What is fluorescence

What is fluorescence

• What is fluorescence

• Use of fluorophores

• How can you make a molecule fluorescent

• Protein synthesis

• Protein folding

Stokes Shift

• Stokes Shift

• is the energy difference between the lowest energy peak of absorbance and the highest energy of emission

Tracking

• Tracking

• Qualitative
• Imaging
• in vitro
• in vivo
• Quantitative
• DNA, protein, lipids, ions, signaling molecules
• Relative amts, absolute amts, environment, interactions
• Nearly as sensitive as radioactivity, and a lot safer

FITC 488 525

• FITC 488 525

• PE 488 575

• APC 630 650

• PerCP™ 488 680

• Cascade Blue 360 450

• Coumerin-phalloidin 350 450

• Texas Red™ 610 630

• Tetramethylrhodamine-amines 550 575

• CY3 (indotrimethinecyanines) 540 575

• CY5 (indopentamethinecyanines) 640 670

Resonance Energy Transfer

• Resonance Energy Transfer

Chemically add

• Chemically add

• Not always specific
• Perturbing
• Direct vs Indirect

• Synthetically incorporate

• Limited to small molecules

• Biosynthetically incorporate

• Genetically engineer
• GFP and derivatives large (>20kD)

Stages

• Stages

• Components

• How can the system be altered to incorporate unnatural amino acids