Freezing without Ice Crystal Damage: Semithin and Ultrathin Frozen Sections of Ethanol-Infiltrated Tissue for Microscopy, with Applications to Immunocytochemistry

1995 ◽  
Vol 1 (5) ◽  
pp. 217-230
Author(s):  
A. Kent Christensen ◽  
Terry B. Lowry
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light And Electron Microscopy ◽  
Ice Crystal ◽  
Freezing Damage ◽  
Frozen Sections ◽  
Glass Slides ◽  
Frozen Tissue ◽  
Crystal Damage ◽  
Ultrathin Frozen Sections

Ethanol (ethyl alcohol) has long been a standard reagent used in preparing tissues for light and electron microscopy. After fixation, tissues are usually dehydrated with ethanol before being embedded in paraffin or plastic. In this study we show that the ethanol-infiltrated tissue can be frozen and sectioned directly without embedding. When tissue impregnated with ethanol is cooled below about −117°C with liquid nitrogen, the ethanol solidifies without appreciable crystallization. The frozen tissue can then be sectioned in a commercial cryoultramicrotome that is set at −155 to −170°C to produce semithin frozen sections (0.5 to 3 μm thick) for light microscopy or ultrathin frozen sections (50 to 100 nm thick) for electron microscopy. Sections are picked up and mounted on glass slides or EM grids by means that are in current use for ice ultrathin frozen sectioning. Because there is no apparent freezing damage, the morphology in these ethanol frozen sections of unembedded tissue appears generally quite good, often resembling that obtained by conventional EM techniques. Examples are provided that illustrate the use of this material for immunocytochemistry at the light and electron microscope levels.

Simultaneous observations of immunolabeled frozen sections in lm and em

1978 ◽  
Vol 36 (2) ◽  
pp. 164-165
Author(s):  
K. T. Tokuyasu ◽  
J. Slot ◽  
S. J. Singer
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Microscopy ◽  
Light And Electron Microscopy ◽  
Frozen Sections ◽  
Cover Glass ◽  
Rat Pancreas ◽  
Light Microscopic Observation ◽  
Rabbit Igg ◽  
Ultrathin Frozen Sections ◽  
Number Of Cells

Immunofluorescent microscopy is more suitable for the analysis of a large number of cells, often greater in the sensitivity for the detection of antigens, and more readily applicable for the identification of multiple antigens than immunoe1ectron microscopy. For combining these features of fluorescent microscopy with the superior resolution of electron microscopy, we attempted to observe the same immunolabeled ultrathin frozen sections with both light and electron microscopy.Ultrathin frozen sections of rat pancreas fixed in a mixture of 2% formaldehyde and 0.2% g1utaraldehyde for 1 hr at 4°C were first immunostained with rabbit anti-rat amylase antibodies, then very lightly with ferritin-goat anti-rabbit IgG conjugates and heavily with rhodamine-goat anti-rabbit IgG conjugates. For light microscopic observation, grids were suspended underneath the cover glass with a very thin layer of 50-90% glycerol and the cover glass was separated from the slide glass by a spacer to avoid the contact of the grid with the slide glass. After the light microscope observation, the grids were floated on 0.1 M phosphate buffer by dissolving glycerol into the buffer and processed for electron microscopy.

scholarly journals An Electron Microscope Study of Basophile Substances of Frozen-Dried Rat Liver

1958 ◽  
Vol 4 (3) ◽  
pp. 291-300 ◽  
Author(s):  
Henry Finck
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Rat Liver ◽  
Microscope Study ◽  
Electron Microscope Study ◽  
Freeze Drying ◽  
Light And Electron Microscopy ◽  
Enzyme Treatment ◽  
Dense Granules ◽  
Homogeneous Matrix

Small pieces of liver from rats subjected to different dietary regimes were fixed by freeze-drying, and postfixed by in vacuo heating and denaturation with alcohol. Specimens were digested with ribo- or deoxyribonuclease, and stained with gallocyanin-chromalum, azure II, the Feulgen procedure or alcoholic platinic tetrabromide. Some specimens were reserved as controls of the effects of enzyme treatment. Stained and unstained specimens were embedded in methacrylate and examined by light and electron microscopy. Basophilic and Feulgen-positive substances, after contact with watery reagents, were found by electron microscopy to exist as small dense granules embedded in a less dense homogeneous matrix, forming the walls of submicroscopic vacuoles. These granules were absent after digestion with nucleodepolymerases. In specimens (unstained, or stained with platinic tetrabromide) which had not passed through water, the dense (basophile) substances in nuclei and cytoplasm were found to exist, not as granules, but as ill defined submicroscopic concentrates which blended imperceptibly into the homogeneous matrix of the vacuolar walls. Objections to the use of stains for improving contrast conditions in electron microscopy of tissues are discussed, and it is concluded that the reagents do not necessarily produce the observed increases in contrast by selectively stabilizing certain structures. The concept of microsomes as pre-existing distinct morphological entities in intact (unhomogenized) cells is thought to be inconsistent with the distribution of basophile substances in frozen-dried liver.

