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Fluorescence Spectroscopy : History, Principle & Application

By: Pharma Tips | Views: 27593 | Date: 23-Mar-2014

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 f

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.

Fluorescence Spectroscopy

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  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.


image1

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  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). 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.

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