Fluorescence spectroscopy

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Fluorescence spectroscopy

  1. 1. Fluorescence Spectroscopy and Application By:- Shubham maurya Anubhav sharma
  2. 2. Spectrum and Spectroscopy ▪ Spectrum:  (a). Different colors observed when the white light was dispersed through the prism  (b). The changing of light intensity as a function of frequency ▪ Spectroscopy: Study of spectrum, to identify substances
  3. 3. Introduction ▪ Absorption: When an incident photon hits a dye molecule in its ground state, the dye may be brought into its excited state. The photon is absorbed during this process, as its energy is used to excite the dye. This process only takes place if the photon energy equals the energy gap between the ground and excited state: Ephoton = E. ▪ fluorescence: If an excited dye molecule returns to its ground state the energy E’ has to be deposited somewhere. One possible process is the emission of a (fluorescence) photon, carrying the energy E’. Although there are other possibilities to deposit E’, there is a class of dyes where fluorescence is the dominant path ▪ .
  4. 4. Absorption and Fluorescence Spectroscopy
  5. 5. The three processes involved in fluorescence are: Excitation: A photon of energy hnex is supplied by an external source such as an incandescent lamp or a laser and absorbed by the fluorophore. The energy is used to push an electron from a ground state (S0) niveau to an excited state (S1) niveau. Non-radiating transitions: The electron spends a finite time (typically 1-10ns) in the excited state. During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment (collisions ...). These processes have two important consequences. First the energy of the initial S1 sub state is partially dissipated, yielding a relaxed singlet excited state from which fluorescence emission originates. Second, not all the molecules initially excited by absorption return to the ground state by fluorescence. Other processes such as collisional quenching, fluorescence energy transfer and intersystem crossing may also depopulate S1 without emitting a photon.
  6. 6. Fluorescence emission: Finally with a certain probability (see discussion above) a photon of energy hnfl is emitted, returning the fluorophore to its ground state S0. Due to energy dissipation during the excited state lifetime (non-radiative relaxation), the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon hnex. The energy difference is related to the Stokes shift, which is the wavelength difference between the absorption and emission maximum:
  7. 7. Instrumentation
  8. 8. APPLICATIONS ▪ There are many and highly varied applications for fluorescence despite the fact that relatively few compounds exhibit the phenomenon. ▪ The effects of pH, solvent composition and the polarisation of fluorescence may all contribute to structural elucidation. Nonfluorescent compounds are often labelled with fluorescent probes to enable monitoring of molecular events. This is termed extrinsic fluorescence as distinct from intrinsic fluorescence where the native compound exhibits the property. ▪ Some fluorescent dyes are sensitive to the presence of metal ions and can thus be used to track changes of these ions in in vitro samples, as well as whole cells.
  9. 9. Intrinsic protein fluorescence ▪ Proteins possess three intrinsic fluorophores: tryptophan, tyrosine and phenylalanine,although the latter has a very low quantum yield and its contribution to protein fluorescence emission is thus negligible. Of the remaining two residues, tyrosine has the lower quantum yield and its fluorescence emission is almost entirely quenched when it becomes ionised, or is located near an amino or carboxyl group, or a tryptophan residue. ▪ Intrinsic protein fluorescence is thus usually determined by tryptophan fluorescence which can be selectively excited at 295–305 nm. Excitation at 280nm excites tyrosine and tryptophan fluorescence and the resulting spectra might therefore contain contributions from both types of residues. ▪ The main application for intrinsic protein fluorescence aims at conformational monitoring. We have already mentioned that the fluorescence properties of a fluorophore depend significantly on environmental factors, including solvent, pH, possible quenchers, neighbouring groups, etc.
  10. 10. Extrinsic fluorescence ▪ Frequently, molecules of interest for biochemical studies are nonfluorescent. In many of these cases, an external fluorophore can be introduced into the system by chemical coupling or non-covalent binding. Some examples of commonly used external fluorophores are shown in Fig. ▪ Three criteria must be met by fluorophores in this context. Firstly, it must not affect the mechanistic properties of the system under investigation. ▪ Secondly, its fluorescence emission needs to be sensitive to environmental conditions in order to enable monitoring of the molecular events. ▪ And lastly, the fluorophore must be tightly bound at a unique location
  11. 11. Fluorescence resonance energy transfer (FRET) ▪ Fluorescence resonance energy transfer (FRET) was first described by Fo¨rster in 1948. ▪ The process can be explained in terms of quantum mechanics by a nonradiative energy transfer from a donor to an acceptor chromophore. The requirements for this process are a reasonable overlap of emission and excitation spectra of donor and acceptor chromophores, close spatial vicinity of both chromophores (10–100 A˚ ), and an almost parallel arrangement of their transition dipoles. Of great practical importance is the correlation showing that the FRET effect is inversely proportional to the distance between donor and acceptor chromophores, R0.
  12. 12. IMPORTANCE OF FRET ▪ The FRET effect is particularly suitable for biological applications, since distances of 10–100A˚ are in the order of the dimensions of biological macromolecules. ▪ The relation between FRET and the distance allows for measurement of molecular distances and makes this application a kind of ‘spectroscopic ruler’. If a process exhibits changes in molecular distances, FRET can also be used to monitor the molecular mechanisms. ▪ The high specificity of the FRET signal allows for monitoring of molecular interactions and conformational changes with high spatial (1–10nm) and temporal resolution (<1ns). Especially the possibility of localising and monitoring cellular structures and proteins in physiological environments makes this method very attractive
  13. 13. FRET APPLICATIONS IN DNA SEQUENCING AND INVESTIGATION OF MOLECULAR MECHANISMS ▪ BigDyes™ are a widely used application of FRET fluorophores (Fig. 12.12). Since 1997, these fluorophores are generally used as chain termination markers in automated DNA sequencing. As such, BigDyes™ are in major parts responsible for the great success of genome projects. ▪ In many instances, FRET allows monitoring of conformational changes, protein folding, as well as protein–protein, protein– membrane and protein–DNA interactions. For instance, the three subunits of T4 DNA polymerase holoenzyme arrange around DNA in torus-like geometry.
  14. 14. CONT. ▪ Other examples of this approach include studies of the architecture of Escherichia coli RNA polymerase, the calciumdependent change of troponin and structural studies of neuropeptide Y dimers
  15. 15. THANK YOU

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