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

Fluorescence spectroscopy has been applied to numerous analytical, bio-analytical, environmental, clinical and forensic investigations. There is a need for a highly sensitive detection tool that can replace the expensive and difficult to handle radioactive tracers, but at the same time the tool/method has to be of low cost, easy to handle and can detect analytes in rapid time. Fluorescence spectroscopy has answer to all these. Fig1_Fluoro_part 3To explain the highly sensitive detection capacity of fluorescence as a tool, Professor J.R. Lakowicz discussed an example in his book “The Principles of Fluorescence Spectroscopy”. He mentioned that since fluorescence intensity is measured directly in relatively dark background (see the inset figure) without the presence of bright reference beam as in case of absorbance, it becomes easy to measure even in low level of light, and electronic impulses of the single photon can be read by the most photomultiplier tubes.1 On the other hand, if we try to measure the absorbance of a solution of concentration 1 nanomolar  (10-10 M) with molar extinction coefficient (e) of 10-5 M-1 cm-1, the absorbance will 10-5 per cm (%transmission= 99.9977). It is very difficult to measure only 0.0023% absorbed light even with highly sophisticated optical system. Following two schematic diagrams represent very basic model of UV-vis and fluorescence spectrophotometer which will help us to understand the technical difference between these two techniques regarding the sensitivity in measurement as explained above. This explains the high sensitivity of fluorescence spectroscopy as a detection tool.

Fig2_Fluoro_part 3

Fluorescence based sensing technologies have been constantly growing with the invention of innovative methods and materials. I will discuss various applications based on fluorescence detection/sensing. Before that, we need to understand the different characteristics of fluorescence emission such as Stokes shift, fluorescence lifetime and quantum yield, steady and time-resolved fluorescence, fluorescence anisotropy, fluorescence quenching, fluorescence resonance energy transfer (FRET), and the molecular information obtained from these.

Fluorescence emission spectrum and Stokes shift

Stokes shift is the difference between the position of absorption band maximum and emission maximum of the same electronic transition in frequency or in wavelength unit (inset figure below). Fig3_Fluoro_part 3Fluorescence always occurs at the higher wavelength than the absorption. The reason can be attributed to the relaxation of the excited electron from the higher vibration energy level to lower vibrational level of S1 and further decay to higher vibrational energy level to S0. Thus, the excitation energy is lost by the thermalization of excess vibrational energy. Irish Physicist, Sir G. G. Stokes first reported this phenomenon in 1852. In addition to this, further Stokes shift can be observed due to solvent effect, pH, excited state reaction, complex formation and energy transfer. From the measurement of Stokes shift, different molecular information can be obtained. As fluorophores are generally sensitive to the environment, by examining the position and intensity of the emission spectrum location of moleculer probe (here the fluorophore attached to some macromolecules) inside a macromolecule can be identified. The property of certain fluorophore being weakly Fig4_Fluoro_part 3fluorescent in aqueous environment but strongly while binding to target biomolecule accompanied by Stokes shift has been widely used. Moreover, utilizing the environment sensitivity of certain flurophores for example indole group of tryptophan residue in protein may reveal whether the protein is in folded or unfolded (denatured) state. Emission from a residue shifts to longer wavelength once it is exposed to the surrounding solvent (here water) due to unfolding. In the folded state, the protein shields it from the solvent. Therefore, conformation of proteins can be obtained from emission intensity and Stokes shift (see the inset figure at the left).

I will talk about the other characteristics along with applications in the future posts. Continued……………


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.


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

Fluorescence spectroscopy, a very sensitive analytical tool, has wide ranges of application in various disciplines of scientific/medical research. I am going to write a series of blog-posts discussing its numerous applications. To begin with, let me first go back to the history; how “FLUORESCENCE” was discovered, and evolved as a primary research tool in diverse fields of scientific research such as chemistry, biochemistry, biophysics, biotechnology, genetics, forensic, medical diagnostics, etc. to name a few.

