Lecture 1: Basic Concepts on Absorption and Fluorescence
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(2) The Goal . • The emission of light after absorption of an outside energy source. Incandescence. Chemiluminescence. Bioluminescence. Photo excited luminescence, specifically fluorescence. Light is the energy input.
(3) Electromagnetic Radiation. • • • . -27. ε = hν = hc/λ, planks constant (h) = 6.62 x 10 ergs*sec, speed of light (c) = 3.00 x 10 m/sec. 8. Wavelength (λ) = c/ν = 1/ν. Wavenumber (ν) = ν/c.
(4) Properties of light. • I =. I0sin(2πνt + ϕ) = I0sin(ω0t + ϕ) . • Dispersion is. fundamental . • Dependence of the phase. velocity upon medium and/or frequency.
(5) Polarization of Light.
(6) Energy in moles. • Einstein (E) = N ε = N*h*(c/λ), Avogadro’s number (N) . • E = N ε = 28.6/λ kcal = 1.24/λ eV. • Having such an equation allows to calculate the kcal or eV for each λ.. • We will see how this critical knowledge for luminescent species .
(7) Quantum Mechanics. • Although light is. continuous, it’s interaction/ absorption is in discrete amounts or quanta. • In the case of light this called photons. • . The photoelectric effect demonstrated wave-particle duality.
(8) Absorption : Interaction of light with matter.
(9) Absorbance is the first step in Fluorescence. • FLUORESCENCE is the light emitted by an atom or molecule after a finite duration subsequent to the absorption of electromagnetic energy. . • Is an electronic transition that promotes an electron from the ground state to an unoccupied orbital after absorption of a photon.
(10) Wavelengths associated with Absorbance/ Fluorescence. • Focus on. wavelength range 200 - 1000 nm. • Why?. • • . Chemical bond energies are in the range of ~ 100 kcal per mole. Using the equations defined in the previous lecture, this gives a wavelength of 286 nm.
(11) Electronic Transitions. • Which transitions will we focus on?. • . What electrons/bonds will be encounter. • C, N, O, H and S are the important elements. σ orbitals. π orbitals.
(12) Electronic Transitions.
(13) Molecular Structure. • Saturated HC show no abs in the UV. • Less tightly bound π e. n = 2 = 217 nm. simplest case. • However, electronic transition. n = 5 = 447 nm. Closed. n = 5 = 305 nm. • Ethylene is the. is at 165 nm and 200 nm. • . How can we shift it higher wavelengths or lower energies? . • Conjugation of π e.
(14) Common Fluorophores. Chemical structure of GFP chromophore. Prasher, (1992) Gene.
(15) Beer’s Law. Deuterium/ Tungsten Lamp. Monochromotor. PMT Sample. I. Sample. PMT Reference I0. Blank. Molecular cross-section (σ). dI = σdN = Naσcdl. S. I. I0. log = 1 Naσcl = εcl. I 2.303. Dynamic range of Absorption. An OD of 1.0 - for every 100 photons entering the sample, 10 leave without being absorbed. An OD of 2.0 - for every 100 photons entering the sample, only 1 leaves without being absorbed. OD =3? - measuring the difference between 999 and 1000 photons is difficult!.
(16) Deviations of Beer’s Law. • Broad band illumination. • Scattering. • . Although some scattering needs to occur, large particles that scatter and do not absorb will increase the absorbance non-linearly. • Fluorescence . • . Part of the absorbed light is re-emitted at a different wavelength and it will decrease the measured absorbance. • Molecular Aggregation. • . Intrinsic case in which self association increases the particle size leading to an increase in scattering.
(17) Transitions moments and oscillation. • Transient dipole due to displacement. • • • . Absorbed light becomes potential energy with the change in position of the nuclei and electron (oscillator). Relationship between oscillator strength (f) and transition integration . Critical for experiments with polarized light. • Transitions of hydrocarbons are in the plane of the molecule.
(18) Frank-Condon Principle. • An electronic. transition occurs without changes in nuclei of an entity. -15. • Quick promotion (10 -10-12. sec) vs. molecular vibration (10 sec). • This produces the. Frank-Condon state. • The transition is called a vertical transition. Valeur, B. (2002) Molecular Fluorescence, . Wiley-VCH, Ch. 2 Pg 32.
(19) Frank-Condon Principle. • Several vibronic. transitions and absorption need not be 0-0. • Homogeneous and. inhomogeneous broadening. • Homogeneous is when there is a continuos set of subleves . • . Inhomogeneous is a fluctuation of structure, likely due to changes in the solvent shell. Valeur, B. (2002) Molecular Fluorescence, . Wiley-VCH, Ch. 2 Pg 32.
