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

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

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.

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

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

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

Reflection and Transmission: Brewster’s Angle and TIR

Scattering elastic inelastic

Quantum Mechanics of the Atom

Molecular Orbitals

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

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)

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

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

Macroscopic Theory of Absorption Beer-Lambert Law

Jablonski Diagram TransitionProcessTimescale (seconds) S(0)  S(1) or S(n) Absorption S(n)  S(1)Internal Conversion to S(1)  S(1)Vibrational Relaxation to S(1)  S(0)Fluorescence10 -9 to S(1)  T(1)Intersystem Crossing to S(1)  S(0)Non-Radiative Relaxation / Quenching to T(1)  S(0)Phosphorescence10 -3 to 100 ST1)  S(0)Non-Radiative Relaxation / Quenching to 100

Measuring Fluorescence

Fluorescence Saturation

Chromophores and Labeling

Absorption and Emission Spectra

Quantum Yield

Fluorescence Lifetime

Solvent Effects

Quenching

Forster Resonance Energy Transfer (FRET)

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