Frequency-domain FLIM

In frequency-domain FLIM, the optics and detection system (MCP image intensifier and slow scan CCD camera) are similar to that of time-domain FLIM, except for the light source, which consists of a CW laser and an acousto-optical modulator instead of a pulsed laser. The principle of lifetime measurement is the same as that described in Chapter 6 (Section 6.2.3.1). The phase shift and modulation depth are measured relative to a known fluorescence standard or to scattering of the excitation light. There are two possible modes of detection heterodyne and homodyne detection. 

In the homodyne mode of detection, the modulation frequency of the excitation light is the same as that of the image intensifier. An example of data is shown in Box 11.1. 

In the case of a single exponential decay, the lifetime can be rapidly calculated by either the phase shift D or the modulation ratio M by means of Eqs (6.25) and (6.26) established in Chapter 6 (Section 6.2.3)  

If the values calculated in these two ways are identical, the fluorescence decay is indeed a single exponential. Otherwise, for a multi-component decay, t tm. In this case, several series of images have to be acquired at different frequencies (at least 5 for a triple exponential decay because three lifetimes and two fractional amplitudes are to be determined), which is a challenging computational problem. 

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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... 

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. 

Two different approaches for measuring fluorescence lifetimes are commonly employed to study FRET-related phenomenon the frequency-domain FLIM (see Chapter 2) and the time-domain... 

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) .

Buranachai C, Kamiyama D, Chiba A et al (2008) Rapid frequency-domain FLIM spinning disk confocal microscope lifetime resolution, image improvement and wavelet analysis. JFluoresc 18 929-942... 

A.H.A. Clayton, Q.A. Hanley, D.J. Arndt-Jovin, V. Subramaniam, T.M. Jovin, Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (FLIM), Biophys. J. 83, 1631-1649 (2002)... 

With frequency domain FLIM the light source is a continuous wave laser as opposed to a pulsed laser. The continuous wave laser is modulated via an acousto-optical modulator and the sample is excited by a sinusoidally modulated light. The fluorescence response is also sinusoidally modulated at the same frequency but it is delayed in phase and is partially demodulated. For a single exponential decay the lifetime of the donor chromophore can be quickly calculated by either the phase shift (j) (rp) or the modulation ratio M (r ,) using the following equations ... 

The above relations show how the phase shift and the relative modulation are related to the decay time. Although single frequency-domain FLIM has the advantage of... 

Biological examples of RET monitored by frequency-domain FLIM... 

In frequency-domain FLIM, the intensity of the excitation light is continuously modulated. Due to the (non-instant) fluorescence decay, the fluorescence emission will display a phase shift and a decrease in modulation. This can be understood if we look at the fluorescence intensity of a mono-exponentially decaying fluorochrome after an infinitesimal short pulse of excitation light at t=0 as described by Eq. (1) ... 

To use this type of microscope for FLIM, the camera has to be able to measure time resolved. Typically, a gain-modulated image intensifier is used for this, although recently the use of a modulated CCD camera for this has been reported [22,23]. The light source has to be intensity-modulated. This can be pulsed, for time-domain FLIM, or continuously, for frequency-domain FLIM. If a laser is used for excitation, the beam will have to be expanded to illuminate not a single point in the specimen but the entire field of view in order to obtain wide-field images. For references to wide-field FLIM instrumentation see Table 1, A1 and Bl. 

Comparing the frequency-domain and the time-domain approach, it was found that one is not fundamentally better than the other in terms of signal to noise ratio, when measuring the fluorescence lifetime of a mono-exponentially decaying fluorochrome [33]. In both systems, it is crucial, however, to use the optimal settings for each particular measurement. In frequency-domain FLIM, the... 

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