(20) Absorbance Fluorescence.
(21) Origin of Fluorescence.
(22) How did he do this?. Quinine fluorescence.
(23) What is Fluorescence?. • . FLUORESCENCE is the light emitted by an atom or molecule after a finite duration subsequent to the absorption of electromagnetic energy. Perrin-Jablonski Diagram.
(24) Fluorescence vs. Phosphorescence.
(25) Fluorescence characteristics. • Stokes’ shift. • . The energy (wavelength) associated with emission is typically lower (higher) than the excitation light. • Energy independence of emission. • . Typically the emission spectrum is the same, regardless of the excitation wavelength. • Emission mirrors that of absorption. • . Emission spectrum is a mirror image of the absorption spectrum.
(26) Stokes’ Shift. E. hν.
(27) Stokes’ shift. Fluorescein. Quinine Sulfate. Stokes shift can be calculated from λmax of Abs and Fluorescence spectrum. Convert Abs λmax and Fluo. λmax to wavenumbers to get Δν. Fluorescein Δν = 1444 cm. Quinine sulfate = 6100 cm.
(28) Fluorescence characteristics. • Stokes’ shift. • . The energy (wavelength) associated with emission is typically lower (higher) than the excitation light. • Energy independence of emission. • . Typically the emission spectrum is the same, regardless of the excitation wavelength. • Emission mirrors that of absorption. • . Emission spectrum is a mirror image of the absorption spectrum.
(29) Energy independence of emission. • What does this mean?. • No matter the. excitation wavelength, the emission spectrum is the same. • The only difference is the intensity.
(30) Energy independence of emission. • How can this be?. • Thermal. relaxation occurs rapidly (10 - 10 sec). -12. -15. • Almost all. emission comes from S1.
(31) Excitation Spectrum. • Describes the efficiency. of different wavelengths to excite fluorophores. • Isn’t it the same as the. absorption spectrum? . • If the system is “well behaved” then they will overlap, however, it is not always the case.
(32) Fluorescence characteristics. • Stokes’ shift. • . The energy (wavelength) associated with emission is typically lower (higher) than the excitation light. • Energy independence of emission. • . Typically the emission spectrum is the same, regardless of the excitation wavelength. • Emission mirrors that of absorption. • . Emission spectrum is a mirror image of the absorption spectrum.
(33) Mirror image rule. • It is common to note the. emission is a mirror image of excitation. • Emission mirrors the S S absorption. 0. 1. • Same transitions involved in both absorption and emission and the similarities of vibrational levels.
(34) Emission intensity. • How bright are fluorophores?. Are all. fluorophores identical?. Intensity of ANS . in Ethanol is ~ 100-200 x greater than in water!.
(35) Emission intensity. • How bright are fluorophores?. Are all. fluorophores identical?. kr. S1. kr + knr + kq + ksr + ket. hv. S0. kr. kTr. kq. ket. kr. kr + knr.
(36) • • . Quantum Yield (Φ). Efficiency of a fluophore to convert absorbed light into fluorescence. Φ of 1 means all light absorbed is converted to fluorescence. Φ = 0.79. At pH 12. Φ = ~1.00. EtOH + HCl. Φ = 0.14. In solution. In proteins?. Φ = 0.00 - 0.3.
(37) Other characteristics of fluorescence. • Lifetime - amount of time electron remains in S1 before returning to ground state. • Polarization - depolarization of emission due to ration of molecule. • FRET.
(38) Factors affecting fluorescence.
(39) Förster Resonance Energy Transfer (FRET). Slide adapted from Enrico Gratton, LFD Workshop.
(40) Conclusion. • Light as our source of energy . • . Fluorescence is a photo excited luminescence process. • . Energy dependence for fluorescence. • Absorbance. • • • . Promotion of an electron to an excited state. Low energy π-π* transitions. Highly conjugated, cyclical hydrocarbons that function as oscillators. • Fluorescence. • • • . Occurs at lower energy than absorbance. Takes place over a short time window (ns). Comes from S1 state.
(41) References . • Valeur, B. (2002) Molecular Fluorescence Whiley-VCH. • Hammes, G. G. (2005) Spectroscopy for the Biological Sciences, Whiley-VCH. • Lakowicz, J. R. (2006) Principles of Fluorescence Spectroscopy, .
(42)
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