Epifluorescence Microscopes: Functions and Uses
Fluorescence is an important technique in microscopy, typically used for viewing biochemical and physiological changes in live specimens. This is the type of microscopy utilized for many medical studies and medical-related fields. This makes epifluorescence microscopes, a type of fluorescence microscopy, important tools in many biochemical and physiological researches and experiments.
How does epifluorescence work?
To understand epifluorescence, it is important that the technique of fluorescence microscopy is discussed first. Fluorescence is quite simply the ability of certain molecules and atoms present in certain organisms to absorb light and emit it using a specific wavelength. The period between light absorption and emission occurs within a short interval and is referred to as the fluorescence lifetime. One fluorescence lifetime can be extremely short – about 1/100,000th of a second, even less – or it could last for several hours. A short period is called a fluorescence while a long period is called a phosphorescence. Either way, the light energy that the specimens produce is generally less than the energy of the excitatory light that started the fluorescence.
Fluorescence occurs when the atoms in a specimen are excited by light energy. This excitation makes the electrons jump to a higher level of energy. These electrons then return to their original ground state and release a photon, which causes a short pulse of light. As a result, the atom – and the specimen – fluoresces.
Unlike other types of microscopy, fluorescence microscopy uses the inherent illumination of the specimen in order to observe it. These instruments typically make use of xenon, tungsten or mercury lamps to produce UV light. This is the same UV light that is used as excitatory light.
Epifluorescence microscopy
Fluorescence microscopy is both an observation and illumination technique that has given rise to various improvements in the observation of minute organisms and cells and produced very sophisticated instruments. The epiflouorescence microscope is one of these.
Epifluorescence is also known as incident light fluorescence. ‘Epi’ means above, which refers to the light source that is located above the specimen. Although it is basically a light microscope, the epifluorescence microscope is also equipped with other capabilities which help process images of specimens differently. In epifluorescence microscopy, light energy is still used to excite the atoms in a specimen to encourage fluorescence. However, instead of allowing the excitatory light to be transmitted through the object, it is allowed to pass first through the microscope’s objective before it hits the specimen. This process filters out the transmitted light, which produces images of high intensity. To encourage this, fluorochrome or fluorescent stains are often used to allow observers to view the specimen in greater detail. This stain helps produce a visible enough view to allow scientists and medical personnel to perform such tasks as a physical count of the bacteria in a sample.
How is the epifluorescence microscope set up?
Epifluorescence refers to the type of fluorescence microscopy where the set up of the objective lens makes a difference. Instead of using a separate condenser or lens to focus UV light on the object or specimen being observed, epifluorescence microscopy uses the objective lens to concentrate the UV light on the target and collect whatever fluorescent light the specimen bounces back.
Using a dichroic mirror, the paths of the excitatory light and the emission light are separated. This then produces a visualization of the specimen’s fluorescence emission, which the observer views through the eyepiece.
Uses of the epifluorescence microscope
Epifluorescence microscope is considered a more efficient type of microscopy compared to transmitted light fluorescence. It is especially effective in viewing live cells or organisms which produce their own fluorescent light. However, it is more commonly used to observe specimens using fluorescent dyes, one of which is the calcein/AM. This is the dye that produces a green fluorescence in living cells when used under UV light.
Epifluorescence microscopy is extremely useful for the observation of living cells. It is often used in many toxicology studies where cell populations are observed upon treatment of certain types of chemicals. It is also excellent for use in the observation of cell cultures, both live and treated. This type of microscopy allows observers to view structures in living cells and take note of ongoing biochemical and physiological changes. This is the same microscopy technique that is used to observe enzymes, substance alkalinity or acidity, DNA and antibodies, most of which are difficult to view using other types of light microscopy.
The epifluorescence microscope is the most commonly used type of fluorescence microscopy, especially in biology, chemistry and other live sciences. Thanks to its development, other more sophisticated and highly valued microscopy techniques have been created, including the TIRF or total internal reflection fluorescence microscope and the confocal laser scanning microscope.
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