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……………

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