Figure 4.

The Time Domain (TD) and Frequency-domain (FD) FLIM Methods.

A: TD FLIM requires a pulsed excitation source with a femtosecond pulse width. The pulsed laser is coupled to the scanning system of the microscope. The photons emitted from the sample are recorded by a fast detector, which is connected to a time-correlated single photon counting (TCSPC) device. The TCSPC records the arrival time each photon relative to the excitation pulse, and a `photon counts' histogram is built for each pixel of an image. The fluorescence lifetime, determined as the time require for the fluorescence to decay to 37% of its initial intensity, is estimated by fitting the corresponding decay data with either single- or multi-exponential models. B: The excitation source for the FD FLIM system is a diode laser that is modulated at high radio frequencies. The emission signals from the specimen are routed to the detector, and the phase delays (Φ) and modulation ratio (M = AC/DC) of the emission (Em) relative to the excitation (Ex) are used to estimate the fluorescence lifetime.