Absorption of energy by fluorochromes occurs between the closely spaced vibrational and rotational energy levels of the excited states in different molecular orbitals. The various energy levels involved in the absorption and emission of light by a fluorophore are classically presented by a Jablonski energy diagram, named in honor of the Polish physicist Professor Alexander Jablonski. This tutorial explores how electrons in common fluorophores are excited from the ground state into higher electronic energy states, and the events that occur as these excited molecules relax by photon emission and other mechanisms to ultimately fall back into the ground-level energy state.

A typical Jablonski diagram illustrates the singlet ground state (S(0)), in addition to the first (S(1)) and second (S(2)) excited singlet states as a stack of horizontal lines. In the tutorial, the thicker lines represent electronic energy levels, while the thinner lines denote the various vibrational energy states (rotational energy states are ignored). Transitions between the states are illustrated as straight or wavy arrows, depending upon whether the transition is associated with absorption or emission of a photon (straight arrow) or results from a molecular internal conversion or non-radiative relaxation process (wavy arrows). Vertical upward arrows are utilized to indicate the instantaneous nature of excitation processes, while the wavy arrows are reserved for those events that occur on a much longer timescale.

The tutorial initializes with a classical Jablonski diagram appearing in the window, which shows the ground state (S(0)), as well as first (S(1)) and second (S(2)) excited singlet energy states. Positioned to the right of the singlet states are the vibrational energy levels of the excited triplet (T(1)) state. In order to operate the tutorial, click on the Start button to initiate a sequence of excitation and relaxation events dictated by the process listed in the pull-down menu (Fluorescence Emission is the default). Once the electron arrives at an excited state vibrational energy level, it will slowly relax to the lowest vibrational level of the first excited state. From this level the electron will behave according to the selected process. The approximate lifetimes of electronic transitions appear in the tutorial window while each transition is occurring.

Absorption of light occurs very quickly (approximately a femtosecond, the time necessary for the photon to travel a single wavelength) in discrete amounts termed quanta and corresponds to excitation of the fluorophore from the ground state to an excited state. Likewise, emission of a photon through fluorescence or phosphorescence is also measured in terms of quanta. The energy in a quantum (Planck's Law) is expressed by the equation:

where E is the energy, h is Planck's constant, n and l are the frequency and wavelength of the incoming photon, and c is the speed of light. Planck's Law dictates that the radiation energy of an absorbed photon is directly proportional to the frequency and inversely proportional to the wavelength, meaning that shorter incident wavelengths possess a greater quantum of energy. The absorption of a photon of energy by a fluorophore, which occurs due to an interaction of the oscillating electric field vector of the light wave with charges (electrons) in the molecule, is an all or none phenomenon and can only occur with incident light of specific wavelengths known as absorption bands. If the absorbed photon contains more energy than is necessary for a simple electronic transition, the excess energy is usually converted into vibrational and rotational energy. However, if a collision occurs between a molecule and a photon having insufficient energy to promote a transition, no absorption occurs. The spectrally broad absorption band arises from the closely spaced vibrational energy levels plus thermal motion that enables a range of photon energies to match a particular transition. Because excitation of a molecule by absorption normally occurs without a change in electron spin-pairing, the excited state is also a singlet. In general, fluorescence investigations are conducted with radiation having wavelengths ranging from the ultraviolet to the visible regions of the electromagnetic spectrum (250 to 700 nanometers).

With ultraviolet or visible light, common fluorophores are usually excited to higher vibrational levels of the first (S(1)) or second (S(2)) singlet energy state. One of the absorption (or excitation) transitions presented in Figure 1 (left-hand green arrow) occurs from the lowest vibrational energy level of the ground state to a higher vibrational level in the second excited state (a transition denoted as S(0) = 0 to S(2) = 3). A second excitation transition is depicted from the second vibrational level of the ground state to the highest vibrational level in the first excited state (denoted as S(0) = 1 to S(1) = 5). In a typical fluorophore, irradiation with a wide spectrum of wavelengths will generate an entire range of allowed transitions that populate the various vibrational energy levels of the excited states. Some of these transitions will have a much higher degree of probability than others, and when combined, will constitute the absorption spectrum of the molecule. Note that for most fluorophores, the absorption and excitation spectra are distinct, but often overlap and can sometimes become indistinguishable. In other cases (fluorescein, for example) the absorption and excitation spectra are clearly separated.

