Perhaps the most beautiful pictures in the world are those created using immunofluorescence. Fluorescence is a mechanism of luminescence, wherein energy released by a molecule is perceived in the form of light. The mechanism of fluorescence is very useful in the field of biology because it is used in immunofluorescence. All immunofluorescence does is attach the fluorophore (a molecule which possesses the property of fluorescence) to a molecule of biological importance.
Mechanism of Fluorescence and Phosphorescence
The basic principles of fluorescence occur at to the atomic level of any element. An atom consists of a positively charged nucleus, surrounded by negatively charged particles called electrons. These electrons are placed at different energy levels. It so happens that an electron in a lower energy state absorbs energy from a photon i.e. a packet of light energy, thereby increasing its energy to a higher level. However, an electron at a higher energy state is not stable.
Unstable electrons need to release that extra energy to come down to a more stable ground state. This energy released appears in the form of light. The mechanism is called fluorescence, if the emission of light is very rapid, following the removal of the source of energy; or else known as phosphorescence, if the emission continues for a few seconds to minutes after the removal of light source.
Using Stokes Shift
Light emitting molecules absorb light of a particular wavelength to emit radiation, which falls in a particular range of wavelength. There exists an optimum wavelength at which emission of light would be at a maximum. The shift in wavelength from short to long between absorbed and emitted light is known as Stokes Shift, after the name of the founder of fluorescence, Sir George Stokes. Many fluorescent molecules have a small Stokes Shift whereas others molecules have a larger Stokes Shift.
- The molecule fluorescein absorbs blue-green light and emits green light. Hence it has a small Stokes Shift.
- The molecule erythropoietin emits yellow-orange light even though it absorbs the same blue-green light, displaying a large Stokes Shift.
Mechanisms of Immunofluorescence
In immunofluorescence, one antibody, known as a primary antibody, binds to a protein molecule of interest, commonly referred to as antigen. A secondary antibody, which is attached to a fluorophore, goes and binds to the primary antibody. This fluorescent molecule emits light and thus, binding of the antibody to the specific antigenic site can be detected.
The binding of antigen-antibody is much like matching a key to a lock. The antibody has a forked structure, in which the forked arms are known as Fab region and the single base is called the Fc region.
Direct and Indirect Methods
- With direct fluorescence, only one antibody is used, which attaches to both the antigenic sites and the fluorophore with its Fab and Fc regions respectively. This takes less time for detection than indirect methods.
- With indirect method, the Fab of the primary antibody binds to the antigenic site, and the Fc region binds to the Fab region of a secondary antibody. The Fc region of the secondary antibody is attached to the fluorophore. This mechanism is very practical and useful since one secondary antibody can go and bind to several different primary antibodies.
- In both methods, the fluorescence emitted is detected by auto imaging instruments
Disadvantages of Immunofluorescence
Photobleaching- Destruction of fluorophores by reactive oxygen species
Autofluorescence- Caused by the presence of co-enzymes in mammalian cells
Emission signaling- When emission signals overlap causing false detection of fluorescence
Immunofluorescence is widely used to study the cellular construction of known antigens in frozen tissues, or localization of specific DNA sequences on chromosomes. It is a highly developed field that incorporates biology, chemistry and physics in ways no other imaging process can.
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- The Molecule That Made the Universe – University of Arizona – Astrobiology Magazine (richarddawkins.net)
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