Mouse ovarian tissue vitrification on copper electron microscope grids versus slow freezing: a comparative ultrastructural study

2015 ◽  
Vol 27 (7) ◽  
pp. 1020 ◽  
Author(s):  
Ferda Topal-Celikkan ◽  
Sinan Ozkavukcu ◽  
Deniz Balci ◽  
Sibel Serin-Kilicoglu ◽  
Esra Atabenli-Erdemli
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Cancer Therapy ◽  
Ovarian Tissue ◽  
Light And Electron Microscopy ◽  
Slow Freezing ◽  
Ovarian Tissue Cryopreservation ◽  
Control Group ◽  
Primordial Follicles ◽  
User Friendly

There are many reasons, including cancer therapy, for premature ovarian failure and infertility. Oocyte, embryo and ovarian cryopreservation are current options for fertility preservation. Ovarian tissue cryopreservation is essential in patients whose cancer therapy cannot be delayed, including prepubertal girls, and is mostly performed using slow freezing. In the present study, mouse ovarian tissues were vitrified on copper electron microscope grids (n = 18) or conventionally slow frozen (n = 18). Post-thaw tissues were examined histologically using light and electron microscopy and compared with the control group. According to light microscopy observations, antral follicles were found to be better preserved with the slow freezing technique rather than vitrification. Electron microscopy revealed swollen mitochondria in the oocyte cytoplasm, condensations in the zona pellucida, breakages in the junctions of granulosa cells and vacuolisation in the extracellular space in pathologic follicles, which were relatively more frequent, in the vitrification group after thawing. These results indicate that ovarian slow freezing is preferable than vitrification on copper electron microscope grids, especially for larger follicles. Conversely, vitrification of ovarian pieces using cooper grids is user-friendly and provided good protection for primordial follicles and stromal cells. There is a need for further studies into advanced tissue vitrification techniques and carriers.

Electron microscopic analysis of objects in light microscopic sections

1978 ◽  
Vol 36 (2) ◽  
pp. 80-81
Author(s):  
Arvid B. Maunsbach
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Microscopic Analysis ◽  
Electron Microscopic Level ◽  
Semithin Section ◽  
Cover Glass ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Glass Slides ◽  
Ultrathin Sections

Structural studies in experimental biology or in pathology are frequently extended from the light to the electron microscopic level. This is often done by cutting both semithin (about 1 μm) and thin sections from the same tissue block after embedding for electron microscopy. However, in many studies it would be of great value to analyse the same structure both by light and electron microscopy, i.e. to be able to study by electron microscopy an object which is first detected by light microscopy in a semithin section. To achieve this, a method has been developed by which ultrathin sections are cut directly from the semithin section containing the object of interest.Semithin sections, about 1 μ in thickness, are cut from Epon or Vestopal embedded tissue. The sections are placed on ordinary glass slides and stained with toluidine blue. The sections are studied in the light microscope without a cover glass or mounted in water.

XVIII.—The Spermatozoon of the Spider BeetlePtinus tectusBoieldieu as seen by Light and Electron Microscopy

1951 ◽  
Vol 64 (3) ◽  
pp. 353-366 ◽  
Author(s):  
Jozef Dlugosz ◽  
John W. Harrold
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscope ◽  
Light And Electron Microscopy ◽  
Sperm Head ◽  
Central Axis

SynopsisThe mature Ptinid sperm examined under the light microscope is found to be specialised in that the chromatin is not contained within a sperm head but is distributed along a central axis. The migration of chromatin resembles that found in Coccids by Hughes-Schrader (1948). Surrounding the axis is a more flexible helical membrane extending the whole length of the sperm.Under the electron microscope the membrane appears to consist of eighteen or twenty thin fibres and two thick fibres with striated sheaths. Near the posterior end of the membrane the fibres are surrounded by a ring. The structure is simpler than that of mammalian and avian sperms examined by other workers with similar techniques. Under the electron microscope, stages in the migration of chromatin in the immature sperm show a number of discrete opaque bodies which may be chromosomes. The approximate dimensions of the various structures are given.

scholarly journals Electron Microscope Studies on Normal Human Myeloid Elements

Blood ◽  
1964 ◽  
Vol 23 (3) ◽  
pp. 300-320 ◽  
Author(s):  
ROBERT J. CAPONE ◽  
EVA LURIE WEINREB ◽  
GEORGE B. CHAPMAN
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Chromatin Condensation ◽  
Light And Electron Microscopy ◽  
Direct Connection ◽  
Granule Formation ◽  
Specific Granules ◽  
Normal Human

