 |
 |
 |
 |
home I what is...? I precisExcite I application notes I distributors I news I benefits I endorsements I filters I gallery I contact
What is Fluorescence Microscopy?Fluorescence illumination and observation is the most rapidly expanding microscopy technique employed today, both in the medical and biological sciences, a fact which has spurred the development of more sophisticated microscopes and numerous fluorescence accessories.
Introductory Concepts
Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which is formally divided into two categories, florescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway. Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state lifetime.
Fundamental Aspects of Fluorescence Microscopy
The modern fluorescence microscope combines the power of high performance optical components with computerized control of the instrument and digital image acquisition to achieve a level of sophistication that far exceeds that of simple observation by the human eye. Microscopy now depends heavily on electronic imaging to rapidly acquire information at low light levels or at visually undetectable wavelengths. These technical improvements are not mere window dressing, but are essential components of the light microscope as a system.
Anatomy of the Fluorescence Microscope
In contrast to other modes of optical microscopy that are based on macroscopic specimen features, such as phase gradients, light absorption, and birefringence, fluorescence microscopy is capable of imaging the distribution of a single molecular species based solely on the properties of fluorescence emission. Thus, using fluorescence microscopy, the precise location of intracellular components labeled with specific fluorophores can be monitored, as well as their associated diffusion coefficients, transport characteristics, and interactions with other biomolecules. In addition, the dramatic response in fluorescence to localized environmental variables enables the investigation of pH, viscosity, refractive index, ionic concentrations, membrane potential, and solvent polarity in living cells and tissues.
Practical Aspects of Fluorescence Filter Combinations
Microscope manufacturers provide proprietary filter combinations (often referred to as “cubes” or “blocks”) that contain a combination of dichroic mirrors and filters capable of exciting fluorescent chromophores and diverting the resulting secondary fluorescence to the eyepieces or camera tube. A wide spectrum of filter cubes is available from most major manufacturers, which now produce filter sets capable of imaging most of the common fluorophores in use today.
Light Sources
In the past, powerful light sources were needed to generate enough excitation light intensity to furnish secondary fluorescence emission capable of detection. These were usually either mercury or xenon arc (burner) lamps, which produce high-intensity illumination powerful enough to image faintly visible fluorescence specimens. With the introduction of precisExcite®, it is now possible to select high intensity LED / Light Emitting Diodes supplying light fluxes of similar intensities without any of the drawbacks of conventional arc systems.
 |
||||||||
|
||||||||
|
