Lecture 1: Basic Concepts on Absorption and Fluorescence

Transcription

1 Lecture 1: Basic Concepts on Absorption and Fluorescence Nicholas G. James Cell and Molecular Biology University of Hawaii at Manoa, Honolulu

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, 8 speed of light (c) = 3.00 x 10 m/sec 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/ Focus on wavelength range 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 Fluorescence

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 Ethylene is the simplest case However, electronic transition is at 165 nm and 200 nm How can we shift it higher wavelengths or lower energies? Conjugation of π e n = 2 = 217 nm n = 5 = 447 nm Closed n = 5 = 305 nm

14 Common Fluorophores Chemical structure of GFP chromophore Prasher, (1992) Gene

15 Beer s Law Deuterium/ Tungsten Lamp Monochromotor Sample PMT Sample I Molecular cross-section (σ) di = σdn = Naσcdl I S I0 log I = Naσcl = εcl Blank PMT Reference I0 Dynamic range of Absorption An OD of for every 100 photons entering the sample, 10 leave without being absorbed An OD of 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 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 What does this mean? No matter the excitation wavelength, the emission spectrum is the same The only difference is the intensity emission

30 Energy independence of How can this be? Thermal relaxation occurs rapidly (10-10 sec) Almost all emission comes from S1 emission

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 S0 S1 absorption 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 ~ x greater than in water!

35 Emission intensity How bright are fluorophores? Are all fluorophores identical? S1 kr kr + knr + kq + ksr + ket hv kr ktr kq ket kr S0 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 =

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,