Frequency domain FLIM data

To analyze frequency domain FLIM data, first the phase shift and demodulation of the fluorescence light with respect to the excitation light are estimated. In the case of single frequency data, this reduces the FLIM data to only three parameters phase shift, demodulation, and total intensity. This step can be done in various ways as described in the following sections. From these parameters, the lifetimes can be estimated either by Eqs. (2.6 and 2.7), or by more elaborate approaches as described below. 

The second chapter by Peter Verveer and Quentin Hanley describes frequency domain FLIM and global analysis. While the frequency domain technique for fluorescence lifetime measurement is sometimes counterintuitive, the majority of the 10 most cited papers using FLIM have taken advantage of the frequency domain method as stated by these authors. The global analysis of lifetime data in the frequency domain, resolving both E and /d has contributed significantly to this advantage. 

Frequency domain FLIM theory, instrumentation, and data analysis... 

The data in Figure B11.1.1 were acquired with a frequency-domain FLIM instrument working in homodyne mode. In this mode, the modulation frequency of the intensifier (40 MHz) is identical to that of the excitation light (40 MHz). The phase and modulation are calculated from a series of images taken at different phase delays between the excitation light and the intensifier. 

Fig. Bll.1.1. FLIM data of a 3T3 cell (stained with BODIPY FL C5-ceramide) obtained by a frequency-domain FLIM instrument (see text) .

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. 

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