Fluorescence Spectroscopy: History, Principle and Application – Part-II

In this part, I will discuss some basic theories behind Fluorescence spectroscopy. In order to realize the potential of this particular spectroscopic technique, one must aware of the principle based on which this technique works. This will allow one to take complete advantage of this sensitive technique in applying in various scientific research.

Spectroscopy, in general, is applied quantum mechanics. Without going deep into the mathematical part, I will try to explain the basic principle of fluorescence spectroscopy rather qualitatively using Jablonski diagram.

Basic Principle of Fluorescence Spectroscopy:

Professor Jablonski, known as the father of fluorescence spectroscopy presented us with a diagram which describes various molecular processes in the excited state. As mentioned in my last blog that fluorophores play the central role in fluorescence. Prior to excitation with light (or photon), the electronic configuration of the fluorophore molecule is described as ground state. Upon absorbing  Jablonski diagram_finalphoton the electrons of the fluorophore molecule get raised to higher energy electronic level. The phenomenon of fluorescence occurs when the excited electron comes back to the ground state from the higher electronic energy level by emitting photon. A typical Jablonski diagram is basically an energy level diagram which illustrates electronic states of a molecule and transitions between them. The electronic states are arranged vertically by energy and grouped horizontally by spin multiplicity (see the inset diagram). Radiative transition is depicted by solid arrows, while the nonradiative transition is shown by squiggly arrows. Within each electronic state there are multiple vibrational energy levels (electronic levels are depicted with thicker lines and the vibrational levels are with thinner lines). As shown in the inset figure, the singlet ground, first and second electronic excited states are depicted by S0, S1 and S2, respectively, while first and second triplet excited states are depicted by T1 and T2, respectively. In singlet state, all the electrons of a molecule have their spin paired, while in triplet state, one set of electron spin becomes unpaired. These two states differ in properties as well as in energies; the triplet states always lie in lower energy than its corresponding singlet state. The transition between singlet to triplet state is forbidden. The probability of singlet-triplet process is 10-6 of the singlet-singlet and triplet-triplet processes.

The first transition in the Jablonski diagram is ABSORPTION. When a fluorophore molecule (or any molecule of interest) absorbs photon of definite energy the electrons in the ground state (S0) is excited to a higher energy level (S1 or S2) depending on the amount of energy absorbed. The process is very fast, and the time scale of absorption is in the order of 10-15 seconds. Once the electron is excited, there are multiple processes by which it dissipates energy and return to the ground state. First through VIBRATIONAL RELAXATION (VR), a non-radiative by which the electron gives away the energy in vibrational mode in the form of kinetic energy, and returns to lowest vibrational level of the corresponding excited electronic state. The Time scale Table time scale of VR is in the order of 10-14-10-11 seconds. Another process of energy dissipation occurs via INTERNAL CONVERSION (IC). IC is mechanistically similar to VR, and it occurs when vibrational level strongly overlaps with the electronic level, the electron in the vibration level of higher excited electronic state may relax to the vibrational level of the lower excited electronic state. However, due to lack of overlap between the vibrational and electronic levels and a large energy difference between ground state and the first excited electronic state, the probability of an electron to return to ground state via IC is very less. FLUORESCENCE (Fl) is another path through which an electron can dissipate energy and return to ground state. The time scale of fluorescence is in the order of 10-9-10-7 seconds. From the lifetime, one can tell that IC is generally complete before emission. Fluorescence emission generally results from thermally equilibrated lowest energy vibration level of S1 to the highest energy vibration level of ground state (S0), Anthracene abs and emission which then quickly thermally equilibrated (VR), and returns to the lowest energy vibration level of ground state. This singlet-singlet transition is allowed. Since emission involves the transition to highest energy vibrational level of ground state, the emission spectrum is typically a mirror image of absorption spectrum of the S0 → S1 transition. Electronic transition does not alter much the nuclear configuration, so the spacing between the vibrational energy level of the excited state remains almost the same as in ground state. This is the reason behind the similar vibrational structure of absorption and emission spectrum of a fluorophore molecule. However, there exists many exception of this mirror image rule. In case of proton dissociation, excited state reactions, charge-transfer complex formation, dimerization, one can observe deviation from the mirror symmetry rule.

Another process of non-radiative energy dissipation is known as INTER SYSTEM CROSSING (ISC) which involves a forbidden transition, where the electron changes spin multiplicity from excited singlet state (S1) to excited triplet state (T1). The emission from T1 to singlet ground state (S0) is known as PHOSPHORESCENCE and this forbidden transition is associated with several order smaller rate constant than that of fluorescence. The lifetime of phosphorescence is quite longer, in the order of 10-4 second to 1 minute.


1. Jihad René Albani. Principles and Applications of Fluorescence Spectroscopy. Blackwell Science Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK. Edition 2007.

2. Joseph R. Lakowicz. Principles of Fluorescence Spectroscopy.Third Edition. Springer.