How Fluorescence Works Step by Step Easily Explained

How fluorescence works may seem mysterious, but it’s a simply step by step process. You don’t need to know much about chemistry to actually understand what happens.

We just have to be comfortable imagining the inside of a molecule, where busy bee electrons buzz with their respective energies. They are easily influenced by incoming light, and have their own reactions to being disturbed by certain kinds of light.

How Fluorescence Works: the Basics

The smallest component of fluorescent material is the fluorophore, a single molecule that can exhibit fluorescence. But, when we talk about fluorescence in dyes and fluorophores, remember we do not have a single molecule isolated. It is a clump of at least hundreds or thousands of individual molecules.

The smallest component of light, though not something we can really isolate at all, is the photon. A photon has a unit of energy that we can measure and see the effects from. If we think about a single photon, its character and behavior is determined from its energy state. Energy of light and photons is related to its wavelength, so when I talk about the wavelength of a photon (unit of light), you can also think of this as its energy.

Wave-coupling: Striking the Fluorophore’s Secret Personality

The photon will excite the fluorophore only under the condition that its wavelength is just right. The electrons of the fluorophore can absorb the energy from the photon, effectively stealing the energy from the photon and thus lowering the photon’s energy and raising the fluorophore’s energy.

Each dye has its own secret world, its absorption spectra, which colors it likes and dislikes. Really this is the map of wavelengths likely to wave-couple (resonate with) the outermost electrons. A dye’s electronic structure and absorption spectra is its secret personality that can be unlocked by scientists’ tests and probing. Coupling occurs when the photon has the same energy as the electron needs, it’s like people at a party with similar interests naturally gravitating towards one another and then their voices amplify in conversation.

Redshifting: The Hidden Changes that Happen to Light

Ground state S₀ –> excited state S₁ :

This is the technical excitation event we will soon delve into. The entire time the electron is excited for lasts on the order of 1 to 10 billionths of a second (nanoseconds). During that short time, some of the energy the electron stole from the photon is also dispersed, randomly leaking out without emitting light.

The electron cannot stay excited for long, because the ground state is more energetically stable. So the energy will release from the electron naturally, dropping it back to the ground state, S₀ from S₁. The release of that exact energy is the “fluorescence” phenomenon. This energy started as light, and when it releases, it has no choice but to become light again. However, there is an important change the light has undergone since entering the electron.

Remember when some energy was randomly dispersed on account of entering the electron? Well, that energy lost means the wavelength of the energy packet released by the electron when it falls from the excited state is NOT going to be the exact same wavelength as when it entered. Lower energy means a longer wavelength, and shorter to longer wavelength of visible light is what our eyes detect as a blue to red shift. That’s called the Stoke’s shift, resulting from non-radiative transitions we discuss below.

Step by Step: How Fluorescence Works in Diagrams

So what confers some molecules with the ability to fluoresce? Every molecule has a structure of electron orbitals, and energy thresholds between the energy levels that the electrons can occupy. Chemicals that can fluoresce have a lower band of electrons, the pi or p orbital electrons, under the “S” ones discussed above. These are very stable and their energy is a bit smeared and shared throughout them, known as a conjugated pi system. Alongside this, fluorescent molecules have electrons that are able to “donate” themselves to a higher energy level. The electrons have mobility and “communicate” with one another.

For example, quinine, known for its anti-inflammatory properties, is present in tonic water. Its conjugated system is circled in blue and its electron donating group circled in red. Fluorescence from transitions of electrons from that blue circled group rarely ever happens. The transitions from the lower, pi states gives the fluorescent signature energy release. This is from the lowest vibrational level, the ground state, to the first excited state. The yield will be much greater for these lower states because probabilistically, more electrons inhabit them. In the higher levels, there are also processes that tend to compete and deactivate the fluorescence.

Another fluorescent molecule is fluorescein. Here is its structure, spectra, and under UV.

Step 1: Excitation

S₀ typically denotes the ground state. This is the energy the outermost electrons hold when the molecule is “just chillin.” An incoming photon has energy expressed by h/λ, Planck’s constant divided by the wavelength. Planck’s constant is just a number that physicists had to start including in their equations to make the numbers with our systems of measurements work out. After excitation of the fluorophore with the photon of energy h/λ, the energy is transferred to those outermost electrons, exciting them to the first level above the ground state, S₁.

Every photon absorbed does not result in a photon emitted. Remember that when we shoot a laser beam of diffuse light, there are very many photons hitting the molecule. Some of these will photobleach, rendering them inactive and unable to emit. The ratio of the emitted photons to absorbed photons is called the quantum yield, and is physically interpreted as proportional to the brightness, or intensity of emission. Compounds having quantum yields of at least 0.1 are in the realm of fluorescence.

Step 2: Excited State Life-time (it flies by)

In the excited state the electron is a bit uncomfortable and tends to fidget and jostle. The electron here will undergo both non-radiative transitions, which do not emit light, and radiative transitions. The important radiative transmission results in the fluorescence light. The non-radiative transitions result in the redshifting of the emitted color.

The time spent in the excited state is about 10^-15 to 10^-9 seconds. So, to our eyes, fluorescence happens instantaneously. Only with special instruments can the lifetime in the excited state be determined. Scientists do like to find the exact excitation lifetime in order to characterize the dyes, and also because some processes they image occur close to those time scales as well!

Jablonski diagram

Below is a typical Jablonski diagram, which shows the energy levels the electron inhabits. Researchers make specific Jablonski diagrams with the energy levels labelled characteristic to the molecule they are studying.

The intervening vibrational relaxation redshifts the emitted photon, since some energy lost is non-radiative. This energy goes mainly towards the actual kinetic energy of the electron, what actually moves it and gives it an acceleration to physically change states in the first place.

Step 3: Emission & Stokes Shift

The energy released is according to how far the electron drops in energy levels. The energy, related to the wavelength, determines the color of the fluorescence and the signature of a particular fluorescent dye. Remember, the energy loss that redshifts the photon results from vibrational relaxation while in the excited state. After dropping levels again, the electron can undergo the fluorescent process again and again.

Rules and Principles

Here are some of the jargon and useful principles scientists have expanded on since discovering the basics of fluorescence.

First, the Stoke’s shift, named for GG Stokes who first observed it. This rule I have already explained and referenced, since it is vital to the phenomenon of fluorescence itself. The light emitted has a longer wavelength, aka “redshifted” compared with the absorbed photons. The energy loss (longer wavelength, lower energy) is due to the non-radiative transitions such as vibrational relaxation the electron experiences while in S₁.

A more recent and more often violated rule is Kasha’s rule. Kasha’s rule depicts that the quantum yield (related to brightness we see) doesn’t depend on the wavelength of absorbed light. The fluorescence spectrum is the same regardless of excitation color, as long as that color can couple with the electrons sufficiently.

The Mirror Image rule applies for many common fluorophores, stating the absorption and emission spectra are reflections of each other.

Now You Know How Fluorescence Works

Basically, the dye absorbs light, the electrons are excited, and then they settle down and release the light. So now this black box of mystery makes sense, and you can think of ideas to play with this phenomena in your own life. Check out examples of fluorescence in everyday life and you can have a greater appreciation for the mundane.