In a follow-up to our introduction to fluorescence, we wanted to discuss a somewhat confusing(at least for us) detail of fluorescence spectroscopy: the fact that emission spectra and excitation spectra of the same fluorophore are mirror images of each other. It wasn’t until we drew out the diagram pictured above that we truly “got it.”
- First, excitation spectra are obtained when you excite a fluorophore with several wavelengths of light and measure the emitted light at a ONE wavelength (Jabloski Diagram). In the resulting fluorescent spectra, the “finger-like” is caused by each vibrational energy level slightly, increasing the energy the possible excitation light.
- Second, emission spectra are obtained when you excite a fluorophore with ONE wavelength and measure the emitted light at several wavelengths (Jabloski Diagram). In the resulting fluorescent spectra, the “finger-like” is the mirror image of the excitation spectra because each vibrational energy level slightly DECREASES the possible excitation light.
- Lakowicz, J. R. in Principles of Fluorescence Spectroscopy, Springer, 2006
- McQuarrie, D. A.; Simon, J. D. Physical Chemistry: A Molecular Approach., University Science Books, 1999
- Skoog, D. A., Holler, F. J. & Nieman, T. A. Principles of Instrumental Analysis (5th ed), Saunders College Publishing, 1998
This work by Eugene Douglass and Chad Miller is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.