TCSPC for FLIM and FRET in - Boston Electronics Corporation

It has been shown that diode lasers can be used for time-resolved microscopy with good results [33,33a]. Unfortunately the beam quality of diode lasers is not very good. Therefore it can be difficult toobtain a diffraction-limited resolution. However, if only the central part of the beam is used, theresult can be quite acceptable. Discarding a large fraction of the beam causes a considerable loss ofpower. This loss is, however, not substantial because 50 µW in the focal plane are sufficient toexcite the sample.Modulated CW LasersFor signal processing techniques based on phase measurement of modulated signals CW Ar+ andHeNe lasers in conjunction with an acousto-optical modulator can be used. However, care must betaken to avoid any crosstalk of the modulation frequency into the detection system. The smallestamount of crosstalk makes an accurate phase measurement impossible.Using modulated CW lasers in conjunction with photon counting techniques is not recommended.These techniques require pulses rather than sinewave signals. Acousto-optical modulators areresonance systems unable to deliver sufficiently short pulses with high on/off ratio.Mode-locked CW LasersAr+ lasers can be actively mode-locked. By introducing a modulator into the laser cavity pulses asshort as 100 ps with 80 to 120 MHz repetition rate are obtained. The pulses can be used directly orto pump a jet-stream dye laser that delivers ps pulses at a wavelength tuneable from 500 to 600 nm .Although the light from these lasers can be used for fluorescence excitation [65] the systems areoften unstable and require permanent maintenance, checking and re-adjustment. We stronglydiscourage to use these lasers as excitation sources for time-resolved microscopy.Pulse Pickers and Cavity DumpersPulse pickers and cavity dumpers are used to obtain a lower repetition rate from lasers running at ahigh, fixed repetition rate. For fluorescence measurements they are sometimes used to measurelifetimes longer than the original period of the laser. The problem of the devices is the poor on-offratio and the electrical noise they often produce. It is by far better to take some incomplete decayinto regard in the data analysis than to cope with electrical noise and satellite pulses. Furthermore, alow repetition rate reduces the useful count rate in photon counting setups and possibly increasesphotobleaching. Don’t use pulse pickers if they are not absolutely necessary.DetectorsPhotomultiplier Tubes (PMTs)The most common detectors for low level detection of lightare photomultiplier tubes. A conventional photomultipliertube (PMT) is a vacuum device which contains aphotocathode, a number of dynodes (amplifying stages) andPhotoananode which delivers the output signal (fig. 9).CathodeD2D1D3D4D6D5D7D8 AnodeThe operating voltage builds up an electrical field thataccelerates the electrons from the photocathode to the firstFig. 9 Principle of a conventional PMTdynode D1, from D1 to D2, further to the next dynodes, andfrom D8 to the anode. When a photoelectron emitted from the photocathode hits D1 it releasesseveral secondary electrons. The same happens for the electrons emitted by D1 when they hit D2.The overall gain reaches values of 10 6 to 10 8 . The secondary emission at the dynodes is very fast,therefore the secondary electrons resulting from one photoelectron arrive at the anode within a few9