PowerPoint Presentation - Intro to Optics

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George Stokes (1819 - 1903)
Max Born (1882 - 1970)
E. Schroedinger (1887 - 1961)
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Historical Perspective

• Fluorescence described by Sir George G.
  Stokes
• 1913 Fluorescence microscope developed
• Heinrich Lehmann
• August Köhler Carl Reichert (Zeiss)
• 1930s Secondary fluorescence technique
• Max Haitinger
• 1945 Quinine based fluorophore
• John Hershel
• 1950s Indirect Immunofluorescence
• Albert Coons Nathan Kaplan

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Solar Spectrum
Frauenhofer Lines (1814)
Kurucz et al (1984)
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Visible Spectrum
Solar Spectrum
H2
He2
O2
C
N2
Si
(Kirchhoff , Bunsen, others 1860s)
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Fluorspar
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Quantum Yield (photons emitted / photons
absorbed) Dye Solvent Exc Em QY Acridine
Orange Ethanol 493 535 0.46 Benzene Ethanol 248
300-350 0.04 Chlorophyll-A Ethanol 440 685 0.23
Eosin Water 521 544 0.16 Fluorescein Water 4
37 515 0.92 Rhodamine-B Ethanol 555 627 0.97
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Solvent Effects Triplet Blinking Fluorescence
Quenching Photobleaching
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Transition to the Dark Side

• Triplet Blinking
• stable triplet state
• S1 T1 due to solvent
• halogens
• transition metals

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Quenching
Collision with solutes Resonance energy transfer
Energy transferred to non-fluorescent solute
molecule
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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)
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Reflected Light (Epifluorescence) Microscopy
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Transmission Fluorescence Microscopy
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AOTF
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What to look for in a fluorophore
Fluorescence Spectrum Quantum Yield Extinction
Coefficient Stability (Photobleaching) Sensitivity
to Environment Toxicity Reactivity Solubility