An example of a college level introduction from a fluorescence in C. elegans lab.
Caenorhabditis elegans is a free living soil nematode that is very advantageous for studying cell and molecular biology. The short life span of C. elegans leads to easy formation of mutant strains as well as studies of multiple generations. Adult C. elegans have about one thousand somatic cells which are all transparent. As a result, these nematodes are useful for descriptive studies of cell fate, morphogenesis, and physiology. The transparent cells of C. elegans makes the entire digestive tract visible, so the nematodes are great specimens for studies of cellular digestion. Cellular digestion is the manner in which cells break down molecules into useable substances.
C. elegans carry out cellular digestion through the use of vesicular transportation to take up exogenous materials. Vesicular transportation is the use of vesicles to move materials into the organism and into its cells. Vesicular transport can be carried out in two different ways, either selective or non-selective. In order to differentiate between the two it is easiest to observe C. elegans in fluorescence experiments. Fluorescence, which is a form of visible radiation that is produced by a certain substance in response to other radiation of higher energy. Cells that have incorporated the substance are able to fluoresce, while cells without the substance are not. This fluorescence can be observed through fluorescence microscopes, which excite electrons within the sample by using high intensity light. This light excites electrons from their ground state into a higher energy excitation state. When electrons drop back down from the excitation state, energy is released and can be viewed as visible light. This light can also be filtered using filter cubes in a fluorescent microscope in order to show only the desired fluorescence.
Fluorescence microscopy can reveal the location of fluorescently labeled molecules so that they can be observed and tracked. However, this proves difficult because C. elegans, as well as other eukaryotes, contain naturally occurring fluorescent molecules. These molecules cause autofluorescence. In C. elegans, one of these autofluorescing molecules is a lipofuscin. Lipofuscins are lipopigments, which are also known as aging pigments because they accumulate over time. Lipofuscins are made of non-functional proteins cross-linked as a by-product of oxidative stress. Autofluorescence, such as that caused by lipofuscins, is equivalent to white noise. In order to separate desired fluorescence from autofluorescence, filter cubes can be used in the fluorescence microscope to “cancel out” autofluorescence.
This investigation utilizes four different treatments to follow the digestive activity of C. elegans. Fluorescence microscopy can be used to track three fluorescent labels, Acridine orange, FITC-microspheres and RITC-Dextran. The different sizes of the fluorescently labeled molecules allow for the determination of the different mechanisms that C. elegans use to uptake exogenous materials.
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