Projects / Programmes
Ultrastructure preservation of eucariotic and procariotic cells and viruses using chemical and cryomethods for transmission electron microscopy.
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B230 |
Biomedical sciences |
Microbiology, bacteriology, virology, mycology |
| B235 |
Biomedical sciences |
Protozoology |
Researchers (5)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
03174 |
PhD Borut Drinovec |
Microbiology and immunology |
Researcher |
1999 - 2001 |
158 |
| 2. |
10514 |
Vesna Golob |
Microbiology and immunology |
Researcher |
1999 - 2001 |
4 |
| 3. |
12555 |
PhD Jošt Kuret |
Microbiology and immunology |
Researcher |
1999 - 2001 |
27 |
| 4. |
03172 |
PhD Jožefa Marin |
Microbiology and immunology |
Researcher |
2000 - 2001 |
237 |
| 5. |
08755 |
PhD Mateja Poljšak-Prijatelj |
Microbiology and immunology |
Head |
1999 - 2001 |
234 |
Organisations (1)
Abstract
Due to its great resolution, transmission electron microscope enables observation of small objects. All the samples must be adequately prepared for observation. In the course of preparation it is of great importance to preserve the samples in as near as life-like state as possible. Currently we can choose between chemical and physical procedures. Chemical methods are being used since the beginning of electron microscopy. They are widely distributed and well adapted to different biological samples. Physical procedures are essentially reduced to cryo methods, as alternative methods (e.g. microvawe irradiation) remain in relatively restricted use. Cells, eukaryotic as well as procaryotic are highly organized structures. The structural organization is in direct relationship to function. By employing different methods for preparation we try to preserve the cell close to its in vivo state. Nevertheless, we always cause some damage and alterations to the cell. Many of these changes cannot be avoided. In our study we plan to analize and compare electron microscopical images of different cells (eukaryotic from animal tissues and cell cultures; HeLa, CV1; plant tissues; prokaryotic cells: Borrelia burgdorferi s.l.; virus infected cell cultures). For fixation we will use chemical methods as well as cryomethods, the latter being introduced and employed in our lab for the first time in Slovenia. Analysis of the sample in the electron microscope enables us to determine the final quality of methods used. As most important parameters size and shape of the cells, distribution and number of of observed organelles and other cell and tissue elements are taken in consideration. The ideal method of fixation should preserve each molecule, ideally each single ion, in its original position. The quality of fixation, which is the first step in the preparation sequence, is very important. Adequate fixation preserves a great deal of information, while choosing a wrong method can destroy the sample at the very beginning. Another very important factor is time between sampling and fixation. This interval should be as short as possible.
Fixation is followed by dehydration and embedding. Both steps can have great influence on chemical and structural composition of the cell. The alterations accompanying chemical fixation and the following steps are loss of epytopes, extraction of moleculs and larger structures, changes in cell shape and volume, redistribution of organelles, steric hindrance caused by chemical reaction with fixative and denaturation of proteins. By using cryo methods, all the alterations caused by chemical reactions can be avoided. Sample is stabilized within miliseconds without any chemical reactions. Such samples can be brought to life if thawed properly, while chemical fixation always means killing of the cell. Inadequate freezing can cause the growth of ice crystals and thus damage the cell. Such alterations can be readily recognized in the electron microscope. Progressive Lowering of Temperature or PLT method is considered as in-between step between chemical and cryo procedures. The sample is still chemically fixed, during the process of dehydration the temperature is gradually lowered. This way, the alterations caused by dehydration at room temperature should be eliminated and the ultrastructure and the antigenicity of the sample better preserved.
If we want to study the presence and distribution of antigens in the sample ie by immunocytochemical methods, we have to use a method that enables us to optimally preserve antigens. In this case the advantages of cryo methods over chemical are even more pronounced.
