1
|
- Light and Fluorescence
- J.Paul Robinson, PhD
- Professor of Immunopharmacology and Bioengineering
- Purdue University
- www.cyto.purdue.edu
|
2
|
- Basic quantum mechanics requires that molecules absorb energy as quanta
(photons) based upon a criteria specific for each molecular structure
- Absorption of a photon raises the molecule from ground state to an excited
state
- Total energy is the sum of all components (electronic, vibrational,
rotational, translations, spin orientation energies) (vibrational
energies are quite small)
- The structure of the molecule dictates the likely-hood of absorption of
energy to raise the energy state to an excited one
|
3
|
- Absorption associated with electronic transitions (electrons changing
states) occurs in about 1 femptosecond (10-15 s)
- The lifetime of a molecule depends on how the molecule disposes of the
extra energy
- Because of the uncertainty principle, the more rapidly the energy is
changing, the less precisely we can define the energy
- So, long-lifetime-excited-states have narrow absorption peaks, and short-lifetime-excited-states
have broad absorption peaks
|
4
|
- Using Beer’s law (Beer-Lambert law) for light travelling through a
curvette thickness d cm containing n molecules/cm3
- ln (Io/I) = snd
- where Io and I are the light entering and leaving and s is the molecular
property called the absorption cross section
- Now we can state that
- ln (Io/I) = and where
C is the concentration and a is the absorption coefficient which
reflects the capacity of the absorbing substance to absorb light
- If there are n (molecules/cm3 ; d in cm, s must be in cm2 so if a is in cm2/mol,
C must be in mol/cm3 do C=a/103
- giving
- log10 (Io/I) = ead = A
- where A is the absorbance or optical density
- and e is the
decadic molar exctinction coeficient in dm3mol-1cm-1
|
5
|
- O.D. units or absorbance is expressed in logarithmic terms so they are additive.
- E.g. an object of O.D. of 1.0 absorbs 90% of the light. Another object
of O.D. 1.0 placed in the path of the 10% of the light 10% of this light
or 1% of the original light is transmitted by the second object
- It is posssible to express the absorbance of a mixture of substances at
a particular wavelength as the sum of the absorbances of the components
- You can calculate the cross sectional area of a molecule to determine
how efficient it will absorb photons. The extinction coefficient
indicates this value
|
6
|
- Extinction Coefficient
- e refers to a single wavelength
(usually the absorption maximum)
- Quantum Yield
- Qf is a measure of the integrated photon
emission over the fluorophore spectral band
- At sub-saturation excitation rates, fluorescence intensity is
proportional to the product of e and Qf
|
7
|
|
8
|
- Photon emission as an electron returns from an excited state to ground
state
|
9
|
- Excitation Spectrum
- Intensity of emission as a function of exciting wavelength
- Chromophores are components of molecules which absorb light
- They are generally aromatic rings
|
10
|
- The wavelength of absorption is related to the size of the chromophores
- Smaller chromophores, higher energy (shorter wavelength)
|
11
|
- Stokes Shift
- is the energy difference between the lowest energy peak of absorbance
and the highest energy of emission
|
12
|
- The longer the wavelength the lower the energy
- The shorter the wavelength the higher the energy
- eg. UV light from sun - this causes the sunburn, not the red visible
light
|
13
|
|
14
|
|
15
|
|
16
|
|
17
|
|
18
|
|
19
|
- Dye molecules must be close to but below saturation levels for optimum
emission
- Fluorescence emission is longer than the exciting wavelength
- The energy of the light increases with reduction of wavelength
|
20
|
|
21
|
|
22
|
- The rate of emission is dependent upon the time the molecule remains
within the excitation state (the excited state lifetime tf)
- Optical saturation occurs when
the rate of excitation exceeds the reciprocal of tf
- In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1
second requires a dwell time per pixel of 2 x 10-6 sec.
- Molecules that remain in the excitation beam for extended periods have
higher probability of interstate crossings and thus phosphorescence
- Usually, increasing dye concentration can be the most effective means of
increasing signal when energy is not the limiting factor (i.e. laser
based confocal systems)
|
23
|
- Following absorption, molecules can relax via a non-radiative transition
to the T1 rather than the S1 state - this is
called an intersystem crossing,
- While it is forbidden it does happen and has a low probability and takes
a longer time - the energy dissipated is called phosphorescence
- Phosphorescence has a longer lifetime than fluorescence (milliseconds
rather than femptoseconds
- Phosphorescence generally occurs at longer wavelengths than fluorescence
because the energy difference between S0 and T1 is
lower
|
24
|
- Resonance energy transfer can occur
when the donor and acceptor molecules are less than 100 A of one
another
- Energy transfer is non-radiative which means the donor is not emitting a
photon which is absorbed by the acceptor
- Fluorescence RET (FRET) can be used to spectrally shift the fluorescence
emission of a molecular combination.
|
25
|
- Resonance Energy Transfer
|
26
|
- A molecule may undergo a vibrational transition (not an electronic
shift) at exactly the same time as scattering occurs
- This results in a photon emission of a photon differing in energy from
the energy of the incident photon by the amount of the above energy -
this is Raman scattering.
- The dominant effect in flow cytometry is the stretch of the O-H bonds of
water. At 488 nm excitation this would give emission at 575-595 nm
|
27
|
- Quenching is when excited molecules relax to ground stat5es via
nonradiative pathways avoiding fluorescence emission (vibration,
collision, intersystem crossing)
- Molecular oxygen quenches by increasing the probability of intersystem
crossing
- Polar solvents such as water generally quench fluorescence by orienting
around the exited state dipoles
|
28
|
- Light and Matter
- Absorption
- Fluorescence
- From this lecture you should understand:
- The nature of fluorescence molecules
- How fluorescence is generated
- Why molecules have different excitation and emission
- What Resonance Energy Transfer is
- What quantum yield is
|