Viewing molecules through photons
Fluorescence is one of the most widely used spectroscopic measurements. Its advantage over other methods is in its sensitivity. Both in the sense that less material can be used (low concentration) and small changes can be detected. MicroLab’s FASTscan™ spectrophotometer simultaneously measures the fluorescence, transmittance, turbidity, scatter and nephelometry, and calculates absorbance during each scan.
Fluorescence is a mechanism for molecules to release energy by emitting light. The molecule acquires this energy through absorption of light energy (photons), from heat or from a chemical reaction. Let’s approach it with a baseball analogy. A pitcher throws a baseball at high velocity to a catcher. The pitcher is a laser (or other light source) and the catcher is our fluorescent molecule (also known as a fluorophore) and the baseball is a photon. The catcher catches the ball, then transfers it to their throwing hand (using some energy), then throws the photon back to the pitcher at a much lower velocity. Correlating this to what happens in fluorescence: the photons from a laser collide with fluorophores in solution. The fluorophores capture this energy and use it to transfer an electron from a ground state orbital (S0) to an excited state orbital (S1* and/or S2*). This is absorption and is depicted by red arrows in the Perrin-Jablonski diagram shown below.
Figure 1: Perrin-Jablonski diagram
There is a short time delay before the electron relaxes back to the ground state. During this delay some energy goes into molecular vibrations and the electron relaxes to the lowest energy electronic state (S1). This is known as internal conversion and is represented by the black dashed arrows below. Complete relaxation to the ground state then occurs, and a photon is emitted (a.k.a. fluorescence, thick green arrows). Because some energy is consumed during internal conversion, the energy gap associated with fluorescence is less than the energy gap of absorption and the resulting emission is seen at longer wavelengths (known as Stoke’s Shift). As shown in the excitation chart below, chlorophyll fluoresces when struck with photons in the 400 and 635 nm regions – the absorption maxima for chlorophyll in a green plant. The inset graph, taken with our fiber-optic spectrophotometer adaptor, shows the fluorescent emission peak at 676 nm demonstrating how emission is always at a longer wavelength than excitation, known as Stoke’s Shift.
Therefore, in the absence of interfering processes there are 3 events occurring during the production of fluorescence: (1) photon absorption, (2) internal conversion and (3) photon emission. Photon absorption is the catcher catching the ball, internal conversion is the catcher transferring the ball to his throwing hand, and fluorescence is the catcher throwing the ball back to the pitcher. Any event that disrupts this process can cause quenching of the fluorescence signal. Quenching is often used to track binding or conformational changes. Additional energy is sometimes lost in ‘intersystem crossing’ resulting in a delayed emission which is called ‘phosphorescence’.