Phase-modulation method

The fluorescent lifetime of chlorophyll in vivo was first measured in 1957, independently by Brody and Rabinowitch (62) using pulse methods, and by Dmitrievskyand co-workers (63) using phase modulation methods. Because the measured quantum yield was lower than that predicted from the measured lifetime, it was concluded that much of the chlorophyll molecule was non-fluorescent, suggesting that energy transfer mechanisms were the means of moving absorbed energy to reactive parts of the molecule. 

Prior to describing the possible applications of laser-diode fluorometry, it is important to understand the two methods now used to measure fluorescence lifetimes these being the time-domain (Tl)/4 5 24 and frequency-domain (FD) or phase-modulation methods.(25) In TD fluorometry, the sample is excited by a pulse of light followed by measurement of the time-dependent intensity. In FD fluorometry, the sample is excited with amplitude-modulated light. The lifetime can be found from the phase angle delay and demodulation of the emission relative to the modulated incident light. We do not wish to fuel the debate of TD versus FD methods, but it is clear that phase and modulation measurements can be performed with simple and low cost instrumentation, and can provide excellent accuracy with short data acquisition times. 

There are two widely used methods for measuring fluorescence lifetimes, the time-domain and frequency-domain or phase-modulation methods. The basic principles of time-domain fluorometry are described in Chapter 1, Vol.l of this series(34) and those of frequency-domain in Chapter 5, Vol. 1 of this series.<35) Good accounts of time-resolved measurements using these methods are also given elsewhere/36,37) It is common to represent intensity decays of varying complexity in terms of the multiexponential model... 

The principal requirements for photomultipliers in both pulse and phase-modulation methods of measuring fluorescence lifetimes are as follows. 

Fig. 1. Set-up of the PTB laser system. The Nd YAG laser is frequency stabilized onto a selected iodine absorption line using the phase modulation method. The probe beam is modulated at 2.05 MHz by an electro-optic modulator (EOM), the pumb beam is frequency shifted by an acousto-optical modulator (AOM). The driving AOM rf power is chopped in order to cancel frequency offsets introduced by the Doppler background using a lock-in detection scheme. The transmitted probe beam signal is detected by a photodiode (PD) and mixed with the EOM rf in a double balanced mixer (DBM)...

Because of advances in the technology for measuring lifetimes, particularly by the phase-modulation method (i2-i7), lifetime-based sensing (Figure 1) offers new opportunities for chemical sensing. This is because the fluorescence lifetimes of probes can be sensitive to a variety of factors or chemicals. Moreover, lifetime measurements are insensitive to probe... 

There are two methods that have been used to determine fluorescence lifetimes in DNA sequencing. In the first method, known as the frequency-domain or phase-modulation method, the excitation beam is intensity modulated. The a.c. portion of the resulting emission is phase-shifted relative to the laser modulation this phase-shift contains information about the fluorescence lifetime, or lifetimes if more than one fluor is present [140]. McGown and coworkers [144,145] used this method for four-color sequencing. In that work, 488 nm or 514 nm laser light was electronically modulated with a Pockels cell before being focused onto a capillary column. Detection, made normal to the laser direction, was optically filtered to reduce laser scatter and was focused onto the detector of a... 

Jurdana LE, Ghiggino KP, Leaver IH, and Cole-Clarke P (1995) Application of FT-IR step-scan photoacoustic phase modulation methods to keratin fibres. Applied Spectroscopy 49 361-366. 

---