- Light microscopy is the oldest and most widely used form of microscopy
- Specimens are illuminated with light, which is focused using glass lenses and viewed using the eye or photographic cells.
- Specimens can be dead or alive, and often need to be stained to me made visible. Iodine, which stains starch blue/black, is used for plant cells, and methylene blue used for animal cells.
A couple of important definitions:
Magnification – refers to the microscope’s power to increase an object’s apparent size.
Resolution – refers to the microscope’s power to show detail clearly, so that the use is able to distinguish two points close together.
The resolving power of a microscope is limited by the wavelength of light (400-600 nm). If objects in the specimen are smaller than the wavelength of radiation being used, they do not interrupt the waves and so are not detected.
Preparation of Slides
Slides need to be made so that the specimen can be viewed time after time for a number of years.
- Fixation – Chemicals are used to preserve the specimen so that they are not distorted over time.
- Dehydration – Water is removed from the specimen using a solvent, generally ethanol. This is because water molecules with deflect the beam of electrons away from the specimen.
- Embedding – Specimens are put into wax/resin to hold the structures in place so that thin slices can be made.
- Staining – Most biological material is transparent. Therefore different stains are used to highlight different organelles.
- Mounting – Mounting on a slide will protect the specimen so that it is suitable for viewing over a long period of time. A coverslip is generally placed over the specimen.
Eukaryotes:
These are cells that contain a double-membrane bound nucleus and other membrane bound organelles, such as mitochondria, endoplasmic reticulum and the golgi apparatus.
Nucleus – This is the most visible structure in a non-dividing cell, and contains most of the cell’s genetic material. The membrane bound area that surrounds the nucleus is known as the nuclear envelope. The nuclear envelope is a double-membrane, part of the endomembrane system, consisting of an inner and outer membrane, seperated by a gap of 20-40 nm. The two membranes fuse at the tips of the nuclear pores, which allow ribosomes and RNA out of the nucleus. Within each nucleus is one (or more) nucleolus, which is an area of concentrated DNA, organised by histones.
The nucleolus is roughly spherical and functions in the synthesis of ribosomes. It consists of nucleolar organisers (specialised chromosomes) with multiple copies of the genes for ribosome synthesis. They will have considerable amounts of rRNA and protein, representing ribosomes in various stages of construction. Generally, only one nucleolus is present, however there may more more than one, dependent on the cell species and the stage of cell cycle.
Ribosomes – These are essential for the process of protein synthesis. They are built up of two subunits; a large one with two tRNA binding sites, and a smaller one which associates with mRNA in a binding groove. They are the smallest eukaryotic organelles, and they do not have a membrane. Free ribosomes are located within the cytosol and produce the proteins needed in the cell. Bound ribosomes are attached to the ER; these produce the proteins needed for secretion.
Endoplasmic Reticulum – This a network of flattened sacs, called cisternae, and membranes throughout the cell. The membrane is continuous with that of the nucleus. There are two parts the the ER: the smooth endoplasmic reticulum and the rough endoplasmic reticulum. The SER is involved in synthesising lipids and steroids, and also carbohydrate metabolism. It also plays a part in the detoxification of drugs and poisons (e.g. alcohol). It is called “smooth” as there are no ribosomes bound to it. The RER has a rough surface, due to the ribosomes bound to the cytosolic side. As the ribosomes feed polypeptide chains into the RER’s lumen, it folds them into a functional proteins. Often carbohydrates are attached to form glycoproteins. Once the ribosome has finished feeding in the polypeptide, it detaches from the ER and moves back through the cytosol.
Golgi Apparatus – Anything made the ER is transported here in vesicles, where it is modified and then sent on to another destination. The golgi, similar to the ER, is made up of a series of folded cisternae, which have a cis face, and a trans face. The cis face is the receiving side of the golgi, and this is where transport vesicles from the ER attach to the apparatus. The trans face is the shipping face; here, vesicles bud off to carry molecules to other destinations, often to be secreted out of the cell. Secretory cells, such as those in the pancreas, have larger, more prevalent golgi.
Lysosomes – These are hydrolytic enzyme complexes surrounded by a single wall membrane. They bud off from the golgi, which produces the enzymes used to break down macromolecules. The lysosomes are capable of maintaining a high concentration of H+ ions by actively pumping them into the lysosymal lumen. Due to the anabolic nature of the enzymes, excessive leakage of the lysosome can result in autodigestion. It is protected from self digestion by the inner surface of its membrane. Lysosomes play a major part in cytosis.
Prokaryotes
Prokaryotes are the most primitive cell, appearing on Earth ~3.5 billion years ago as the first sign of life. They have no membrane mound organelles, most particularly, they have no double-membrane bound nucleus. They still contain DNA, however their genetic material is shaped in a circular plasmid, rather the the eukaryotic double helix, these strands are left lying free in the cytosol. The ribosomes in prokaryotes are a lot smaller than those in eukaryotes, but the cells do have a similar metabolism. Generally they are much smaller in size, typically between 0.5-10 microns in diameter. In bacteria is the only place that nitrogen fixation occurs. Nitrogen is absorbed from the atmosphere and converted into nitrates by the organisms.
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