Lambert Instruments

FLIM can be measured in two ways: by the time domain method that is mainly applied on confocal laser scanning microscopes and by the frequency domain method that is used on widefield fluorescence microscopes.
The Lambert Instruments FLIM system (LIFA) operates in the frequency domain and can be attached to any wide field fluorescence microscope.

Frequency domain method
This determination requires a modulated light source and a modulated image intensifier as detector. The excitation light is modulated in intensity at the frequency of 10 - 100 MHz. Therefore, the induced fluorescence emission will be intensity-modulated as well. Due to the decay of the emission, the emitted light will show a phase-shift (delay in time) and a decrease in modulation-depth (lower maximum intensity and higher minimum intensity than the excitation light, while the average intensity remains the same) with respect to the excitation light. This phase-shift and decrease in modulation-depth depend on the decay constants of the fluorescent material and the modulation frequency. The lifetime can be calculated from each of these two parameters.

To extract the phase shift and the decrease in modulation depth from the emission signal relative to the excitation signal, the sensitivity of the image intensifier (coupled to the CCD camera) is modulated with the same radio frequency. Several phase steps are introduced in the sensitivity of the image intensifier. The resulting signal is detected at each phase step during a small integration period of the CCD camera. In the phase step that the emission signal is in phase with the sensitivity of the intensifier, a high detector signal level is measured. Similarly, when the emission signal is out phase with the sensitivity of the intensifier, a low detector signal level is measured.

These measurements are also done with a reference of known lifetime, so the phase shift and the decrease in modulation depth can be calculated by comparing the measurements of the sample with those of the reference. The lifetime is calculated out of both of these parameters, so that two values of the lifetime are given.

Time domain method
This determination requires elaborate fast excitation pulses and fast detection circuits. Each point in the sample is excited sequentially by a very short light pulse. The emitted fluorescence from each point is measured in many time windows by fast PMTs (photo multiplier tubes). The relative intensity measured in the different time windows is used to calculate the decay characteristics. When sufficient channels (time windows) are used, multi-exponential lifetimes can be extracted.

More information
"Advances in Biochemical Engineering / Biotechnology", chapter "Fluorescence Lifetime Imaging Microscopy (FLIM)" by E.B. van Munster & T.W.J. Gadella (2005) 95:143-175