Biomod/2014/Fluorescence
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Electrons are known to have discrete levels of energy. Without any stimulation the electrons are in the so called ground state. If the electron is excited by irradiation of visible light, the electron absorbs energy to reach higher – so called excited states – levels. These energy package can be described as photons of different wavelengths. The electron can also relax back into the ground state while emitting a photon, this process is known as emission. The energy of a photon is described by the Planck´s law. [1]
This equation uses c and h which are the constants for speed of light in vacuum and the Planck constant. Further λ is used as wavelength. <img src="http://openwetware.org/images/thumb/8/83/Jablonski.png/800px-Jablonski.png" width="" height="" > Figure 1: Jablonski-Diagramm with radiative crossings like absorption (light-blue), fluorescence (violet) and phosphorescence (orange) and radiationless crossing like internal conversion (dashed grey) and intersystem crossing (dashed red). Figure 2 shows excitation (green) and emission spectrum (dashed green) of the dye Atto 532. <img src="http://openwetware.org/images/thumb/6/62/Atto_532.png/800px-Atto_532.png" width="" height="" > Figure 2: Excitation (green) and emission spectrum (dashed green) of the dye Atto 532.[3]
The emission spectrum is the mirror image of the excitation spectrum. This could be explained by the Stokes-Shift. The Stoke-Shift depends on to different effects the variation of the vibronal states and the solvent relaxation.
The first effect describes that an excitation not only changes the ground state to an excitation state but also changes the vibronal state. To determined fluorescence it is necessary that the lowest vibronal state of the singlet-state is reached. The internal conversion makes this possible. The lost energy results in a higher wavelength.
The reorganization of a polar solvate in a polar solvent after excitation is described by the solvent relaxation. The solvent is aligned by the dipole moment μ of dissolved dyes. After an excitation the dipole moment could stabilize (μ ≤ μ*) or destabilize (μ > μ*) the dyes which results in a higher or lower energy and although in a lower or higher wavelength.[4] <img src="http://openwetware.org/images/a/a4/Weitfeld.png" width="" height="" > Figure 3: Schematic of a widefield microscope. The sample is irradiated with laser light, while the excitation- and the fluorescence-light are separated by a dichroic beamsplitter. A CCD camera is used for detection.[3]
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