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
Luminescence
Photoluminescence
Thermoluminescence
Chemiluminescence
Electroluminescence
Bioluminescence
etc.
Fluorescence
Phosphorescence
• 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
Basic Properties of Light
c = 299,792,458 m/s
c2 = (ε0μ0)-1
ε0 ≈ 8.85 × 10−12 F·m−1
μ0 = 4π×10 −7 N·A−2
v = c/n
Photons = light quanta
Interaction of Light with Matter: Dispersion
Sellmeier Equation:
Transmission
Reflection
Scattering
Absorption
Reflection and Transmission: Fresnel Equations
θr = θi
Incidence = Reflection
ni sin θi = nt sin θt 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
Laporte Symmetry Rule
g: σ*, π
u: s, p, d, σ, π* (Wiki)
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)
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
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 vibrational wavefunctions of the two
states that are involved in the transition
Vibronic coupling: in reality, nuclear and electronic
motions are coupled  can explain some symmetryforbidden 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
Transition
Process
Timescale
(seconds)
S(0)  S(1) or
S(n)
Absorption
10-15
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)
Fluorescence
10-9 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)
Phosphorescence
10-3 to 100
ST1) S(0)
Non-Radiative
Relaxation /
Quenching
10-3 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.
2.
3.
4.
5.
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