FLIM Measurement System

In the present study, a FLIM measurement system was constructed and applied to Halobacterium salinarum (Hb. salinarum) loaded with 2, 7 -bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to obtain information on the intracellular environment as well as the intracellular pH in each ofthe cells [9-12]. Hb. salinarum belongs to the family of extreme halophilic archaebacteria, and considerable attention has been paid to this bacterium in relation to proton transport, phototaxis or the adaptation of an organism to extreme environments [13-15]. Intracellular pH is an essential parameter for Hb. salinarum in the regulation of intracellular processes [14, 16, 17], and fluorescence intensity ratio methods have been used to measure the intracellular pH [18,19]. The... 

Examination of Eqs. (2.9-2.11) suggests that having frequency domain lifetimes measured at a variety of frequencies is desirable, as it will allow a mixture of fluorophores to be determined. With this in mind, two approaches may be taken to obtain multifrequency results. The first of these is simply to make a series of FLIM measurements while stepping through a predetermined set of frequencies. In practice, this is of limited utility for biological systems because of photo-induced damage to the specimen. 

FLIM measurements were carried out using a four-channel time-gated detection system [9,11, 34]. The experimental system is shown in Figure 31.2. A mode-locked... 

A detailed description of a TCSPC-FLIM-FRET system is given in [147]. The system is used for FRET between ECPF-EYFP and FMl-43 - FM4-64 in cultured neurones. FRET between ECFP and EYFP in plant cells was demonstrated in [68]. FRET measurements in plant cells are difficult because of the strong autofluorescence of the plant tissue. It is possible to show that two-photon excitation can be used to keep the autofluorescence signal at a tolerable level. 

Acquisition times for TCSPC FLIM measurements can vary widely. In vivo lifetime measurements of the human ocular fundus in conjunction with an ophthalmic scanner delivered single exponential lifetimes for an array of 128 x 128 pixels within a few seconds [451, 452, 454]. High-quality double exponential lifetime images of microscopic samples were obtained within 10 seconds by a four-module TCSPC system (see Fig. 5.84) [39]. On the other hand, for the double exponential decay data of FRET measurements in live cells (see Fig. 5.87), acquisition times ranged from 5 to 30 minutes [32, 37]. In practice the acquisition time depends on the size and the photostability of the sample and the requirements for accuracy rather than on the counting capability of the TCSPC device. 

Instrumentally, spectral FLIM generates a spectrally resolved set of lifetimes by either introducing filters to provide spectral resolution or a spectrograph between the sample and image intensifier. The first such system was created for looking at the long lifetimes of lanthanide dyes [37]. Later, a spectral FLIM system was described for measuring from a two-dimensional (2D) area of a microscope field... 

The accuracy with which a system can measure lifetimes depends on a number of different factors including calibration of the instrument, the number of detected photons and also the efficiency of the analysis routines. In addition, sources of background and scattered light should be eliminated. Emission filters should be chosen with great care to make sure that no scattered laser light reaches the detector. Detection of scattered excitation light results in a spurious fast component in the decay and complicates the interpretation of the data. The choice of emission filters is much more critical in FLIM than in conventional fluorescence intensity imaging methods. 

The lifetime resolution is the smallest variation in lifetime that can be detected. If external noise sources are ignored, the lifetime resolution depends essentially on the photon-economy of the system. For instance, if a 2 ns lifetime is measured with a 4 gate TG single-photon counting FLIM (F = 1.3) and 1000 photons, variations of about 80 ps can be resolved. However, for reasons discussed earlier, in biological samples these values could be higher. 

Measuring FRET by fluorescence lifetime imaging microscopy (FRET-FLIM) offers the ability to see beyond the resolution of the optical system ( 10-100 times that of modern far field microscopes [5]). FRET efficiency can be used as a proxy for molecular distance, thereby allowing the easy detection and somewhat more challenging quantification of molecular interactions. Although many types of assay exist, FRET-FLIM is a highly suitable technique that is capable of in situ measurements of molecular interactions and conformation in living and fixed cells. 

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. 

As described in the previous section, the femtosecond fluorescence up-conversion microscope enabled us to visualize microscopic samples based on position-depen-dent ultrafast fluorescence dynamics. However, in the imaging measurements using the fluorescence up-conversion microscope, XY scanning was necessary as when using FLIM systems. To achieve non-scanning measurements of time-resolved fluorescence images, we developed another time-resolved fluorescence microscope. 

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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