FLIM measurements

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. 

However, FLIM measurements by both frequency and time domain using commercially available software suffers from variation in the measured lifetime from region to region in cells [36], which can be confusing. In the light of the potential pitfalls associated with each of the above-mentioned FRET techniques, potential protein associations should ideally be tested independently by a combination of two or more FRET-based methods in addition to biochemical techniques such as co-immunoprecipitations. 

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... 

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... 

FLIM measurements were applied to Hb. salinarum loaded with BCECF. At least two halobacteria species that exhibit different fluorescence lifetimes from each other are found to exist in the cell suspension, suggesting that the cells have different intracellular environments. The difference in the fluorescence lifetime may reflect the difference in activity of halobacteria. It is suggested that strong electric fields inside a cell play a significant role in the determination of the fluorescence lifetime of BC ECF. 

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. 

Fluorescence lifetime imaging microscopy (FLIM) measures a chromophore s fluorescence lifetime, allowing spatial resolution of biochemical processes. The fluorescence lifetime of a donor dye decreases under FRET conditions, independent of fluorophore concentration or of excitation intensity (35). 

Fluorescence Lifetime Imaging Microscopy (FLIM) Measurement... 

For FLIM determinations, cells are plated and transfected as in Subheading 3.7.1, FLIM measurements are performed using a confocal microscope with a High-Speed Lifetime Module and a 60x PlanApo 1.4 objective or equivalent equipment. Fluorescence lifetime is determined after excitation with a pulsed laser (picosecond pulses) and a bandpass emission filter appropriate for the fluorochrome used, and quantitated using LIMO (Nikon) or similar software. Avoid the use of mounting solutions, which increase autofluorescence. 

Time-resolved image acquisition is also achievable by fluorescence lifetime imaging microscopy (FLIM). FLIM measures the fluorescence decay of each pixel of an image, on the basis of which time-resolved imaging can be performed. FLIM has two main variants the time-resolved information is established either in the time... 

Typically, pulsed light sources are used for time-domain measurements, but frequency-domain measurements are possible as well, since the pulse rate can be made very constant and usually is in the range used for frequency-domain FLIM measurements (around 80 MHz). An added advantage is that a pulsed signal is very rich in harmonic-content, when compared to a modulated signal coming out of an AOM, or EOM, enabling multi-frequency measurements. 

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