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Fluorescence Spectroscopy

Page history last edited by Sarah Matteazzi 13 years, 7 months ago

Fluorescence Spectroscopy

Table of Contents

  1. Introduction

  2. Quantum Theory

  3. Application

  4. Fluorescence Quenching

Fluorescence is a very useful technique that deals with the emission of light. When light is shined on an object, photons are promoted to an excited state and then return to a ground state. The process of returning directly to the ground state is called fluorescence. Fluorescence spectroscopy then measures the amount number of photons that are emitted during this process, called the emission intensity. This can be translated into an intensity versus wavelength graph showing the wavelengths where a molecule or substance has the highest emission intensity, this is called an excited spectrum. Another type of measurement involving fluorescence is fluorescence efficiency. Fluorescence efficiency is found by calculating the quantum yield. The quantum yield is proportionality constant that that measures how effectively photons absorbed by a molecule are converted into luminescence, which is the emission of light from a body and a type of fluorescence. Basically, the higher the quantum efficiency, the more electrons are released as photons instead of other forms. Fluorescence spectroscopy can be used to find the wavelength of light that allows for the highest yield of electrons being promoted and returning to the ground state. This can then be used as an analytical method to determine many different things, such as identifying bacteria and diagnosing viruses.

Theory

In fluorescence spectroscopy, the molecule being analyzed is exposed to light of a certain wavelength within the ultraviolet and visible regions (approximately 400nm to 800nm), causing the molecule to be excited from its ground electronic state to a vibrational energy level within its excited electronic state. Through collisions with its surroundings, the molecule loses vibrational energy over time. This process is known as vibrational relaxation. When the molecule relaxes from the lowest vibrational state of the exited electronic state to a vibrational level within the ground electronic state, the result is the release of energy in the form of a photon. This is known as fluorescence.

Figure 1:Molecules are exited with a photon from the ground electronic state to an excited vibrational state within an excited electronic state. Over time, the molecules relax in energy levels, and eventually drop back to the ground electronic state, releasing a photon.

Since the molecule can drop in energy to any of the vibrational levels within the ground electronic state, a wide range of photons with different frequencies are emitted by a single analyte. This provides a unique set of frequency combinations for each molecule that undergoes fluorescence. By analyzing the frequencies and relative intensities of the emitted photons, the energy differences between each vibrational energy state can be determined. Also, since the frequencies of the emitted photons are unique for every molecule, observing a single known fluorescence frequency for a molecule allows for detection of that specific analyte without interference from different molecules.

The emitted photon is lower in energy than the energy of the light to which the molecule was initially exposed to. The overall energy gap between the initial photons and the incident photons can be described through the following equation:

E fluor= E abs− Evib− E solv.relax.

E fluoris the energy of the incident photon, E absis the energy lost from the initial photon upon absorption, Evibis the energy lost by the molecule upon relaxation from its excited state, and E solv.relax.is the energy taken up by reorientation of the molecular structure between transition between the exited and ground states.

Applications

There is a lot of research currently being done on the use of fluorescence spectroscopy as a diagnostic tool in the medical field, specifically in medical microbiology. Different types of bacteria have different spectral signatures. There are enough differences in the fluorescence spectra of medically significant bacteria that it is possible to identify and classify the bacteria into their respective genus, species, and family. The studies discovering this suggest that fluorescence spectroscopy can be an extremely accurate diagnostic tool for differentiating between microorganisms. Currently, physicians typically prescribe broad spectrum antibiotics to patients with a bacterial infection. Bacteria are becoming more and more immune to the effects of these antibiotics. If fluorescence spectroscopy can be used to quickly and accurately determine the specific bacterium causing the infection, the use of broad spectrum antibiotics would be diminished.

Additionally, fluorescence spectroscopy has promising applications in virus detection and diagnostics. Tryptophan is a fluorophore, a molecular component that allows a molecule to be fluorescent, which is present in viruses and host bacterial proteins. In different proteins, tryptophan is in different structural environments and the different environments are responsible for different fluorescence spectra. With this in mind, fluorescence spectra can be used to monitor virus-receptor interactions. As the virus binds to the host, the tryptophan emission spectra will change as the structural environment changes. Analyzing these spectra can provide valuable information about how a virus interacts with the host receptor site. Fluorescence spectroscopy is thus a viable method in monitoring the process of viral infections inside a cell.

Another interesting use of fluorescence is the creation of fluorescent puppies. In this study, the fluorescence is a convenient marker that makes it obvious if the transplantation and cloning process was successful. Researchers in South Korea genetically engineered the dogs to produce a fluorescent gene normally produced by sea anemones. The puppies, all named Ruppy, glow red under UV light. The scientists inserted fluorescent genes into the nucleus of a beagle cell and then placed the nucleus into egg cells of a surrogate mother that had the nucleus removed. The fluorescence serves as a marker to indicate that the genes that the scientists were intending to implant were successfully transferred in the cloning process. The fluorescence gives immediate feedback to the scientists on whether their experiment was successful. If the genes were not transferred properly the dogs would not glow red under UV light. The immediate feedback reduces the need for potentially lengthy and expensive tests to determine the success of the experiment. The scientists on the team that cloned the puppies say that successfully cloning dogs with fluorescent genes is a step toward implanting disease-related genes into dogs. This may help scientists further study human diseases.

Figure 2: Researchers in South Korea genetically engineered the dogs to produce a fluorescent gene normally produced by sea anemones.

Fluorescence Quenching

Quenching refers to the process during which the fluorescence intensity of a certain substance is decreased. There are multiple techniques that can lead to this including excited state reactions, energy transfer, complex-formation and collisional quenching. We introduce three mechanisms for dynamic quenching here which we can use to explain why the transfer of nonradioactive energy can occur between two substances.

The first one is called the Fluorescence resonance energy transfer (abbreviated FRET) which is used to describe the energy transfer between two fluorescent chromophores. The donor in its electronic excited state may transfer energy to an acceptor through dipole–dipole coupling.

The second way is known as exchange or collisional energy transfer, which occurs when excited state fluorophore is deactivated upon contact with other molecule in the solution, which is called quencher. The decrease in intensity can be expressed in Stern-Volmer equation:

is the rate of fluorescence without a quencher, I is with a quencher, is the quencher rate co-efficient, is the fluorescence lifetime of A, without a quencher present and [Q] is the concentration of the quencher.

Exciplex is another mechanism which deals with the decay of excimers. Excimers are short-lived dimeric or heterodimeric molecule formed from two species, at least one of which is in an electronic excited state. When the excimer returns to the ground state, its components dissociate and often repel each other. The wavelength of an excimer's emission is longer than that of the excited monomer's emission. An excimer can thus be measured by fluorescent emissions.

There are other mechanisms that are static or contact quenching which occurs when the donor and acceptor molecules are in the ground state.

Works Cited

http://www.oswego.edu/~kadima/CHE425/CHE425L/FLUORESCENCE_SPECTROSCOPY_08.pdf

http://www.translational-medicine.com/content/7/1/99

http://www.newscientist.com/article/dn17003-fluorescent-puppy-is-worlds-first-transgenic-dog.html

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