The ability of molecules to fluoresce, by undergoing constant cycles of energy absorption and emission, has proven to be an invaluable tool in cell biology research. From western blotting to immunocytochemistry or proximity ligation assays, fluorophores or fluorescent dyes can be used to detect, identify, and semi-quantify proteins at the sub-cellular level, over many orders of expression magnitude. Furthermore, their ability to emit specific wavelengths of light enables multiple proteins to be detected simultaneously.
The Jablonski diagram demonstrates the process of decay from an excited state to a more stable, lower energy state. An example can be viewed here.
The excitation (Ex) and emission (Em) maxima determine the key characteristics of fluorophores. Ex and Em refer to the wavelengths at which molecules absorb and emit the maximum relative intensity of light. Since the instrument that is being used to detect or image the biological sample relies heavily on these properties, it is imperative that the fluorophores in use possess spectra that are appropriate for the application.
Firstly, it is important that the excitation maxima is compatible with the excitation source such that the emission intensity is not compromised. You can view example graphs of optimal and sub-optimal excitation and emission spectra in this comprehensive guide by Bio-Rad.
Secondly, the spectral overlap should be minimal. When wavelength is plotted against relative intensity (of excitation or emission) for each molecule, a curve is generated, and spectral overlap is the area shared between both curves. The distance between Ex and Em is termed the ‘Stoke’s Shift’. Some fluorophores possess narrow spectra at high intensity whilst others have wider, flatter absorption and emission sprectra.
When detecting more than one protein, the spectra of individual fluorophores must not overlap significantly, or be excited by the source in similar ways. Spectral separation enables cleaner, more reproducible data to be achieved.
Read the full article by Bio-Rad to find out more detailed information on the excitation and emission process, as well as the products that will help streamline or multiplex applications involving fluorescence.