Two-photon FLIM

As described above, two-photon excitation microscopy provides several advantages (reduced photobleaching, deeper penetration into the specimen). A fluorescence microscope combining two-photon excitation and fluorescence time-resolved 

A great deal of information is available when lifetimes are imaged. It is indispensable for the user to have as much information as possible presented in images and plots that convey multiple parameters simultaneously and conveniently, especially when the images are available in real time. The example presented in this box illustrates how to achieve this. 

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Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

Another important lesson from Table 8.1 is that all three of the methods tested yielded virtually the same FRET efficiencies for the same samples. The sRET method as implemented used two-photon excitation on a Zeiss 510 META/NLO microscope, as did the FLIM-FRET method, but FLIM-FRET used auxiliary time... 

Another difference in these FRET methods is the cost of the microscopes. The two-photon microscope and its mode-locked laser used for sRET and FLIM-FRET cost approximately an order of magnitude more than the E-FRET system. Clearly, if cost is a limiting factor then the E-FRET approach is superior. 

A system specialised for diagnostie autofluorescence imaging of skin is de-seribed in [282, 283]. Typieal results are shown in Fig. 5.67, page 126. Other applieations of two-photon TCSPC FLIM with nondescanned deteetion are de-seribed in [7, 15, 16, 45, 46, 62, 68, 93, 147, 161, 372]. 

A two-photon microscope with multispectral FLIM and nondescanned detection is described in [60]. An image of the back aperture of the microscope lens is projected into the input plane of a fibre. The fibre feeds the light into a polychro-mator. The spectrum is detected by a PML-16 multianode detector head, and the time-resolved images of the 16 spectral channels are recorded in an SPC-830 TCSPC module. Spectrally resolved lifetime images obtained by this instrument are shown in Fig. 5.82. 

A potential application of multimodule systems is high-speed two-photon multibeam scaiming systems [53, 77]. FLIM systems with 4, 8 or even 16 beams and the same number of parallel TCSPC channels appear feasible. The problem is to direct the fluorescence signals from the individual beams to separate PMTs or separate charmels of a multianode PMT. If this problem is solved, two-photon lifetime images can be recorded with unprecedented speed and resolution. 

An application of TCSPC FLIM to CFP-YFP FRET is shown in Fig. 5.87 and Fig. 5.88. The microscope was a Zeiss LSM 510 NLO two-photon laser scanning microscope in the Axiovert 200 version. An excitation wavelength of 860 nm was used. The nondescanned fluorescence signal from the sample was fed out of the... 

A detailed description of a TCSPC-FLIM-FRET system is given in [147]. The system is used for FRET between ECPF-EYFP and FMl-43 - FM4-64 in cultured neurones. FRET between ECFP and EYFP in plant cells was demonstrated in [68]. FRET measurements in plant cells are difficult because of the strong autofluorescence of the plant tissue. It is possible to show that two-photon excitation can be used to keep the autofluorescence signal at a tolerable level. 

Excitation and detection geometry. The sample volume from which the fluorescence is detected can differ considerably. In two-photon imaging the excited volume is of the order of 0.1 pm. Confocal imaging with a wide pinhole detects from a considerably larger sample volume. Consequently, the fluorescence comes from a larger number of molecules, and a correspondingly higher intensity is available. The majority of FLIM experiments are performed in two-photon systems with a small focal volume and low intensity. 

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...

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. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

A nondescanned ( direct ) FLIM detection module with two wavelength channels was developed for the Radiance 2000 microscope from Biorad. The detector module contains computer-controlled dichroic beamsplitters and filters, preamplifiers, and overload shutdown of the detectors. The preamplifiers simultaneously deliver photon pulses to a Becker Hickl SPC-830 TCSPC and intensity signals to the standard steady-state recording electronics. Unfortunately all Radiance scanning microscopes were discontinued in 2004. 

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