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Instrumentation

IRF

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The temporal response of the FLIM instrument itself, which must be accounted for in lifetime analysis.

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Definition
The instrument response function (IRF) represents the temporal resolution limit of a FLIMLoading... system—what the instrument measures when presented with an infinitely short pulse. Real measurements are the convolution of the IRF with the true decay. Accurate lifetime determination requires either deconvolution of the IRF or using measurement approaches (like phasor analysisLoading...) that account for it.
Finite time resolution
No instrument is infinitely fast
Convolution effect
Measured = IRF ⊗ True decay
Measurement methods
Scatter or reference compound
Correction required
Deconvolution or calibration

Understanding IRF Effects

Consider measuring a 2 ns lifetime with an instrument having a 0.5 ns IRF:

  • The measured decay is broadened by the IRF
  • Without correction, the apparent lifetime would be overestimated
  • The effect is larger for short lifetimes (approaching the IRF width)

For typical fluorophore lifetimes (2-5 ns) and modern TCSPC systems (IRF ~100-200 ps), the effect is manageable but must be accounted for.

Simplified

The Problem: The instrument isn't infinitely fast. It blurs the measurement slightly. For very short lifetimes, this blur matters more.

The Solution: Measure the blur (IRF) and mathematically remove it from your data.

Handling the IRF

Several approaches handle IRF effects:

Deconvolution Fitting: Include the measured IRF in the fitting model. The algorithm finds the decay parameters that, when convolved with the IRF, best match the measured data.
Tail Fitting: Fit only the later portion of the decay where IRF effects are minimal. Loses some data but avoids deconvolution artifacts.
Phasor Calibration: Use a reference compound with known lifetime to calibrate the phasor position, implicitly accounting for IRF.
Simplified

Three Approaches:

  1. Mathematically remove the IRF blur (deconvolution)
  2. Only look at data where IRF doesn't matter (tail fitting)
  3. Use a known sample to calibrate (reference method)

Clinical Implications

  • System characterization: Regular IRF measurement ensures consistent performance
  • Short lifetime detection: Important for detecting FRET-quenched donors with reduced lifetimes
  • Standardization: IRF differences between instruments affect cross-site comparisons
  • QF-Pro approach: Calibration protocols account for IRF to ensure quantitative accuracy

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