Physical Properties that Define Fluorescence

Fluorescent molecules by definition absorb light at one color (wavelength) and emit it at another. The difference in colors is called the Stokes shift. The cameras used in fluorescence microscopy allow the detection of signal beyond the wavelengths our eyes can see.

Learn about the physical properties that define fluorescence, including wavelength, how energy relates to fluorescence and fluorescent colors, and what defines a fluorescent molecule's spectra.

The range for fluorescence imaging extends a bit beyond where our eyes can see. Using this extended range is no problem because the CCD cameras that collect light emitted from our sample in a typical fluorescence microscopy setup have a wider range than our eyes do. In practical terms, imaging wavelengths for cell biology are usually in the range of 300–800 nm.

Figure 2. The range and efficiency of light detection for a CCD camera compared to that of the human eye.

But what is special about the term fluorescence? Fluorescence refers to the physical property of an object absorbing light at one wavelength and then reemitting it at another wavelength. If a molecule absorbs the light of one wavelength and emits it in another (i.e., fluoresces), we call that molecule a fluorophore. Usually the wavelength the molecule emits will be lower energy than what it absorbed, so in simpler terms we could say that something absorbs blue light and emits green, or absorbs green light and emits red.

Further study: Watch "Introduction to Fluorescence"

Figure 3. The inverse relationship between energy and wavelength in the visible spectrum.

Figure 4. Simplified Jablonski diagram showing the energy state change of a fluorophore’s electron as it undergoes fluorescence, with the corresponding change in the color of light.

To understand this at a deeper level, we need to think about photons, the packets of energy that make up light. The magnitude of energy that a photon contains determines its color or, in physical terms, its wavelength. When the light (or photon) hits a fluorophore, the energy is transferred to the fluorophore’s electrons. The electrons are excited, but then rapidly lose that extra energy (that sounds like people too, doesn’t it?). The end result of this loss of energy is the emission of a photon of light, but that photon will have less energy than the original photon, so it will have a longer wavelength and be a different color. The emitted photons are the signal you need to collect as data during your imaging experiment.