Fluorescence Spectra

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Fluorescence Spectra
Wavelength
c 3 ? 108 m/s ?????400 700 nm ??(Wave
number) ??? ???
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Principles of Fluorescence
Aromatic molecules Delocalized electrons in
conjugated double bonds
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Stokes Shift
1852, Sir G. G. Stokes in Cambridge
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Kashas Rule
1950 The same fluorescence emission spectrum is
generally observed irrespective of the excitation
wavelength. The vibrational relaxation 1 ps
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Mirror Image Rule
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Intrinsic Biochemical Fluorophores
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Extrinsic Fluorophores
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Green Fluorescent Protein
Jellyfish Aequorea victoria The spontaneous
cyclization and oxidation of the sequence Ser65
(Thr65) Tyr66 Gly67 11-stranded ? barrel
with a coaxial helix 238 residues, 27 kDa
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GFP
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Spectra of GFP
Absorption maxima 395 and 475 nm Ex 395 nm Em
508 nm with QE 0.72 0.85 QE (Quantum
Yield) the ratio of the no. of photons emitted
to the no. absorbed.
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Microscope
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Filter Set
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Spatial Resolution
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Green Fluorescent Protein Imaging of Tumour
Growth, Metastasis, and Angiogenesis in Mouse
Models
Robert M. Hoffman The Lancet Oncology, vol. 3,
546 556, 2002
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Tumour Progression Metastasis

• High specificity
• Strong signal
• High resolution
• Good physiological conditions
• No other proteins, substrates, cofactors
• Stability ( 24 days )
• Cytoplasmic marker into any cell type
• Unaffected by the external environment
• Excitation wavelength is quite long ( 490 nm )

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GFP in Veins Capillaries
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Metastasis in Brain
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Bone Metastasis
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Single Cancer Cell
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Angiogenesis of Pancreatic Tumour
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Lymphoma Dissemination
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GFP and Luciferase