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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?.

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Presentation Transcript
slide1

Journal Club:

Introduction to Fluorescence Spectroscopy and Microscopy

Avtar Singh

4/5/11

slide2

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
slide3

What is Fluorescence?

  • definition: absorption of light by molecules and subsequent re-emission from excited singlet states

Fluorescence

Phosphorescence

Photoluminescence

Thermoluminescence

Chemiluminescence

Electroluminescence

Bioluminescence

etc.

Luminescence

  • 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
slide4

Basic Properties of Light

c = 299,792,458 m/s

c2 = (ε0μ0)-1

ε0 ≈ 8.85× 10−12F·m−1

μ0 = 4π×10 −7 N·A−2

v = c/n

Photons = light quanta

slide5

Interaction of Light with Matter: Dispersion

Sellmeier Equation:

Transmission

Reflection

Scattering

Absorption

slide6

Reflection and Transmission: Fresnel Equations

θr = θiIncidence = Reflection

ni sin θi = nt sin θt Snell’s Law

slide8

Scattering

elastic

inelastic

slide11

Selection Rules

Symmetry: electric dipole moment of the transition must be nonzero

Spin: total spin of the system must remain unchanged (photons have no magnetic moment)

Nuclear overlap: probability of a transition depends on nuclear overlap integral squared

Transition Oscillating Strength

slide12

Transition Dipole Moment

Laporte Symmetry Rule

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)

g: σ*, π

u: s, p, d, σ, π* (Wiki)

slide13

Wigner’s Spin Selection Rule

Partial allowance of spin-forbidden transitions due to spin-orbit coupling, typically weak with f ~ 10-7

slide14

Nuclear Wavefunctions and Franck-Condon Factors

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

Franck-Condon principle: intensity of a vibronic transition is proportional to the square of the overlap integral between the vibrationalwavefunctions of the two states that are involved in the transition

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

slide26

References

  • Lakowicz, Joseph. Principles of Fluorescence Spectroscopy. Springer, 2006.
  • CR Cantor and PR Schimmel. Biophysical Chemistry, Part 2: Techniques for the Study of Biological Structure and Function. WH Freeman and Co., 1980.
  • Olympus microscopy website http://www.olympusmicro.com/primer/techniques/fluorescence/fluorhome.html
  • Warren Zipfel Biomedical Optics Lecture Slides (BME 6260)
  • EPFL Photochemistry Course Lecture Notes http://photochemistry.epfl.ch/PC.html