Florescent Microscopy
Biochemistry 3401
Alyssia Sloan
Dallas Baptist University

September 20, 2011 Science has made incredible advancements in ways that one can see very small particles. One of these new advancements is in fluorescent microscopy. Biologists have just recently started to discover the new wonders of fluorescent microscopy, and even though it is a newer technique, it has been evolving at a very rapid rate and has helped scientists to discover so much. Fluorescent microscopy in its technique is similar to just a regular conventional light microscope, only with a little augmentation. Conventional microscopes magnify very small objects using visible light. Because visible light is about 400 to 700 nanometers, this is the limit of conventional microscopes. Fluorescent microscopes use an elevated strength of light source. This technique is often used to see very precise features on really small samples like microbes. Fluorescent microscopy is useful in that it can visually enhance the 3D features of very small objects. The method that is used to do this is to attach small fluorescent tags, known as a fluorophore, to antibodies that are then attaching on to target features that can then be seen. The researcher can then visualize the unique features of that target, for example an organelle. Many times, a very powerful light source is necessary to be able to be absorbed by the fluorophores that are attached on to the target, therefore lasers are used. The result of this process is that it will emit a longer and lower energy wavelength light, creating fluorescent light. Then the researcher can gather up all the data and construct a 3D image. Because of the highly advanced fluorescent microscopes, they are able to produce the sharp images that are therefore used for constructing the structures of small specimens, conducting viability studies on cell populations, imaging DNA and RNA, and viewing specific cells in a population (Bradbury, 1996). This technique is very useful in biology and has given answers to many questions scientists have had on imaging. However, even though many biologists use this technique, many have very little experience in how it actually works. There are many proteins that are used when tagging the sample that is to be observed. Of the most noteworthy is the green fluorescent protein, which allows molecular biologist to genetically tag cell components of living systems, a new opening for this technique. As previously mentioned, fluorophores are the molecules that are used for their fluorescent properties. This is possible because when they are in their ground state they absorb photons (light energy), vibrational and rotational states of the molecule. The transition into an excited state happens very quickly and this will set in motion molecular vibrations. In order to let out this “excited” energy, it will release it by creating an emission of fluorescents and thus returning to its ground state (Litchman, 2005).
There are many organic substances that have auto-fluorescence (intrinsic fluorescents). The technique of fluorescent microscopy takes advantage of these and synthesizes compounds that will have some degree of conjugated double bonds, usually aromatic molecules with pi bonds. These compounds are the most favorable for fluorescent microscopy because of the small difference between the ground state and the excited state. This can be seen in the Green Fluorescent Protein (GFP) that has an alteration of three amino acids (Ser-Try-Gly). The protein forms an imidazolidone ring with conjugate double bonds. The use of fluorescent microscopy is expanding very quickly to all fields of cell and molecular biology and understanding its basic chemical functions is crucial to be able to take full advantage of what it has to offer. These genetically engineered fluorescent proteins are one of the most powerful tools scientists have in order to look into