Fluorescence lifetime/steady state mode

Based on relaxation studies (Tritton and Mohr, 1971), fluorescence lifetime, steady-state fluorescence studies (Jones et al., 1978), and, especially, H-NMR studies (Jones and Kearns, 1975 Jones et al., 1978), the major mode of binding of Et to tRNa is believed to be intercalation between base pairs AUg and AU7 (or AUj-AUg) of the acceptor stem. The interpretation has received wide acceptance among many authors. However, the X-ray diffraction studies of crystals of tRNA into which Et have been allowed to diffuse indicate another mode of binding. The ethidium is... 

In addition to chemical reaction, weak fluorescence was detected from 50 at room temperature (acxc 460 nm, Xem 552 nm, cj)f = 0.04). Temperature effects on reaction and fluorescence from 77-310 K have been studied 68). A steady decrease in quantum yield for reaction (r) and a complementary increase in fluorescence quantum yield (< )f) were observed down to about 150K where a sharp increase in f occurred. Photochemical reaction was negligible at 77 K (436 nm). The fluorescence lifetime at 77 K was a few nanoseconds and the estimated value at room temperature is on the order of 60 ps. Detailed analysis of the data showed that two thermally-activated processes are involved (1) chemical reaction of the singlet state with an Arrhenius activation energy of 1.5 kcal/mol and (2) radiationless decay of the singlet with Eact =1.1 kcal/mol. Both processes would appear to be associated with certain vibrational modes of the excited state which become progressively less populated with decreasing temperature. 

What all FLIM instruments have in common is that 1) the excitation light is intensity-modulated or pulsed, and 2) that the emitted fluorescence light is measured time-resolved. Since the lifetimes that have to be resolved are typically in the nanosecond range, this means that both the modulation of the excitation light and the detection need to be performed at extremely high speeds. Consequently, most of the conventional instrumentation used for steady-state fluorescence microscopy cannot be used. In the next section several modes of implementation of FLIM are discussed. 

Some of these applications are complementary to the steady state methods discussed in section 8.2. Investigations of fluorescence lifetimes and of anisotropy or fluorescence quenching phenomena in the lifetime mode, that is during the decay after a single flash, require more elaborate instrumentation and theory than steady state investigations. On the whole applications rather than detail of methods are discussed here. The use of the lifetime method for the study of molecular rotation, domain movement and more local dynamic events can often, some experts say always, provide additional information even for those problems which can be investigated with considerable success by steady state measurements. 

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