1. Fluorescence Spectroscopy
and Application
By:-
Shubham maurya
Anubhav sharma

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. 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. Absorption and Fluorescence Spectroscopy

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. 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:

8. Instrumentation

9. 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.

10. 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.

11. 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

13. 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.

15. 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

16. 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.

18. 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

19. THANK YOU