Fluorescence and Confocal Microscopy
Fluorescence and Confocal Microscopy
Fluorescence and Confocal Microscopy
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• Physical Process<br />
– Excitation: S 0 + hv → S 1<br />
<strong>Fluorescence</strong><br />
– <strong>Fluorescence</strong> (emission): S 1 → S 0 + hv<br />
• S 0 is the ground state of the fluorophore<br />
• S 1 is the first excited state of the fluorophore<br />
• hν is a generic term for photon energy where:<br />
– h = Planck’s Constant<br />
– ν = Frequency of light<br />
Fluorophores<br />
Giepmans et al., 2006<br />
Green Fluorescent Protein<br />
• GFP was first cloned in 1992 from Aequorea<br />
victoria by Douglas Prasher.<br />
• Prahser also successfully expressed GFP in C.<br />
elegans in 1994.<br />
<strong>Fluorescence</strong><br />
<strong>Fluorescence</strong> Quantum Yield<br />
– the efficiency of the fluorescence process<br />
– Φ = # photons emitted<br />
# photons absorbed<br />
– The theoretical maximum fluorescence quantum<br />
yield is 1.0 (100%) where every photon absorbed<br />
results in a photon emitted.<br />
RCSB Protein Data Bank<br />
Aequorea victoria<br />
3
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