Time-domain FLIM

In principle, lifetime imaging is possible by combination of the single-photon timing technique with scanning techniques. However, the long measurement time required for collecting photons at each point is problematic. 

By this procedure, which requires calculation from only four parameters Dj, D2,h and t2), lifetime images can be obtained very quickly. Time-resolved im- 

The time resolution of the image intensifier is about 3 ns (minimal gate width), which may not be sufficient for fast decaying probes. Moreover, a pixel-by-pixel deconvolution, if necessary, would require excessively long computation times. 

A much better time resolution, together with space resolution, can be obtained by new imaging detectors consisting of a microchannel plate photomultiplier (MCP) in which the disk anode is replaced by a coded anode (Kemnitz, 2001). Using a Ti-sapphire laser as excitation source and the single-photon timing method of detection, the time resolution is 10 ps. The space resolution is 100 pm (250 x 250 channels). 

---

The fourth chapter by James McGuinty et al. describes the more advanced forms of time-domain FLIM. While not immediately available on commercial instruments this chapter should give the reader an idea what the current state-of-the-art is in terms of FLIM instrumentation, and perhaps what to expect on future commercial instruments. Real-time FLIM, combined FLIM-spectral imaging, hyperspectral FLIM-imaging, combined lifetime-anisotropy imaging and some of their applications are covered here. 

Time domain FLIM Theory, instrumentation, and data analysis... 

Point scanning time domain FLIM implementations... 

Implementation of time domain FLIM methods is comparatively straightforward in laser scanning microscopes (LSMs). Here, pointscanning is used so that single channel lifetime detection suffices. In principle, standard fluorescence lifetime detection equipment developed for spectroscopy can be used in combination with point-scanning systems and a pulsed laser. 

Barber, P. R., Ameer-Beg, S. M., Gilbey, J. D., Edens, R. J., Ezike, I. and Vojnovic, B. (2005). Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM. In Multiphoton Microscopy in the Biomedical Sciences V.Vol. 5700. SPIE, San Jose, CA, USA, pp. 171-81. 

Historically, this has been the most constrained parameter, particularly for confocal laser scanning microscopes that require spatially coherent sources and so have been typically limited to a few discrete excitation wavelengths, traditionally obtained from gas lasers. Convenient tunable continuous wave (c.w.) excitation for wide-held microscopy was widely available from filtered lamp sources but, for time domain FLIM, the only ultrafast light sources covering the visible spectrum were c.w. mode-locked dye lasers before the advent of ultrafast Ti Sapphire lasers. 

Padilla-Parra, S, Auduge, N, Coppey-Moisan, M and Tramier, M. (2008). Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells. Biophys. J DOI 10.1529/bio-... 

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. 

There are two ways to collect FLIM data freqnency-domain or time-domain data acqnisition (Alcala et al. 1985 Jameson et al. 1984). Briefly, in freqnency domain FLIM, the fluorescence lifetime is determined by its different phase relative to a freqnency modulated excitation signal nsing a fast Fourier transform algorithm. This method requires a frequency synthesizer phase-locked to the repetition freqnency of the laser to drive an RF power amplifier that modulates the amplification of the detector photomultiplier at the master frequency plus an additional cross-correlation freqnency. In contrast, time-domain FLIM directly measures t using a photon connting PMT and card. 

Fig. 2 Data acquisition for time-domain FLIM. FI fluorescence intensity, h gated image no 1,12 gated image no 2. Left Excitation pulse of the light source and synchronized timegated detection with a CCD camera. Right Lifetime determination by two subsequent time-gates according to Eq. 2...

Biological example - two-photon time-domain FLIM... 

The next example illustrates how two-photon time domain FLIM can be used for detecting the interaction of two protein kinases PDKl (3,4,5-phosphoinositide protein kinase) and PKB (protein kinase B) at the plasma membrane of NIH3T3 cells. Both PDKl and PKB associate with PtdIns(3,4,5)P3 and PtdIns(3,4)P2 via their plecktrin homology (PH) domains. It seems that this mutual interaction with such lipids leads these enzymes to co-localize at the plasma membrane and in turn to activate PKB. However, until recently it had not been shown that these molecules actually associate at the plasma membrane. 

The exploitation of RET by two-photon time domain FLIM has permitted illustration of the association of these two kinases. Figure 11.15 shows that NIH3T3 is... 

Figure 11.15 Interaction of PDKl and PKB detected by two-photon time domain FLIM. NIH3T3 are transfected with GFP-PDKl (upper panel), co-transfected with mRFP-PKB (middle panel) and stimulated by growth factor PDGF (lower panel). The lifetime maps indicate that the GFP-PDKl lifetime changes at the plasma membrane of these cells upon stimulation. In the presence of the acceptor mRFP-PKB there is no variation of the donor lifetime (GFP-PDKl) at the plasma membrane. The lifetime distributions are indicated by the histograms (right panels). It can be clearly seen that, upon stimulation, the GFP-PDKl lifetime at the plasma membrane decreases from 2.5 to 1.9 ns and the GFP-PDKl lifetime at the cytoplasm (2.3 ns) remains the same as when the acceptor is present. The decrease in lifetime at the plasma membrane illustrates that PDKl and PKB associate upon growth factor stimulation...

To use this type of microscope for FLIM, the camera has to be able to measure time resolved. Typically, a gain-modulated image intensifier is used for this, although recently the use of a modulated CCD camera for this has been reported [22,23]. The light source has to be intensity-modulated. This can be pulsed, for time-domain FLIM, or continuously, for frequency-domain FLIM. If a laser is used for excitation, the beam will have to be expanded to illuminate not a single point in the specimen but the entire field of view in order to obtain wide-field images. For references to wide-field FLIM instrumentation see Table 1, A1 and Bl. 

---