The molecular source of signal
Key fluorophore properties for FRET include:
Excitation/emission spectra: Define which wavelengths excite and are emitted.
Quantum yield: Fraction of absorbed photons that are re-emitted (higher = brighter).
Photostability: Resistance to photobleaching during imaging.
Fluorescence lifetimeLoading...: Duration of excited state–critical for FLIMLoading... detection.
Key Characteristics:
• Brightness: How much light it emits
• Photostability: How long it lasts under illumination
• Lifetime: How long the excited state lasts
• Spectra: What colors it absorbs and emits
The ATTO488/Alexa594 pair used in iFRETLoading... was optimized for:
Large Forster radius (5.83 nm) enabling detection of protein-protein interactions.
Distinct lifetimes allowing autofluorescenceLoading... separation by FLIM.
Spectral separation minimizing bleed-through artifacts.
FFPE compatibility for clinical tissue specimens.
QF-Pro's Choices: ATTO488 (donor) and Alexa594 (acceptor) were selected for:
• Good spectral overlap for efficient FRET
• Brightness sufficient for tissue samples
• Lifetime properties suitable for FLIM
• Stability in FFPE tissue environment
