1. George Stokes
( )
Max Born
( )
E. Schroedinger
( )

2. Historical Perspective
Fluorescence described by Sir George G. Stokes
Fluorescence microscope developed
Heinrich Lehmann
August Köhler & Carl Reichert (Zeiss)
1930s Secondary fluorescence technique
Max Haitinger
Quinine based fluorophore
John Hershel
1950s Indirect Immunofluorescence
Albert Coons & Nathan Kaplan

3. Solar Spectrum
“Frauenhofer Lines”
(1814)
Kurucz et al (1984)

4. Visible Spectrum
Solar Spectrum H2
He2 O2 C N2 Si
(Kirchhoff , Bunsen, others; 1860s)

5. Fluorspar

16. Quantum Yield (photons emitted / photons absorbed)
Dye Solvent Exc Em QY
Acridine Orange Ethanol
Benzene Ethanol Chlorophyll-A Ethanol Eosin Water Fluorescein Water Rhodamine-B Ethanol

17. Solvent Effects
Triplet Blinking
Fluorescence Quenching
Photobleaching

18. Transition to the Dark Side
Triplet Blinking
stable triplet state
S1 T1 due to solvent
halogens
transition metals

19. Collision with solutes Resonance energy transfer
Quenching
Collision with solutes
Resonance energy transfer
Energy transferred
to non-fluorescent
solute molecule

20. Oxygen radicals are highly reactive & cytotoxic
Death of a Fluor
Photobleaching
T1 is highly reactive
O2 likes T1 electrons
Oxygen radicals are highly
reactive & cytotoxic
O2 or other solute
New compound
(disrupted conjugation)

23. Reflected Light (Epifluorescence) Microscopy

29. Transmission Fluorescence Microscopy

46. AOTF

49. What to look for in a fluorophore
Fluorescence Spectrum
Quantum Yield
Extinction Coefficient
Stability (Photobleaching)
Sensitivity to Environment
Toxicity
Reactivity
Solubility