scholarly journals Integrating Light and TEM Information with F-TEM images

2005 ◽  
Vol 13 (5) ◽  
pp. 16-19
Author(s):  
P.A. Sims ◽  
C.A. Lockwood ◽  
J.D. Hardin
Keyword(s):  
Tissue Culture ◽  
High Pressure ◽  
Light Microscopy ◽  
Fusion Proteins ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Embedding Method ◽  
Culture Cells ◽  
Tissue Culture Cells

Fluorescent fusion proteins are widely used to visualize the localization of proteins in worms, fish, flies and tissue culture cells. We have used two different methods that use high pressure freezing (HPF) combined with correlative light microscopy (LM) and TEM. The first method uses fluorescence from live organisms immobilized in agarose followed by HPF and standard freeze substitution in dry solvent with osmium. This pre-embedding method optimizes ultrastructural preservation. A second, post-embedding method preserves fluorescence and immunoreactivity from embedded and polymerized thin sections. Here we describe post-embedding fluorescent integrated TEM images (F-TEM).

Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells

2007 ◽  
Vol 226 (2) ◽  
pp. 182-189 ◽  
Author(s):  
P. HAWES ◽  
C. L. NETHERTON ◽  
M. MUELLER ◽  
T. WILEMAN ◽  
P. MONAGHAN
Keyword(s):  
Tissue Culture ◽  
High Pressure ◽  
Freeze Substitution ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Frozen Tissue

Ultrastructure of the Infection of Poinsettia by Oidium SP. using High Pressure Freezing and Freeze Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 690-691
Author(s):  
G. J. Celio ◽  
E. A. Richardson ◽  
C. W. Mims
Keyword(s):  
High Pressure ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Euphorbia Pulcherrima ◽  
High Pressure Freezing ◽  
Thin Sections ◽  
Transmission Electron ◽  
Dextran Solution ◽  
Leaf Disks ◽  
Diamond Knife

Cryofixation is becoming more widely used to study host-pathogen relationships in fungal diseases of plants. This presentation describes results we have obtained using high pressure freezing and freeze substitution to study powdery mildew disease of poinsettia ﹛Euphorbia pulcherrima) caused by Oidium sp.Approximately 0.5 mm leaf disks bearing sporulating colonies of Oidium sp. were excised and placed in a 15% dextran solution contained in brass planchets. Samples were frozen using a Balzer's HPM 010 High Pressure Freezing Machine and substituted according to the procedures of Hoch.6 Thin sections of embedded leaves were cut using a diamond knife, collected on gold slot grids, and placed on formvar-coated racks. Sections were poststained with uranyl acetate and lead citrate and examined using a Zeiss EM 902A transmission electron microscope.Outstanding preservation of haustoria, the specialized nutrient-absorbing structures produced in host epidermal cells by Oidium, was obtained. Both young, unlobed (Fig. 1) as well as mature, highly lobed (Fig. 2) haustoria were observed.

A plunge-freezing method for imaging mammalian tissue culture cells

1991 ◽  
Vol 49 ◽  
pp. 58-59
Author(s):  
E.T. O'Toole ◽  
J.R. McIntosh
Keyword(s):  
Tissue Culture ◽  
Culture Medium ◽  
Freeze Substitution ◽  
Chemical Fixation ◽  
Freezing Method ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Humidity Chamber ◽  
Cellular Components ◽  
Coated Carbon

Ultrarapid freezing of tissue culture cells followed by freeze substitution has been used a method for the optimum fixation of cytoskeletal components that are often sensitive to routine chemical fixation. This is due to the fact that freezing methods such as plunge freezing, result in the almost instantaneous fixation of all cellular components without alteration of the cell's morphology. In addition, we have found that the plunge freezing method is useful for obtaining thin frozen cells for direct cryoimaging. Here we describe how the plunge freezing method can be applied both for freeze substitution analysis and for direct cryoimaging of frozen tissue culture cells.PTK, cells were grown to confluence on formvar coated, carbon stabilized gold grids. Prior to freezing, the grids were blotted in a 37°C, high humidity chamber so that a minimum of culture medium remained on the grid. This blotting step was critical to obtain a sample thin enough for optimum cryopreservation and subsequent cryoimaging.

The Use of High Pressure Freezing and Freeze Substitution to Study Host–Pathogen Interactions in Fungal Diseases of Plants

2003 ◽  
Vol 9 (6) ◽  
pp. 522-531 ◽  
Author(s):  
C.W. Mims ◽  
Gail J. Celio ◽  
Elizabeth A. Richardson
Keyword(s):  
High Pressure ◽  
Cell Walls ◽  
Freeze Substitution ◽  
Fungal Diseases ◽  
Host Cells ◽  
High Pressure Freezing ◽  
Plant Interactions ◽  
Host Pathogen Interactions ◽  
Thin Sections ◽  
Host Pathogen

This article reports on the use of high pressure freezing followed by freeze substitution (HPF/FS) to study ultrastructural details of host–pathogen interactions in fungal diseases of plants. The specific host–pathogen systems discussed here include a powdery mildew infection of poinsettia and rust infections of daylily and Indian strawberry. The three pathogens considered here all attack the leaves of their hosts and produce specialized hyphal branches known as haustoria that invade individual host cells without killing them. We found that HPF/FS provided excellent preservation of both haustoria and host cells for all three host–pathogen systems. Preservation of fungal and host cell membranes was particularly good and greatly facilitated the detailed study of host–pathogen interfaces. In some instances, HPF/FS provided information that was not available in samples prepared for study using conventional chemical fixation. On the other hand, we did encounter various problems associated with the use of HPF/FS. Examples included freeze damage of samples, inconsistency of fixation in different samples, separation of plant cell cytoplasm from cell walls, breakage of cell walls and membranes, and splitting of thin sections. However, we believe that the outstanding preservation of ultrastructural details afforded by HPF/FS significantly outweighs these problems and we highly recommend the use of this fixation protocol for future studies of fungal host-plant interactions.

scholarly journals THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

1965 ◽  
Vol 27 (2) ◽  
pp. 411-422 ◽  
Author(s):  
B. R. Brinkley
Keyword(s):  
Tissue Culture ◽  
Fine Structure ◽  
Osmium Tetroxide ◽  
Chinese Hamster ◽  
Late Prophase ◽  
Thin Sections ◽  
Chinese Hamster Cells ◽  
Culture Cells ◽  
Tissue Culture Cells

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

A Method for Electron Microscopic Study of Tissue Culture Cell Monolayers Grown in Tubes

1967 ◽  
Vol 25 ◽  
pp. 132-133
Author(s):  
R. Stephens ◽  
G. Schidlovsky ◽  
S. Kuzmic ◽  
P. Gaudreau
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Light Microscopy ◽  
Cell Sheet ◽  
Culture Cell ◽  
Tissue Culture Cell ◽  
Electron Microscopic ◽  
Cell Sheets ◽  
Morphological Alterations ◽  
Culture Cells

The usual method of scraping or trypsinization to detach tissue culture cell sheets from their glass substrate for further pelletization and processing for electron microscopy introduces objectionable morphological alterations. It is also impossible under these conditions to study a particular area or individual cell which have been preselected by light microscopy in the living state.Several schemes which obviate centrifugation and allow the embedding of nondetached tissue culture cells have been proposed. However, they all preserve only a small part of the cell sheet and make use of inverted gelatin capsules which are in this case difficult to handle.We have evolved and used over a period of several years a technique which allows the embedding of a complete cell sheet growing at the inner surface of a tissue culture roller tube. Observation of the same cell by light microscopy in the living and embedded states followed by electron microscopy is performed conveniently.

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