The MicroTime 200 confocal TCSPC microscope uses SymPhoTime 64 for data analysis. When fitting fluorescence lifetime decays, you need an Instrument Response Function (IRF) to correctly account for the timing characteristics of the system. There are two ways to obtain this IRF: it can be automatically estimated (calculated) from the decay data itself, or manually measured using a suitable sample.
Automatically estimated (calculated) IRF
SymPhoTime can reconstruct an IRF mathematically from the fluorescence decay histogram, specifically from the steeply rising edge at the beginning of the decay. No separate measurement is required. This is convenient but comes with limitations: the reconstruction is sensitive to noise, and the result can look irregular if photon statistics are low. Importantly, an irregular-looking reconstructed IRF does not necessarily mean the fit results are wrong — but it is a sign that the approach may be reaching its limits.
The automatically estimated IRF works well when:
- Your sample has a lifetime well above ~700 ps (i.e. more than twice the MicroTime 200 IRF width of ~350 ps)
- You have sufficient photon counts in the decay (generally at least several thousand counts at the peak)
- No separate IRF measurement was acquired alongside the fluorescence data
Manually measured IRF
A measured IRF is obtained by recording the instrument’s timing response directly. On a confocal microscope such as the MicroTime 200, the most practical approach is to use the reflection from a glass surface (e.g., the coverslip) at the same excitation wavelength and detection settings used for the fluorescence measurement. Alternatively, a quenched dye with a negligible fluorescence lifetime can be used as a reference sample.
A manually measured IRF is preferable when:
- Your sample lifetime is below ~700 ps — as a conservative rule of thumb, this corresponds to less than twice the MicroTime 200 IRF width (~350 ps) — where accurate deconvolution from the IRF becomes critical
- The automatically estimated IRF looks noisy or shows multiple peaks despite adequate photon counts
- Your decay contains rising components — for example due to energy transfer (FRET), where the acceptor emission builds up over time before decaying. The automatic IRF reconstruction relies on the rising edge of the histogram and cannot distinguish instrument timing from sample photophysics in such cases
- You are measuring time-resolved anisotropy. Anisotropy analysis requires a properly measured IRF for both the parallel and perpendicular detection channels, as the IRF shape and any inter-channel timing offset must be correctly accounted for in the reconvolution
- You need reproducible, publication-quality fits
- You are comparing decays across multiple samples or sessions and want a consistent IRF reference
Practical recommendation
Start with the automatically estimated IRF for routine measurements. Switch to a manually measured IRF in any of the following situations:
- Your fitted residuals look poor or the reconstructed IRF appears irregular despite adequate photon counts
- Your sample lifetime is below ~700 ps — the MicroTime 200 IRF is ~350 ps wide, and the calculated IRF cannot be trusted for reliable deconvolution below roughly twice that value
- Your decay is not a pure fluorescence decay but contains rising components — for example due to energy transfer (FRET), where the acceptor emission builds up over time before decaying. In such cases the automatic IRF reconstruction, which relies on the rising edge of the histogram to infer the instrument response, cannot reliably separate the IRF contribution from the photophysics of the sample. A measured IRF is then the only robust option.
- You are performing time-resolved anisotropy measurements — a measured IRF is always required
- You need reproducible, publication-quality fits, or are comparing decays across multiple samples or sessions and want a consistent IRF reference