Abstract The development of representative myeloid elements is traced by correlated light and electron microscopy. Cytoplasmic changes during maturation of granulocytes from the myeloblast include loss of basophilia, development of the endoplasmic reticulum complex, decrease in number of mitochondria, and granule formation. The endoplasmic reticulum vesicles increase in size and number during the promyelocyte and myelocyte stages, accompanied by the appearance of non-specific and specific granules, and decrease again during the cytosomal maturation of the metamyelocyte. A reduction in number of mitochondria is noted through the metamyelocyte stage. The apparent continuity of the limiting membranes of both the granules and mitochondria with those of the cisternae of endoplasmic reticulum suggests a direct connection among cytosomal organelles. The role of the endoplasmic reticulum in granulogenesis is discussed. Maturation of the nucleus involves a loss of nucleolar differentiation by a loosening of the compact fibrillar aggregates, and progressive chromatin condensation.

Preliminary Observations on the Chemistry and Biology of the Lorica in a Collared Flagellate (Stephanoeca Diplocostata Ellis)

1974 ◽  
Vol 54 (2) ◽  
pp. 269-276 ◽  
Author(s):  
B. S. C. Leadbeater ◽  
I. Manton
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Hydrofluoric Acid ◽  
Light And Electron Microscopy ◽  
Amorphous Form ◽  
X Ray ◽  
Analytical Electron ◽  
Analytical Electron Microscope ◽  
Biological Aspects ◽  
Stephanoeca Diplocostata

Evidence for the presence of silica in the costae of Stephanoeca diplocostata Ellis has been provided by a combination of light and electron microscopy. The solubility of costae in hydrofluoric acid has been demonstrated. The presence of silicon in the costae has been strongly confirmed by means of the X-ray analytical electron microscope (EMMA) but tests for a crystalline substructure by means of electron diffraction were negative; it is concluded that silicon is present in a crystallographically amorphous form. Preliminary observations on other biological aspects of the organism include the presence of a sac-like membrane between the lorica and protoplast, the presence of immature costal strips within the cytoplasm and the nature of ingested food. The phyletic position of the group is briefly discussed with special reference to mitochondrial substructure.

scholarly journals Localization of chitin in algal and fungal cell walls by light and electron microscopy.

1978 ◽  
Vol 26 (10) ◽  
pp. 782-791 ◽  
Author(s):  
N L Pearlmutter ◽  
C A Lembi
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Acetic Acid ◽  
Electron Microscope ◽  
Cell Walls ◽  
Potassium Hydroxide ◽  
Light And Electron Microscopy ◽  
Fungal Cell ◽  
Blastocladiella Emersonii ◽  
Fungal Cell Walls

Chitin was visualized in cell walls after hydrolysis with potassium hydroxide and subsequent postfixation of the deacetylated polysaccharide (chitosan) in OsO4. Areas of chitin deposition appeared dark borwn by light microscopy and electron dense in the electron microscope. With this method, the presence of chitin was demonstrated in the cell walls of the green alga Pithophora oedogonia (Montagne) Wittrock and two fungi, Ceratocystis ulmi Buism. (C. Moreau) and Blastocladiella emersonii Cantino and Hyatt. Most of the chitin in P. oedogonia ws found in the crosswall disk and small amounts occurred in the outer longitudinal walls. The septal disk of C. ulmi also contained chitin, but significant amounts were present in the inner and outer regions of longitudinal walls as well. Chitin was present throughout the walls of B. emersonii. Small amounts of chitin were not easily demonstrated by this technique, but removal of chitosan by exposure to dilute acetic acid before osmium fixation disrupted cell wall integrity, suggesting that small amounts of the structural polysaccharide had been removed.

scholarly journals DETECTION OF DNA BY TRITIATED ACTINOMYCIN D ON ULTRATHIN FROZEN SECTIONS

1972 ◽  
Vol 53 (3) ◽  
pp. 798-808 ◽  
Author(s):  
Roch Bernier ◽  
Roberto Iglesias ◽  
René Simard
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Liver Tissue ◽  
Actinomycin D ◽  
Frozen Sections ◽  
Chromosomal Proteins ◽  
The Future ◽  
Complete Series ◽  
Ultrathin Frozen Sections ◽  
Cellular Components

Ultrathin frozen sections of fresh liver tissue were floated on actinomycin D-3H. Quantitative high resolution radioautography was performed to determine the value of the method for detection of DNA by electron microscopy. A complete series of control experiments involving various treatments of frozen sections with enzymes (pronase, DNase) and 0.1 N HCl were also carried out to determine the specificity of the labeling. The results indicate the value of the method for detection of DNA directly on ultrathin frozen sections. Short treatments with pronase followed by DNase reduce the labeling to zero, whereas removal of chromosomal proteins with HCl increases the amount of radioactivity in the nucleus considerably. The results are discussed in view of the future applications opened by ultracryotomy, since radioautographic detection of various macromolecules and cellular components by labeled compound with specific affinities will now be possible.

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