Chapter 1 Advances in High-Pressure and Plunge-Freeze Fixation

1995 ◽  
pp. 3-19 ◽  
Author(s):  
M.E. Galway ◽  
J.W. Heckman ◽  
G.J. Hyde ◽  
L.C. Fowke
PROTOPLASMA ◽  
1992 ◽  
Vol 170 (3-4) ◽  
pp. 104-114 ◽  
Author(s):  
Ursula Meindl ◽  
Susan Lancelle ◽  
P. K. Hepler

1995 ◽  
Vol 73 (1-2) ◽  
pp. 1-10 ◽  
Author(s):  
Michael W. Hess

The ultrastructure of the vegetative cell in the pollen of Ledebouria socialis Roth (Hyacinthaceae) was investigated from microspore mitosis to anthesis. As a result of the good preservation quality achieved with high-pressure freeze fixation and freeze substitution, novel structural features were observed. Extensive endomembrane compartments emerging at the onset of lipid and starch mobilization, were identified as protein bodies by using video-enhanced contrast light microscopy. Thus, proteins, apart from starch and lipids, represent a third class of important intermediary storage substances in developing pollen. The close spatial relationship between protein bodies, endoplasmic reticulum (ER), and storage lipids suggest that protein bodies and ER contribute to lipid digestion. Immediately prior to anthesis the protein bodies become transformed into unspecialized vacuoles as a result of the gradual dissolution of their contents; the formation of the protein bodies remains still to be elucidated. The ER proliferates extensively during pollen ontogenesis, thereby changing its ultrastructure and spatial organization. Microfilaments were detected during all developmental stages, in particular microtubule-associated single microfilaments. The microfilaments are likely to be composed of actin as shown by immunogold labeling.Key words: angiosperm pollen, freeze substitution, protein bodies, microfilaments, Hyacinthaceae.


Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris

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.


Author(s):  
Robert Corbett ◽  
Delbert E. Philpott ◽  
Sam Black

Observation of subtle or early signs of change in spaceflight induced alterations on living systems require precise methods of sampling. In-flight analysis would be preferable but constraints of time, equipment, personnel and cost dictate the necessity for prolonged storage before retrieval. Because of this, various tissues have been stored in fixatives and combinations of fixatives and observed at various time intervals. High pressure and the effect of buffer alone have also been tried.Of the various tissues embedded, muscle, cartilage and liver, liver has been the most extensively studied because it contains large numbers of organelles common to all tissues (Fig. 1).


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