Is luciferase really all that different from other reporters, like green fluorescent protein (GFP)? To answer that question, we have to dig deeper into how these different luminescent processes work. Let’s get technical.
Luminescence is the process of photon (light) emission. When an electron transitions from an excited state to a ground state, if the conditions are right, a photon is emitted. Luminescent processes are categorized based on how the electron becomes excited.
There are two main kinds of luminescence: photoluminescence and chemiluminescence. In photoluminescent processes, like fluorescence, the electron gets excited by absorbing light. On the other hand, in chemiluminescent processes, such as bioluminescence, a chemical reaction provides the energy to excite the electron. This distinction makes a huge difference in terms of how photon emission is measured—and that’s the important part for experimental scientists, because only what is measurable matters.
Because fluorescence requires putting light in to get light out, there must be a way to distinguish between input light and output light. So, we use filters to keep some wavelengths of light out, and only let through the wavelengths we’re interested in measuring. Unfortunately, no filter is perfect, and some amount of the excitation light will make it through the filter, adding background noise.