A Short History of Fluorescence:

Nicholás Monardes, a Spanish physician and botanist observed a bluish opalescence from water infusion of a wood of a small Mexican tree. In 1565, he described about this observation in the Historia medicinal de lascosasque se traen de nuestras Indias Occidentales. A Franciscan missionary named Bernardino de Sahagún also independently observed similar observation for the wood named “coatli”, around same time. He reported in the Florentine Codex, “Coatli …..patli, yoanaqujxtiloni, matlaticiniayoaxixpatli..“, which means “it is a medicine, and makes the water of blue color, its juice is medicinal for the urine”. In 1574, Charles de L’Écluse, a Flemish botanist named Monardes’s wood as Lignum Nephriticum (kidney wood) because of its therapeutic properties in treating kidney related ailments. Thereafter, many scientists reported this type of luminescence property in various substances such as chlorophyll, barium sulfate, etc. Sir John Frederich William Herchel first observed the fluorescence from a solution of quinine sulfate (in tartaric acid) in sunlight in 1845, and described it as “beautiful celestial blue color”. This was published in Philosophical Translation of the Royal Society of London (1845) 135:143–145. Sir John Herchel termed this phenomenon as “epipolic dispersion”. [Inset figure shows the fluorescence image1 from a quinine sulfate solution.] Later in 1852, G.G. Stokes published a very long article (more than 100 pages), “On the change of Refrangibility of Light”, where he mentioned about his disagreement on Sir John Herchel’s term of “epipolic dispersion”, and wrote; “I confess I do not like this term. I am almost inclined to coin a word, and call the appearance fluorescence from fluor-spar, as the analogous term opalescence is derived from a mineral.” G.G. Stokes was the first person who proposed to use fluorescence as an analytical tool in a lecture “On the application of the optical properties to detection and discrimination of organic substances” in 1864. Following are the important research works done in much earlier days (1904-1942), which immensely contributed to the understanding, improvement and advancement in Fluorescence spectroscopy as a technology.

1905: The first excitation spectrum of a dye – E. Nichols and E. Merrit

1919: Fluorescence quenching – Stern and Volmer

1924: Determination of fluorescence yield -S.J. Vavilov

1925: Theory of fluorescence polarization-F. Perrin

1926: First direct measurement of nanosecond lifetime – E. Gaviola

1935: Jablonskidiagram – A. Jablonski

1948: QM theory of dipole-dipole interaction – T. Förster

Fluorophores are mainly organic compounds which play the central role in fluorescence. They not only absorb light of specific wavelength, but also emit light at specific wavelength. The energy of this emitted light depends on the fluorophore as well as on the surrounding environment of the fluorophore. R.Meyer in 1897 first coined the term “fluorophores” to describe those compounds or the specific image2 functional groups responsible for the phenomenon of fluorescence. A lot of fluorophores has been discovered such as fluorosceine, eosine, quinine, rhodamine, acridine, etc. to name a few. The first fluorometric analysis was performed by F. Goppelsröderin 1867 for the quantitative determination of Al(III) from the fluorescence of its morin chelate. Otto Heimstaedt and Heinrich Lehmann (1911-1913) first developed the fluorescence microscope to investigate the autofluoresecence of biosamples such as bacteria, protozoa, plant, and animal tissues. Later, American Instrument Company (AMINCO) collaborated with Dr. Robert Bowman who designed the instrument and marketed first ever spectrophotofluorimeter (SPF) in 1956 (inset picture) (http://history.nih.gov/exhibits/bowman/HSfluor.htm). Antimalarial research actually initiated the invention of a spectrophotofluorimeter as an analytical instrument which can determine the presence of analytes which fluoresce. The story dated back to 1940, during World War II, when scientists in USA required to determine the amount of drug reached to the malaria parasites in patient’s blood for a clinical trial of antimalarial drugs. Bernard Brodie and Sidney Udenfriend of Goldwater Memorial Hospital in New York City designed a new test using an instrument called fluorimeter which can determine the amount of the drugs in the blood plasma from the intensity of the fluorescence emitting from the drug, since many of the drugs used in the trial fluoresce. This helped them to come up with a critical dose of a drug minimizing the adverse side effects. Atabrine was one such promising drug which destroys malaria parasite effectively. Scientists at Goldwater realized that this technique has immense potential in scientific research, and needed a better instrument to utilize the full potential of this new spectroscopic technique. Dr. James Shannon, the leader of antimalarial research at Goldwater became the first director of NIH (National Institute of Health) at Bethesda, Maryland, USA, and recruited a team of scientists to design a new instrument utilizing the principles of fluorescence. Dr. Robert Bowman led this team and came up with the design of first spectrophotofluorimeter. Invention of spectrophotofluorimeter was indeed an exciting journey which started with a need to destroy the malaria parasite effectively.  This is another example of the famous English proverb “Necessity is the Mother of Invention”.

Come back to know more about fluorescence. In a series of posts, I will explain basic principles of fluorescence spectroscopy and its various applications in a qualitative manner, which may help beginners to understand the potential of this particular spectroscopy in scientific research.