Frequency domain FLIM system

In frequency-domain FLIM, the optics and detection system (MCP image intensifier and slow scan CCD camera) are similar to that of time-domain FLIM, except for the light source, which consists of a CW laser and an acousto-optical modulator instead of a pulsed laser. The principle of lifetime measurement is the same as that described in Chapter 6 (Section 6.2.3.1). The phase shift and modulation depth are measured relative to a known fluorescence standard or to scattering of the excitation light. There are two possible modes of detection heterodyne and homodyne detection. 

Comparing the frequency-domain and the time-domain approach, it was found that one is not fundamentally better than the other in terms of signal to noise ratio, when measuring the fluorescence lifetime of a mono-exponentially decaying fluorochrome [33]. In both systems, it is crucial, however, to use the optimal settings for each particular measurement. In frequency-domain FLIM, the... 

Examination of Eqs. (2.9-2.11) suggests that having frequency domain lifetimes measured at a variety of frequencies is desirable, as it will allow a mixture of fluorophores to be determined. With this in mind, two approaches may be taken to obtain multifrequency results. The first of these is simply to make a series of FLIM measurements while stepping through a predetermined set of frequencies. In practice, this is of limited utility for biological systems because of photo-induced damage to the specimen. 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Since TIRF produces an evanescent wave of typically 80 nm depth and several tens of microns width, detection of TIRF-induced fluorescence requires a camera-based (imaging) detector. Hence, implementing TIRF on scanning FLIM systems or multiphoton FLIM systems is generally not possible. To combine it with FLIM, a nanosecond-gated or high-frequency-modulated imaging detector is required in addition to a pulsed or modulated laser source. In this chapter, the implementation with of TIRF into a frequency-domain wide-field FLIM system is described. 

Described below is how we upgraded our frequency-domain wide-held FLIM system in Amsterdam to incorporate TIRF. A TIRF upgrade to an existing wide-held FLIM setup is marginal both with respect to cost and time. Furthermore, after the upgrade, the system can still be used as a regular wide-held FLIM system. 

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