Lifetime Imaging Techniques for Laser Scanning Microscopy

2 Lifetime Imaging Techniques for Laser Scanning Microscopy 

Frequency-Domain versus Time-Domain Techniques 

Fluorescence lifetime imaging techniques are usually classified into frequency-domain and time-domain techniques (see also Sect. 1, page 1 and Sect. 5.5.1, page 97). 

An overview of frequency-domain detection techniques is given in [88]. Frequency-domain techniques compare the phase shift and the modulation degree of the fluorescence with the modulated excitation. Modulation of the excitation is achieved either by actively modulating the light of a continuous laser or by using pulsed lasers of high repetition rate. With pulsed lasers, phase and modulation can be measured at the fundamental repetition frequency or at its harmonics. 

Heterodyne detection mixes the detection signal with a frequency slightly different from the modulation frequency [67, 73, 469, 470]. The result is that the phase angle and the modulation degree are transferred to the difference frequency signal. The difference frequency is in the kHz range where phase and modulation can be measured more easily. 

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