Fundamentals of fluorescence measurements

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Fundamentals of fluorescence measurements

The use of fiber optic sensors based in fluorescence has become a common practice for applications of detection in areas such as analytical chemistry, biochemistry, cell biology, photochemistry, medical diagnostics or biotechnology during last decades [1]. Fluorescence-based sensors present high sensitivity and specificity, being the design and manufacture of the appropriate sensitive coatings one of the main research areas with the aim to create fast, reproducible and lasting devices.

One of the main disadvantages of fluorescence-based fiber optic sensors is the photodegradation of the indicator after a prolongated excitation time, which shortens considerably the lifetime of the device and prevents from carrying out reliable measurements. This problem can be diminished with the utilization of pulsed excitation light sources (see our COB series LED light sources). Other problem associated with this type of sensors occurs at high fluorophore concentrations, resulting in a decrease in fluorescence, which is known as self-quenching (originated by the collisions between molecules and self-absorption by overlapping the Absorption and emission spectra). On the other hand, when many absorbent molecules are present, only those closest to the source are excited, preventing the excitation light beam from reaching the rest. These factors are strongly influenced by the composition and morphology of the matrix or solution that contains the fluorophores [23].

In the following lines it is introduced a brief theoretical description of the fluorescence phenomenon and its practical use for the manufacturing and interrogation of fiber optic sensors as well as the main problems associated with them.

The most direct way to promote an electron to an excited state is that the molecule absorbs a photon of the appropriate energy. The difference in energy between the two states will correspond to the energy of the absorbed photon, E = hc / l where h is Planck’s constant, c is the speed of light in vacuum and l is the wavelength of light.

Diagram of fluorescence. Jablonski diagram showing the main routes of relaxation of an excited fluorophore

Jablonski diagram showing the main routes of relaxation of an excited fluorophore

The figure above represents the Jablonski diagram that shows the different relaxation pathways of a fluorophore from an excited state. The energy difference between the Sx states is too large to populate them thermally. Therefore, light, instead of heat, is used generally to induce fluorescence. Each of the electronic states has several vibrational levels (represented by the horizontal lines in the figure above). Vibrational levels are associated to the small discrete increments of energy absorbed by a molecule in a defined electronic state while maintains the same electronic configuration. Among the different transitions between excited states only Fluorescence and Phosphorescence cause radiation. Fluorescence is a light emission phenomenon from an excited material. Light emission is associated to the release of the energy collected from the absorption of the incident photons. Fluorescence can be described as the light emitted by a molecule when it returns to the stable state from its excited state [4]. Among the rest of the relaxation transitions we can distinguish three additional cases that do not produce radiation [5]:

– Non-radiant relaxation: consists of the energy dissipation from the excited state to the stable state in the form of molecular vibration (heat).

– Static quenching (or photobleaching) [6]: is produced by a fluorophore combination with different molecules that generate a stable compound, which is not capable of emitting fluorescence. This causes a reduction of the number of molecules capable of emitting fluorescence and a subsequent decrease of the fluorescence intensity. In addition, the generated non-fluorescent compounds may not absorb light at the fluorescence excitation frequency originating an optical absorption change as well.

– Dynamic quenching or self-quenching: is originated when the excited fluorophore collides with the rest of molecules, transferring their energy and returning to steady state without fluorescence emission. In this case the fluorophore can reach the state for fluorescence emission, but it’s quickly brought to the stable state due to this mechanism. This process causes a reduction of the fluorescence emission intensity as well as a reduction of the excited state time of molecules and the fluorophore lifetime. This phenomenon is generally associated to high fluorophore concentrations where the light emitted by a fluorophore is directly absorbed by another. The reabsorbed energy, below the initial excitation energy, is not able to induce fluorescence and it is lost as vibrational energy or phosphorescence at wavelengths less than fluorescence reducing the total fluorescence intensity. Fluorescence is very appropriate for optical sensors due to its high sensitivity and specificity. Fluorescence intensity (If) is generally proportional to the excitation light intensity (Io) and fluorophore concentration ([D]), although the latter is not so clear due to the self-quenching phenomena explained previously. Here is also important to consider the fluorescence efficiency, that is, the ratio between the number of photons absorbed by the material and the number of photons emitted by fluorescence or phosphorescence mechanisms (h). This relationship is shown by Parker’s Law shown in the following expression:

If = Io · d · e ·  h · [D] · K

where d is the path of the light path in the detection layer, e is the molar absorption coefficient, h is the quantum yield of the fluorescent indicator and K is a factor of proportionality that considers the geometry of the measuring instrument.

Some detection systems do not obtain the emission directly from the analyte, but they quantify the concentration of it indirectly thanks to the excitation or deactivation (quenching) that occurs in a determined fluorophore indicator. The emission intensity in the absence (Io) and presence (IQ) of the deactivator (Q) in the sample are directly related by the Stern-Volmer equation [78]:

Io / IQ = 1 + KSV [Q]

being Ksv the Stern-Volmer constant (Ksv = tokq, kq is the constant of bimolecular deactivation rate and to is the duration of the excited state).


[1] O. S. Wolfbeis, “Materials for fluorescence-based optical chemical sensors,” Journal of Materials Chemistry, vol. 15, pp. 2657-2669, 2005.

[2] F. J. García Moreda, F. J. Arregui, M. Achaerandio, I. R. Matias, “Study of indicators for the development of fluorescence based optical fiber temperature sensors,” Sensors and Actuators B, vol. 118, pp. 425-432, 2006.

[3] G. Decher, “Fuzzy Nanoassemblies toward layered polymeric multicomposites,” Science, vol. 277, pp. 1232-1237, 1997.

[4] R. B. Thompson, Fluorescence Sensors and Biosensors, New York, NY.CRC Press, 2005.

[5] D. A. Skoog, D. M. West, F. J. Holler, S. R. Crouch, Fundamentals of Analytical Chemistry. Belmont, CA: Brooks Cole, 2003.

[6] J. Goicoechea, C. R. Zamarreño, I. R. Matias, F. J. Arregui, “Minimizing the photobleaching of self-assembled multilayers for sensor applications,” Sensors and Actuators B, vol. 126, pp. 41-47, 2007.

[7] O. Stern, M. Volmer, Physik. Z., 20, 183, 1919.[8] S-H. Lee, J. Kumar, S. K. Tripathy, “Thin film optical sensors employing polyelectrolyte assembly,” Langmuir, vol. 16, pp. 10482-10489, 2000.

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