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Category
Fluorescence imaging
Summary
Fluorescence imaging is a type of non-invasive imaging technique that can help visualize biological processes taking place in a living organism. Images can be produced from a variety of methods including: microscopy, imaging probes, and spectroscopy.
Fluorescence itself, is a form of luminescence that results from matter emitting light of a certain wavelength after absorbing electromagnetic radiation. Molecules that re-emit light upon absorption of light are called fluorophores.
Fluorescence imaging photographs fluorescent dyes and fluorescent proteins to mark molecular mechanisms and structures. It allows one to experimentally observe the dynamics of gene expression, protein expression, and molecular interactions in a living cell. It essentially serves as a precise, quantitative tool regarding biochemical applications.
A common misconception, fluorescence differs from bioluminescence by how the proteins from each process produce light. Bioluminescence is a chemical process that involves enzymes breaking down a substrate to produce light. Fluorescence is the physical excitation of an electron, and subsequent return to emit light.
When a certain molecule absorbs light, the energy of the molecule is briefly raised to a higher excited state. The subsequent return to ground state results in emission of fluorescent light that can be detected and measured. The emitted light, resulting from the absorbed photon of energy hv, has a specific wavelength. It is important to know this wavelength beforehand so that when an experiment is running, the measuring device knows what wavelength to be set at to detect light production. This wavelength is determined by the equation:
Where h = Planck's constant, and c = the speed of light. Typically a large scanning device or CCD is used here to measure the intensity and digitally photograph an image.
Fluorescent dyes, with no maturation time, offer higher photo stability and brightness in comparison to fluorescent proteins.
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