AssayMetrics www.assaymetrics.com

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AssayMetrics www.assaymetrics.com
Physical and Chemical Properties of
Fluorescence Lifetime Dyes Thursday 4th May
2006 Dr Pierre Graves
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Intrinsic fluorescence encountered in microscopy

• Protein fluorescence (from tyr and trp residues)
  ?300-340nm ? 1-4ns
• Modified protein e.g. elastin collagen
  ?410-420 nm ? 1.5-2.5ns
• Nicotinamide adenine dinucleotide (NADH)
  ? 450nm ? 0.3-0.4ns
• Flavin adenine dinucleotide (FAD)
  ? 540nm ? 2.3-2.85ns

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An ideal Fluorescent Lifetime probe?

• Measure sensitively (pH, enzyme action, ion,
  molecular binding)
• Report accurately and precisely
• Measure robustly
• Uneffecting

Relatively long lifetime ratiometric method
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Physical requirements for an ideal long
fluorescence lifetime dye

• Molecule must be rigid so that the excited state
  does not decay by non-radiative processes
• No intersystem crossing leading to
  phosphorescence
• High quantum yield
• Physically and chemically stable

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PURETIME 325
?max (ab) ?max (em) ?
PURETIME 325 375nm 430nm 325ns

• Diazo bicyclo2.2.2 octene derivative
• N-Fmoc derivative of aspartic acid (for solid
  phase peptide synthesis)
• NHS ester for labelling amino groups
• Ratiometric lifetime change enzyme assays
  possible

in
non-degassed water
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PURETIME 14 PURETIME 20spectral properties
?max (ab) ?max (em) ?
PURETIME 14 410nm 426nm 14.2ns
PURETIME 20 421nm 463nm 19.6ns
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PURETIME 14 PURETIME 20

• Acridone derivatives
• NHS ester for labelling amino groups
• Excite with 405nm laser
• Lifetime NOT pH dependant
• Ratiometric lifetime change enzyme assays
  possible

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PURETIME 17
?max (ab) ?max (em) ?
PURETIME 17 (unconjugated) 405nm 543nm 10.9ns
PURETIME 17 (conjugated) 398nm 450nm 17.1ns

• Fluorescent enzyme substrates
• Ratiometric lifetime change enzyme assays
  possible
• Series of fluorogenic peptides other than
  substrates (e.g. ligands)

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PURETIME 22
?max (ab) ?max (em) ?
PURETIME 22 505/536nm 559nm 21.7ns
Fluorescein (pH dependent) 494nm 521nm 4ns
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PURETIME 22

• Quinacridone derivative
• NHS ester for amino acid labelling
• Excite with 488nm laser and use same filters as
  fluorescein
• Spectra, intensity and lifetime are not affected
  by pH
• Ratiometric lifetime change enzyme assays possible

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Photostability of dyes under intense white light
PURETIME 22
PURETIME 14 20
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PURETIME 1, 3, 5
?max (ab) ?max (em) ?
PURETIME 1 655nm 704nm 1.2ns
PURETIME 3 650nm 698nm 2.8ns
PURETIME 5 688nm 720nm 4.7ns
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PURETIME 1, 3, 5
Live HeLa cells doped with form of PT-3

• derivatives with pKa 6.5 and 4.3 for pH probes

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PURETIME dyes summary
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