The first step in molecular fluorescence is the absorption of light energy by a molecule. The molecule "accommodates" this additional energy by promoting an electron to a higher (excited) energy level. The additional energy can be released several different ways. The molecule could lose some of the energy through enhanced vibrations (red squiggly line). Fluorescence occurs when the molecule releases that remainder of the energy by emitting light. Because of the energy loss due to molecular vibration in between the absorbency and emission processes, the fluorescence is usually lower energy (higher wavelength) than the absorbency.
Instrumentation used to measure fluorescence involves 1) an excitation light source and a means to select the excitation wavelength, 2) a sample holder, 3) a means to select the fluorescence wavelength to be monitored, 4) a detector capable of generating a signal proportional to the intensity of light striking it, and 5) associated electronics and readout devices.
Fluorescence is capable of detecting analyte molecules present in extremely low concentrations (1 fluorescent molecule out of a billion molecules can be detected routinely). The fluorescence of a molecule is often sensitive to its local environment. Therefore, fluorescence signals can report information about the microenvironment surrounding a molecule.
Fluorescence has provided rich information regarding biomolecules and their dynamics. It has been used to monitor polymerization processes, detect bases on DNA, measure diffusion coefficients, investigate binding sites of antibodies, and probe the internal polarity of proteins, just to name a few.
KU Fluorescence Spectrometers
Photon Technology International QuantaMaster Fluorescence Spectrometer
The PTI Quantamaster fluorometer is the sweetest fluorometer that we have. Its modular design offers flexibility in orientation, and the dual-emission monochromators offer us access to just about every type of steady-state fluorescence measurement available. We have added polarizers to perform fluorescence polarization measurements, a 4-position automated sample holder, and fiber optic chucks on the excitation and emission monochromators. A computer controls all instrument functions. We have a HeCd laser that can be substituted for the Xe arc lamp source.
Partial Key to PTI C-61 Diagram
1: Xe Arc Lamp (excitation source)
3: Excitation Monochromator
(selects ex wavelength)
8: Sample Holder
11: Emission Monochromators
(selects fluorescence wavelength)
12: Detector Photomultiplier Tubes
Photon Technology International EasyLife Lifetime Fluorometer
This newest addition to our fluorescence measurement capabilities is capable of measuring fluorescence lifetimes. That is the time lag between absorption and emission of photons. This process typically occurs in the range of nanoseconds. (That's a billionth of a second!) The light source in this fluorometer is an rapidly pulsed LED. This instrument provides much more detailed information regarding molecular environments and interactions than can be obtained with steady-state techniques (where the light source is on continuously).
BioTek Synergy 2 Plate Reader
The Synergy 2 Plate Reader is capable of measuring absorbency or fluorescence of many samples in a fast, automated fashion that requires very little sample amounts. The open drawer seen in the image of the instrument accommodates a well plate.
We typically use plates with 96 separate compartments, so we can analyze up to 96 samples at a time.
Turner Model 112 Fluorometer
The Turner Model 112 is an inexpensive filter fluorometer from the early 1980s. Instead of using monochromators to select the excitation or emission wavelengths, this instrument uses filters. This lowers the cost dramatically, and has the ability to allow more photons to reach the detector. This particular instrument is in excellent shape, and can detect quinine in tonic water at the part per trillion range. By attaching a general purpose computer interface, we can use this instrument to examine kinetics of systems involving a change in fluorescence.
A few student projects have involved the construction of simple, inexpensive fluorometers. These fluorometers are based on LEDs for excitation sources, and silicon photodiodes for detectors. We can use a general purpose computer interface to digitize data, or simply use a voltmeter to monitor the output of the detector after it has been amplified.
Applications of Fluorescence
One of the classic fluorescence experiments that is performed in undergraduate curricula is the determination of quinine in tonic water. Quinine fluoresces when excited with UV light (around 350 nm). The intensity of quinine fluorescence (at around 450 nm) depends upon quinine concentration, providing a means to determine quinine concentration in an unknown.
One can also use fluorescence to determine characteristics of a molecules local environment. PRODAN is sensitive to the polarity of its environment. In a nonpolar solvent PRODAN's fluorescence is blue. In a polar solvent like water, PRODAN glows green. This phenomenon can be used to determine the extent of binding of PRODAN to cyclodextrin, because the binding results in a change in polarity of PRODAN's environment. The interior of CD is nonpolar. Therefore, when PRODAN moves from water to the interior of the CD, the fluorescence changes. One can use this information to determine the equilibrium constant for the binding process.
More sophisticated applications of fluorescence abound. Fluorescence has provided insights into biomolecule dynamics, solvation kinetics, and reached the ultimate detection limit - a single molecule! By measuring the time scale of fluorescence, one opens the door to a treasure trove of dynamic information.
The following list of fluorescence applications is by no means comprehensive.
Protein Dynamics and Denaturation
Fast Solvation Kinetics
Dynamics of Protein Folding
Structure and Flexibility of Membranes
Investigating Antigen-Antibody Binding
Selective Detection in Chromatography and Electrophoresis
Detection in DNA Sequencing
Determination of Ca2+ and Mg2+ Inside Cells
Remote Sensing using Fiber Optics