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Journal Club: Introduction to Fluorescence Spectroscopy and Microscopy Avtar Singh 4/5/11.

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Presentation on theme: "Journal Club: Introduction to Fluorescence Spectroscopy and Microscopy Avtar Singh 4/5/11."— Presentation transcript:

1 Journal Club: Introduction to Fluorescence Spectroscopy and Microscopy Avtar Singh 4/5/11

2 Introduction to Journal Club discuss basic tools / ideas that are useful to all of us ask questions! topic-based or article-based? slides posted on DRBIO site

3 What is Fluorescence? definition: absorption of light by molecules and subsequent re-emission from excited singlet states why useful? other diagnostic imaging tools for biology: MRI, CT, X-ray, EM other optical tools: absorption, phase contrast, DIC, scattering (CARS, SRS) advantages of fluorescence: optical (high-res, in vivo), high contrast, sensitive emission profiles discovered by Stokes (1852) initially a nuisance in microscopy: Kohler and Reichert Fluorescence Phosphorescence Luminescence Photoluminescence Thermoluminescence Chemiluminescence Electroluminescence Bioluminescence etc.

4 Basic Properties of Light c = 299,792,458 m/s c 2 = (ε 0 μ 0 ) -1 ε 0 ≈ 8.85 × 10 −12 F·m −1 μ 0 = 4π×10 −7 N·A −2 v = c/n Photons = light quanta

5 Interaction of Light with Matter: Dispersion Transmission Reflection Scattering Absorption Sellmeier Equation:

6 Reflection and Transmission: Fresnel Equations θ r = θ i Incidence = Reflection n i sin θ i = n t sin θ t Snell’s Law

7 Reflection and Transmission: Brewster’s Angle and TIR

8 Scattering elastic inelastic

9 Quantum Mechanics of the Atom

10 Molecular Orbitals

11 Selection Rules 1.Symmetry: electric dipole moment of the transition must be nonzero 2.Spin: total spin of the system must remain unchanged (photons have no magnetic moment) 3.Nuclear overlap: probability of a transition depends on nuclear overlap integral squared Transition Oscillating Strength

12 Transition Dipole Moment Just because the transition is symmetry –forbidden does not mean it can’t occur  ignored vibrational motion and used approximate wavefunctions (Cantor and Schimmel p 373) Laporte Symmetry Rule g: σ*, π u: s, p, d, σ, π* (Wiki)

13 Wigner’s Spin Selection Rule Partial allowance of spin-forbidden transitions due to spin-orbit coupling, typically weak with f ~ 10 -7

14 Nuclear Wavefunctions and Franck-Condon Factors Franck-Condon principle: intensity of a vibronic transition is proportional to the square of the overlap integral between the vibrational wavefunctions of the two states that are involved in the transition Born-Oppenheimer approximation: In order to simplify the molecular Hamiltonian, assume that the nuclei are stationary (makes sense because they’re much more massive than the electrons)  this allows us to separate the nuclear and electron wavefunctions Vibronic coupling: in reality, nuclear and electronic motions are coupled  can explain some symmetry- forbidden transitions that proceed at low f-values Solvent broadening: local solvent environments smear out the vibrational details of absorption / emission spectra

15 Macroscopic Theory of Absorption Beer-Lambert Law

16 Jablonski Diagram TransitionProcessTimescale (seconds) S(0)  S(1) or S(n) Absorption10 -15 S(n)  S(1)Internal Conversion 10 -14 to 10 -10 S(1)  S(1)Vibrational Relaxation 10 -12 to 10 -10 S(1)  S(0)Fluorescence10 -9 to 10 -7 S(1)  T(1)Intersystem Crossing 10 -10 to 10 -8 S(1)  S(0)Non-Radiative Relaxation / Quenching 10 -7 to 10 -5 T(1)  S(0)Phosphorescence10 -3 to 100 ST1)  S(0)Non-Radiative Relaxation / Quenching 10 -3 to 100

17 Measuring Fluorescence

18 Fluorescence Saturation

19 Chromophores and Labeling

20 Absorption and Emission Spectra

21 Quantum Yield

22 Fluorescence Lifetime

23 Solvent Effects

24 Quenching

25 Forster Resonance Energy Transfer (FRET)

26 References 1.Lakowicz, Joseph. Principles of Fluorescence Spectroscopy. Springer, 2006. 2.CR Cantor and PR Schimmel. Biophysical Chemistry, Part 2: Techniques for the Study of Biological Structure and Function. WH Freeman and Co., 1980. 3.Olympus microscopy website 4.Warren Zipfel Biomedical Optics Lecture Slides (BME 6260) 5.EPFL Photochemistry Course Lecture Notes

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