1	BMS 631: Lecture 3 Light and Fluorescence J.Paul Robinson, PhD Professor of Immunopharmacology and Bioengineering Purdue University www.cyto.purdue.edu
2	Absorption 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	Lifetime 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	Exctinction Using Beer’s law (Beer-Lambert law) for light travelling through a curvette thickness d cm containing n molecules/cm³ ln (I_o/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 (I_o/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 (I_o/I) = ead = A where A is the absorbance or optical density and e is the decadic molar exctinction coeficient in dm³mol^-1cm^-1
5	Absorbance 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	Parameters Extinction Coefficient e refers to a single wavelength (usually the absorption maximum) Quantum Yield Q_f 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 Q_f
7	Fluorescence Quantum Yield
8	Fluorescence Photon emission as an electron returns from an excited state to ground state
9	Fluorescence 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	Fluorescence The wavelength of absorption is related to the size of the chromophores Smaller chromophores, higher energy (shorter wavelength)
11	Fluorescence Stokes Shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
12	Fluorescence 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
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15	Simplified Jablonski Diagram
16	Fluorescence
17	Fluorescence
18	Fluorescence Excitation Spectra
19	Conclusions 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	Allophycocyanin (APC)
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22	Excitation Saturation The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime t_f) Optical saturation occurs when the rate of excitation exceeds the reciprocal of t_f 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^-6sec. 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	Phosphorescence Following absorption, molecules can relax via a non-radiative transition to the T₁ rather than the S₁state - 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 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	Fluorescence Resonance Energy Transfer
26	Raman Scatter 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, Bleaching & Saturation 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	Lecture Summary 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