TCSPC for FLIM and FRET in - Boston Electronics Corporation

A phase fluorometer of this type can be built up by using a commercially available network analyserand a laser diode. The setup is very simple - unless you have to build the network analyser. Theprinciple is shown in fig. 22. The network analyser runs afrequency sweep over a selectable frequency interval. Theoutput signal of the network analyser drives a laser diodethat is used to excite the sample. The detected fluorescencesignal is fed back into the signal analyser. The measurementdelivers the phase and the amplitude of the signal as afunction of the frequency. The network analyser is even ableto correct the results by using reference data recorded with ascattering solution in place of the sample.The mixers used in fig. 20 and 21 can be replaced with amodulated detector (fig. 23). A setup of this type has beenused for lifetime imaging in a two-photon laser scanningmicroscope [54]. A commercially available frequencysynthesiser generates the modulation frequency, theoscillator frequency and the difference of both with a highfrequency and phase stability. Mixing is accomplished bymodulating the gain in a photomultiplier. The mixed signalat the output of the PMT has a frequency of 25 kHz and isdirectly digitised. Further filtering and phase calculation isdone digitally.The benefit of the detector modulation is that the modulationfrequency is not limited by the bandwidth of the gain systemof the PMT. Therefore relatively high modulationfrequencies can be used. The drawback is that the detector isa very poor mixer. This results in a poor efficiency of thedetector gain modulation technique.If a Ti:Sa laser is used in a modulation system, e.g. for twophotonexcitation, the 80 MHz fundamental repetitionfrequency of the laser and its harmonics can be used asmodulation frequency. Due to the short pulse width theAmplifierspectrum of the laser modulation is actually a frequency comb that extends up to THz frequencies.By proper tuning of the frequency synthesiser, any of the harmonics of the fundamental repetitionfrequency within the detector bandwidth can be used for the phase measurement.A heterodyne system with a Ti:Sa laser, two-photon excitation and modulated PMT delivered alifetime accuracy of ± 300 ps for lifetimes between 2.4 and 4.2 ns [54]. This relatively pooraccuracy is probably due to the short acquisition time of only 160 µs per pixel or 10 seconds for a256 x 256 pixel image. Although the photon detection rate was estimated to be more than 10 8photons/s - which is probably an overestimation - the accuracy is of the same order as for TCSPCimaging with 10 6 photons per second and the same acquisition time and pixel number.The efficiency of the single channel modulation technique in terms of photon counts stronglydepends on the operating conditions. Results of Monte-Carlo simulations of the efficiency forsingle-exponential decay are given in [39]. The simulations yield ‘F values’, i.e. the ratio of theideal SNR to the SNR achieved by the investigated method.For a setup with separate detector and mixer, sine-wave-modulated excitation, sine-wave mixingand 100% modulation of the excitation F was found to be 3.7. If the modulation is less than 100% aLaserDiodeNetwork AnalyserRF outDiode BiasSampleRF inDetectorAmplifierFig. 22: Phase fluorometer with a networkanalyserFrequencySynthesiserLaser Modulator Samplef modf oscf mod - f oscdigitiserA/DGainmodulatedDetectorA/Ddigital filterphase calculationf mod - f oscdigitiserFig. 23: Mixing by gain-modulating the detector19