3rd Lecture Medical students Medical Biology
Tools of Cell Biology
As in all experimental sciences, research in cell biology depends on the laboratory methods that can be used to study cell structure and function.
1-Light Microscopy:
Because most cells are too small to be seen by the naked eye, the study of cells has depended heavily on the use of microscopes. Light microscopes are able to magnify objects up to about a thousand times. Since most cells are between 1 and 100 μm in diameter, they can be observed by light microscopy, as can some of the larger subcellular organelles, such as nuclei, chloroplasts, and mitochondria.However, the light microscope is not sufficiently powerful to reveal fine details of cell structure, for which resolution—the ability of a microscope to distinguish objects separated by small distances—is even more important than magnification.
The limit of resolution of the light microscope is approximately 0.2 μm; two objects separated by less than this distance appear as a single image, rather than being distinguished from one another.
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2-Electron Microscopy:Because of the limited resolution of the light microscope, analysis of the details of cell structure has required the use of more powerful microscopic techniques—namely electron microscopy. An electron microscope is a microscope that uses a beam of electrons as a source of illumination. The electron microscope can achieve a much greater resolution than that obtained with the light microscope because the wavelength of electrons is shorter than that of light. Thus, under optimal conditions, the resolving power of the electron microscope is approximately 0.2 nm. Two types of electron microscopy—transmission and scanning—are widely used to study cells.
Transmission electron microscopy (TEM): In this case, the electron beam is passed through the sample. The result is a two dimensional image.
Scanning electron microscopy (SEM): Here the electron beam is projected on the sample. The electrons do not go through the sample but bounce off. This way it is possible to visualize the surface structure of the specimen. The image appears 3 dimensional.
Transmission electron microscope(للاطلاع)
Figure: First 2 images were getting by transmission EM, others by scanning EM- enlarged between 10000-45000 (للاطلاع).3-Cell fractionation
This means separating different parts and organelles of a cell, so that they can be studied in detail. All the processes of cell metabolism (such as respiration or photosynthesis) have been studied in this way. The most common method of fractionating cells is to use differential centrifugation:1.Cut tissue (e.g. liver, heart, leaf, etc) in ice-cold isotonic buffer.
Cold to stop enzyme reactions, isotonic to stop osmosis, and buffer to stop pH changes.
2.Grind tissue in a blender to break open cells.
3. Filter. This removes insoluble tissue (e.g. fat, connective tissue, plant cell walls, etc).
This filtrate is now called a cell-free extract,
and is capable of carrying out most of the normal cell reactions.
4. Centrifuge filtrate at low speed
(1 000 x g for 10 min)5. Centrifuge supernatant at medium speed
(10 000 x g for 30 min)6. Centrifuge supernatant at high speed
(100 000 x g for 1 hour)7. Centrifuge supernatant at very high speed
(300 000 x g for 3 hours)8. Supernatant is now organelle-free cytoplasm
4-Growth of Animal Cells in Culture
The ability to study cells depends largely on how readily they can be grown and manipulated in the laboratory. Although the process is technically far more difficult than the culture of bacteria or yeasts, a wide variety of animal cells can be grown and manipulated in culture.
Animal cell cultures are initiated by the dispersion of a piece of tissue into a suspension of its component cells, which is then added to a culture dish containing nutrient media. Most animal cell types attach and grow on the plastic surface of dishes used for cell culture .
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In addition to salts and glucose, the media used for animal cell cultures contain various amino acids and vitamins, which the cells cannot make for themselves. The growth media for most animal cells in culture also include serum, which serves as a source of polypeptide growth factors that are required to stimulate cell division.The initial cell cultures established from a tissue are called primary cultures .The cells in a primary culture usually grow until they cover the culture dish surface. They can then be removed from the dish and replaced at a lower density to form secondary cultures.
5. Viruses
Viruses are intracellular parasites that cannot replicate on their own. They reproduce by infecting host cells and usurping the cellular machinery to produce more virus particles. In their simplest forms, viruses consist only of genomic nucleic acid (either DNA or RNA) surrounded by a protein coat. Viruses are important in molecular and cellular biology because they provide simple systems that can be used to investigate the functions of cells. Because virus replication depends on the metabolism of the infected cells.