optical spec 6 - fluorescence spectroscopy

8/8/2019 Optical Spec 6 - Fluorescence Spectroscopy

1/5

Physical Biochemistry Fluorescence Spectroscopy

[Page 1]

Emission spectrum = florescence spectrum

Frank-Condon Principle:

As the time scale for electronic transitions is shorter than that of vibrational transitions, it

can be assumed that the inter-nuclear distances will not change during the transition.

The transition that is most likely to occur is the one where the initial and excited

wavefunctions overlap the most.

At room temperature we expect molecules to be in the lowest vibrational state, so a

transition is most likely to occur from this state to a higher state (that overlaps the best).

As electronic energy levels are split into vibrational energy levels, and further into

rotational energy levels, the absorption peaks are broad (many different possibilities).

Fluorescence:

An excited molecule can lose its excess vibrational energy in collisions (internal conversion). Internal conversion takes

around 10-13s, absorption takes around 10-15s. Small amounts of energy will be lost until the electron is in the lowest

vibrational state of the excited electronic state. This low vibrational energy state is relatively stable and can last around

10-9s.

As a result of this, the emitted photon will have a longer wavelength (lower energy) than the absorbed photon

(bathochromic shift).

It is the transmission back to the ground state that is studied in fluorescence spectroscopy. Again, the most likely

transition will be that of most overlapping wavefunction. At ground state internal conversion can occur again.

Fluorescence usually requires flat, rigid molecules with extensive conjugation and delocalisation. In floppy molecules,

internal conversion can take the molecule from the excited state to the ground state without emission of fluorescence.

Spectra will often, but not always have mirror symmetry (absorption & emission)

If 0p n is the most likely vibrational transition for absorption, it will also be the most likely for emission.


2/5


[Page 2]

1. Absorption (should be 1 vertical arrow)

2. Internal conversion

3. Solvent relaxation

Excited state has different electrondistribution / electrostatic dipole

Solvent cage reorganises itself to betterstabilise excited state

This influences the energy and lifetime ofthe excited state

Causes fluorescence to occur at a lowerenergy than expected

4. Fluorescence

5. Internal conversion

Solvent polarity and pH can influence the fluorescence spectra, however it is difficult to define clear cut rules on how

this should occur, due to the short timescales.

Fluorescence Parameters:

Quantum efficiency (Q.E.): Emitted photons / Absorbed photons. If the molecule has been excited, what is the

probability that it will emit a photo, rather than lose its energy through internal conversion. Values between 0-1.

Molar extinction coefficient () [M-1cm-1]: Absorbance of a 1 molar solution in a 1cm flightpath at a specified

wavelength. Simply out, it is the probability that a molecule will undergo a transition from the lower state to an excited

state. Wavelength dependent the closer you are to the energy difference, the better you excite the molecule.

Brightness (sensitivity) = Q.E. . Wavelength dependent due to . concentration must be kept constant.

Changing the absorption wavelength will change the peak intensity of the spectra; the shape of the emission spectra

will stay the same.Absorption and fluorescence spectra are independent.

Lifetime:

Emission of a

photon is based

on probability, not a defined time. An average time for molecules

waiting in the excited state is the lifetime of the molecule.


3/5


[Page 3]

Intensity (y) Vs. Time (x). Exponential decay.

The lifetime of a molecule is defined as being the point at which exponential decay reaches a value of 1/e.

Fluorescence Spectrometer:

The source and detector are at right angles to one another.

Quenching & Photobleaching:

Quenching: Competing process that induce non-radiative relaxation of excited state electrons to the ground state.

Can be used as a tool to probe fluorophore environment Requires contact between fluorophore and quencher No fluorescence Non-permanent

The fact that contact is required can be useful for locating specifically targeted

fluorophores.

Photobleaching (fading): Permanent loss of fluorescence due to photo-induced chemical modification of molecule.

The excited state of a molecule is quite reactive, and can undergo reactions which lead to the molecule being

chemically modified.

Fluorescence Anisotropy: (~45 mins)

Sample is excited with linearly polarised light

If molecule is not rotating or tumbling then its fluorescence will also be linearly polarised. Its polarisation will almost

be parallel to that of the excitation polarisation.

Not identical as the dipole moment of the excited state is different to that of the ground state.


4/5


[Page 4]

If the molecule is rotating quickly, then the molecule will have changed orientation by the time that it has to emit

fluorescence, the plane of polarisation fluorescence will be random.

Anisotropy is the difference

P = parallel

Fluorescence Recovery After Photobleaching (FRAP)

Useful for studying transport proteins within a cell (especially transmembrane protein diffusion.

1. Label molecule with a fluorophore

2. Bleach (destroy) the fluorophore in a well defined area using a high intensity laser

3. Use a weaker beam to examine the recovery of fluorescence in the defined area as a function of time

Can be used to estimate the rate of diffusion, and the proportion of mobile molecules

Can be used to distinguish between active transport and diffusion

Some molecules that are destroyed will not be replaced by active transport or diffusion as they are immobile

(permanent damage)

Fluorescence Resonance Energy Transfer (FRET):

Non-radiative transfer of excited state energy from donor molecule to acceptor molecule (a form of quenching).

Acceptor then fluoresces at a different wavelength. Difficult technique.

Donor and acceptor molecules need to be in close proximity (


5/5


[Page 5]

Intrinsic Fluorescence:

Can be used instead of fluorophores

Some amino acids have intrinsic fluorescence (phenylalanine, tyrosine, tryptophan)

All lie in the UV region

Tyr and Trp spectra are very sensitive to the local environment

e.g. studying protein folding there will be a shift of peaks in folded / unfolded

Summary:

Fluorescence spectroscopy generally allows more sensitive measurement than absorption spectroscopy

optical spec 6 - fluorescence spectroscopy

Documents