Immediately following absorption of a photon, several processes will occur with varying probabilities, but the most likely will be relaxation to the lowest vibrational energy level of the first excited state (S(1) = 0; Figure 1). This process is known as internal conversion or vibrational relaxation (loss of energy in the absence of light emission) and generally occurs in a picosecond or less. Because a significant number of vibration cycles transpire during the lifetime of excited states, molecules virtually always undergo complete vibrational relaxation during their excited lifetimes. The excess vibrational energy is converted into heat, which is absorbed by neighboring solvent molecules upon colliding with the excited state fluorophore.

An excited molecule exists in the lowest excited singlet state (S(1)) for periods on the order of nanoseconds (the longest time period in the fluorescence process by several orders of magnitude) before finally relaxing to the ground state. If relaxation from this long-lived state is accompanied by emission of a photon, the process is formally known as fluorescence. The closely spaced vibrational energy levels of the ground state, when coupled with normal thermal motion, produce a wide range of photon energies during emission. As a result, fluorescence is normally observed as emission intensity over a band of wavelengths rather than a sharp line. Most fluorophores can repeat the excitation and emission cycle many hundreds to thousands of times before the highly reactive excited state molecule is photobleached, resulting in the destruction of fluorescence. For example, the well-studied probe fluorescein isothiocyanate (FITC) can undergo excitation and relaxation for approximately 30,000 cycles before the molecule no longer responds to incident illumination.

Several other relaxation pathways that have varying degrees of probability compete with the fluorescence emission process. The excited state energy can be dissipated non-radiatively as heat (illustrated by the cyan wavy arrow in Figure 1), the excited fluorophore can collide with another molecule to transfer energy in a second type of non-radiative process (for example, quenching, as indicated by the purple wavy arrow in Figure 1), or a phenomenon known as intersystem crossing to the lowest excited triplet state can occur (the blue wavy arrow in Figure 1). The latter event is relatively rare, but ultimately results either in emission of a photon through phosphorescence or a transition back to the excited singlet state that yields delayed fluorescence. Transitions from the triplet excited state to the singlet ground state are forbidden, which results in rate constants for triplet emission that are several orders of magnitude lower than those for fluorescence.

Both of the triplet state transitions are diagrammed on the right-hand side of the Jablonski energy profile illustrated in Figure 1. The low probability of intersystem crossing arises from the fact that molecules must first undergo spin conversion to produce unpaired electrons, an unfavorable process. The primary importance of the triplet state is the high degree of chemical reactivity exhibited by molecules in this state, which often results in photobleaching and the production of damaging free radicals. In biological specimens, dissolved oxygen is a very effective quenching agent for fluorophores in the triplet state. The ground state oxygen molecule, which is normally a triplet, can be excited to a reactive singlet state, leading to reactions that bleach the fluorophore or exhibit a phototoxic effect on living cells. Fluorophores in the triplet state can also react directly with other biological molecules, often resulting in deactivation of both species. Molecules containing heavy atoms, such as the halogens and many transition metals, often facilitate intersystem crossing and are frequently phosphorescent.

It is interesting to note that the emission spectrum of a fluorophore is typically a mirror image of the S(0) to S(1) absorption spectrum transition. This is due to the fact that electronic excitation does not significantly alter the geometry of the nucleus and the spacing of excited state vibrational levels is similar to that of the ground state. The end result is that fluorescence emission spectra recorded with a spectrophotometer often display similar, but reversed, vibrational structures to those observed in the absorption spectra.

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