13-week-old apoE mice, lipid-rich plaques are visible in the aorta. Our study shows that the combination of OsO(4) and micro-CT permits the visualization of the coronary artery wall in intact mouse hearts.
Topics: Animals; Coronary Vessels; Female; Male; Mice; Mice, Inbred C57BL; Osmium Tetroxide; X-Ray Microtomography
PubMed: 22360411
DOI: 10.1111/j.1469-7580.2012.01483.x -
Frontiers in Cardiovascular Medicine 2021Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining...
Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.
PubMed: 34760942
DOI: 10.3389/fcvm.2021.739549 -
The Journal of Histochemistry and... Jun 2022Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM)...
Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with α-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.
Topics: Animals; Gold; Immunohistochemistry; Microscopy, Electron, Scanning; Osmium Tetroxide; Rats; Vacuum
PubMed: 35611640
DOI: 10.1369/00221554221102996 -
Nature Methods Mar 2015Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering...
Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.
Topics: Amino Acid Sequence; Animals; Base Sequence; CHO Cells; Cricetulus; Fluorescence; HeLa Cells; Humans; Luminescent Proteins; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Molecular Imaging; Molecular Sequence Data; Osmium Tetroxide; Photochemistry; Recombinant Fusion Proteins
PubMed: 25581799
DOI: 10.1038/nmeth.3225 -
The Journal of Cell Biology Oct 1974Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO(4)), and osmium tetroxide (OsO(4)) on ion and ATP content, cell volume, vital dye...
Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO(4)), and osmium tetroxide (OsO(4)) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol.69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO(4) or OsO(4) but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO(4) but little or no effect with GA or GA-OsO(4). Osmolality of the buffer had a marked effect on cell volume with OsO(4), whereas with GA or GA-OsO(4) it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO(4) alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO(4). After fixation with GA and/or OsO(4) cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.
Topics: Adenosine Triphosphate; Aldehydes; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Cells, Cultured; Coloring Agents; Freezing; Glutaral; Histological Techniques; Magnesium; Mice; Osmium; Osmolar Concentration; Oxides; Potassium; Sonication; Staining and Labeling; Ultracentrifugation
PubMed: 4138889
DOI: 10.1083/jcb.63.1.197