High-Pressure Freezing and Freeze Substitution for Transmission Electron Microscopy Imaging and Immunogold-Labeling

2020 ◽  
pp. 337-347
Author(s):  
Marisa S. Otegui
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Pressure ◽  
Freeze Substitution ◽  
Immunogold Labeling ◽  
High Pressure Freezing ◽  
Microscopy Imaging ◽  
Transmission Electron

Ultrastructure of the host-parasite interaction in leaves of Duchesnea indica infected by the rust fungus Frommeëla mexicana var. indicae as revealed by high pressure freezing

2001 ◽  
Vol 79 (1) ◽  
pp. 49-57 ◽  
Author(s):  
C W Mims ◽  
C Rodriguez-Lother ◽  
E A Richardson
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Pressure ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Transfer Cells ◽  
Host Cells ◽  
High Pressure Freezing ◽  
Transmission Electron ◽  
Duchesnea Indica

A combination of scanning and transmission electron microscopy was used to examine the host-pathogen relationship in leaves of Duchesnea indica (Andrz) Focke infected by the rust fungus Frommeëla mexicana var. indicae McCain & Hennen. Samples for transmission electron microscopy were prepared using high pressure freezing followed by freeze substitution. This protocol provided excellent preservation of both host cells and fungal haustoria. Each haustorium of F. mexicana var. indicae possessed a long slender neck with a neck band and an expanded body that contained two nuclei positioned close together. The haustorial body was lobed and sometimes even branched but lacked septa. Details of the extrahaustorial membrane that separated each haustorium from the cytoplasm of its host cell were particularly well preserved. Extensive labyrinth cell wall ingrowths developed around haustorial necks, as well as elsewhere, in infected cells. These ingrowths appeared to be identical to those present in plant transfer cells. Transfer cells are thought to be involved in intensive solute transfer over short distances. This appears to be the first report of the development of transfer cells in response to infection by a plant pathogenic fungus.Key words: haustoria, transfer cells, freeze substitution, electron microscopy.

High-pressure freezing and freeze substitution fixation of the plant pathogenic fungus Exobasidium vaccinii

1996 ◽  
Vol 54 ◽  
pp. 78-79
Author(s):  
Elizabeth A. Richardson ◽  
Charles W. Mims
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Plant Pathogens ◽  
Pathogenic Fungus ◽  
Freeze Substitution ◽  
High Pressure Freezing ◽  
Large Samples ◽  
Reproductive Structures ◽  
Transmission Electron ◽  
Fungal Genus

Members of the small fungal genus Exobasidium are all plant pathogens. Most species are noted for their ability to produce large fleshy galls on leaves and flower parts. They produce their hyphae and specialized absorbing structures known as haustoria inside these galls and their sexual reproductive structures (basidia) on gall surfaces. The objective of this study was to examine the feasibility of using high pressure freezing (HPF) followed by freeze-substitution (FS) fixation to prepare Exobasidium induced galls on Rhododendron sp. for study with transmission electron microscopy (TEM). HPF fixation followed by FS fixation is considered to be the best method for preserving large samples of plant tissue for TEM. This approach has shown promise for the examination of host-pathogen relationships in fungal diseases of plants.

Cytology of irregular growth forms of Ophiostoma ulmi and Ophiostoma novo-ulmi growing through millipore filter membranes and sterilized elm wood sections

1995 ◽  
Vol 41 (12) ◽  
pp. 1095-1110 ◽  
Author(s):  
G. B. Ouellette ◽  
C. Côté ◽  
N. Méthot ◽  
H. Chamberland ◽  
J. -G. Lafontaine
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
High Pressure ◽  
Thin Wall ◽  
High Pressure Freezing ◽  
Growth Forms ◽  
Millipore Filter ◽  
Ophiostoma Ulmi ◽  
Transmission Electron ◽  
Gold Labelling

When Ophiostoma ulmi or Ophiostoma novo-ulmi are grown on either 0.22- or 0.45-μm millipore filter membranes placed on impoverished agar medium, the fungus grows through these membranes and takes on various irregular pleomorphic growth forms (P-forms). Links of continuity between these forms and the more regular ones have been shown using light, confocal, and transmission electron microscopy. Tests with labelled probes, such as gold-complexed wheat germ agglutinin for chitin and β-exoglucanase for cellulosic β-1,4-glucans, have indicated that in P-forms deposition of chitin is much altered but is less so in the case of cellulosic glucan. The cytology of these forms compared with the regular fungal ones is also very different, particularly with reference to mitochondria and nuclei. Also, numerous vesiculate structures were noted in the rarely septate P-forms. Similar irregular forms with opaque contents were produced by these fungi when they were grown on sterilized elm wood sections. When these latter samples were fixed by high-pressure freezing, the following main features were noted: fungal cells with a very thin wall, slightly labelled for chitin but more intensely for cellulosic glucans; well-preserved structures, such as plasmalemma and endoplasmic reticulum; and a slightly opaque, fibril-containing extracellular sheath. Differences in labelling for galactose, whether of wall layers or cell contents, were also observed in regular and P-forms. Electron opaque bodies that labelled strongly for galactose were also numerous in P-forms in some samples.Key words: transmission electron microscopy, high-pressure freezing, gold labelling, extracellular sheaths, wall constituents.

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.

scholarly journals Fabrication of a liquid cell for in situ transmission electron microscopy

Microscopy ◽  
2020 ◽  
Author(s):  
Xiaoguang Li ◽  
Kazutaka Mitsuishi ◽  
Masaki Takeguchi
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Cost Effective ◽  
Production Method ◽  
Microscopy Imaging ◽  
Transmission Electron ◽  
In Situ Electron Microscopy ◽  
Liquid Cell ◽  
Si Wafer

Abstract Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 µm × 40 µm) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.

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