Fluorescence Optical Microscopy
For some unique materials, incident ultraviolet light energy will excite the electrons within the material, resulting in the emission of photons in the visible spectrum. This phenomenon is known as fluorescence. Organic compounds which exhibit fluorescence are known as fluorophores. When a fluorophore is exposed to light of a particular wavelength, the electrons in the outermost electronic orbitals are transferred to a higher energy excited state, and then the electrons quickly relax to the original ground state. The energy difference between the higher energy excited state and the lower energy ground state represents a measure of energy that must be conserved within the system. In order to satisfy the law of conservation of energy, a photon of light is emitted from the compound with an energy approximately equal to the difference between the two electron energy states. The relaxation between energy states occurs very quickly (within microseconds), so photon emission is observed as being simultaneous with excitation. The emitted light energy is always less than that of the incident light energy due to a collection of interactions resulting in non-radiative processes, like heat energy loss.
Fluorophores, because of their unique properties, have proven very useful in the imaging of biological specimens. Some fluorophores can be cloned into the DNA of a cell to “tag” certain molecules within the cell, or fluorophores can be used to stain a cell by interacting with certain cellular components. Using fluorophores in such a manner allows a researcher to label specific cellular components or molecules with a fluorophore. Examination of the cell with a fluorescence microscope allows for the visualization of these individual components with high specificity over the rest of the cell. It is not uncommon to use multiple fluorophores within one sample to identify several components of interest.
Within a fluorescence microscope, incident light energy is passed through a filter which shifts the light energy to a particular wavelength, resulting in electron excitation and photon emission within a particular fluorophore. The light emitted from a sample is then passed through an emission filter to ensure that only the longer wavelengths of the emitted light are collected and the shorter wavelengths of the incident light are filtered out. The longer wavelength light is then used to create an image of the sample.
A compound will not fluoresce indefinitely; there is a limited amount of time before the intensity of the fluorescence begins to fade through two processes known as photobleaching and quenching. In order to preserve the life of a fluorescent sample, a user should turn off the shutter when live viewing of the sample is not required.