What causes the IRF to show multiple peaks or irregular shapes?
An Instrument Response Function (IRF) with multiple peaks, shoulders, or irregular shapes is typically caused by optical artifacts. The most common causes are:
Back-reflections in the excitation path: Reflections from optical components (lenses, windows, optical fiber connectors, etc.) can reach the detector and create secondary peaks, often shifted by the optical path length of the reflection.
Raman scattering in immersion media or solvents: If the scatter solution used for IRF measurement contains a component with a strong Raman band close to the excitation wavelength, a Raman peak may appear as an additional feature in the IRF. Water, glycerol, and common immersion media can contribute Raman peaks.
Emission from fluorescent impurities: If the scattering sample is contaminated with fluorescent material (e.g., fluorescent dust, residual dye), this can produce a broadened or asymmetric IRF profile. Very short-lived dye residues may still look similar to the IRF but with slightly modified shape.
Detector afterpulsing: Some single-photon detectors, particularly older PMT types, show afterpulsing — a secondary pulse generated by the detector itself shortly after the primary detection event. This typically appears as a broad, low-amplitude secondary peak in the IRF, independent of the optical setup.
Can these IRF artifacts affect the lifetime fit?
Yes. If an irregular IRF is used as-is in a reconvolution fit, the artifacts will be incorporated into the fit model. This can lead to:
Poor fit quality (elevated residuals, poor χ² values)
Systematic errors in the recovered lifetimes
Apparent short-lifetime components that do not reflect real photophysics
If the IRF artifacts are reproducible and stable, they may still produce acceptable fits — but this should be verified carefully.
How to address an irregular IRF
Identify and remove the source of the reflection or artifact in the optical path, if possible.
Use a different scattering solution that avoids Raman contributions near the excitation wavelength (e.g., switch from water to a dilute colloidal silica suspension such as Ludox, or vice versa).
Clean optical surfaces and check fiber connectors.
Try the automatically estimated IRF option in SymPhoTime, which reconstructs the IRF from the rising edge of the decay — this avoids the need for a scatter measurement and sidesteps optical artifacts in the IRF.
If your sample has a very short lifetime (close to the instrument timing resolution), it is generally preferable to measure the IRF directly rather than reconstruct it.