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Fluorescence Lifetime Imaging Microscopy
Transcript of Fluorescence Lifetime Imaging Microscopy
Average time a molecule stays in its excited state before it emits a photon.
Definition of FLIM
Time domain vs frequency domain
Wide field vs point scanning
Time domain versus Frequency domain
Г = radiative decay
k = non-radiative decay
τ= 1/(Γ +k )
τ = fluorescence lifetime
I(t)= I(0)e^(-t/τ) + c
Does not depend on intensity
Depends on the environment
differentiate fluorophores with similar spectra
Point scanning versus wide field FLIM
High frequency pulsed laser
Single photons are detected
Arrival times detected
Modulated light wave
2 Parameters measured:
Phase shift & amplitude reduction
For discrimination of different fractions of the same fluorophore in different states of interaction with its environment
Förster resonance energy transfer
Changes lifetime donor
More robust than intensity
pH and ion concentration
FLIM to obtain information on the environment
Christoph Biskup, University Jena, Germany
Theodora Mauro, University of San Fransisco, USA
Thomas Gensch, Julich Research Centre, Germany
Skin - lifetime as
indicator of pH
Spinal ganglion - lifetime as indicator of Cl- concentration
Paul French, Imperial College London, UK
Slow versus Fast
High sensitivity versus Low sensitivity
Higher temporal resolution versus Lower temporal resolution
Determine expected fluorescence lifetime changes in tumor specific probes.
- Without cells
- With targeted cells
- With already saturated targeted cells
- With cells that show no/a low target expression