Time-resolved fluorescence apparatus

The probe molecule pyrene (-10"6 M) was used in time-resolved fluorescence quenching experiments using a single photon counting apparatus, cetylpiridinium chloride (CpyC, 10"3 M) being introduced as a quencher of the pyrene fluorescence[ll-13]. All the experiments were performed at 303K. From these fluorescence studies the micelle aggregation number (N) and the pyrene fluorescence lifetime (x) were obtained [14]. 

Due to the short lifetime of the fluorescence decay process (10 — 10 s) time-resolved fluorescence studies provide a number of experimental difficulties and require the use of more sophisticated apparatus. Several tedini(pes have been applied to measure fluorescence decay characteristics but all require the use of a pulsed or modulated excitation source (see reviews by Birks, Ware, Knight and Selinger ). 

Fluorescence techniques were used to study micelles in clear solutions. Time-resolved fluorescence quenching (recording of the fluorescence decay curves on a single photon counting apparatus) was used to determine the pyrene (used as the probe molecule) fluorescence lifetime (t). Micelle aggregation numbers (N), which correspond to the number... 

Harris describe a method for the quantitative estimation of component amplitudes in multiexponential data obtained from time-resolved fluorescence spectroscopy. A design of apparatus which uses time correlated and single photon counting with alternate recording of excitation and emission minimises troublesome lamp... 

As a result of enormous development in the technology and production of pulse lasers, laser diodes, detector systems, and powerful computers in recent decades, steady-state and time-resolved fluorometers now belong to the standard equipment of biochemical and macromolecular laboratories. For example, there are apparatuses combined with microscopes that are suitable for time-resolved fluorescence measurements of individual organelles in living cells. However, the widespread use of fluorescence techniques generates certain danger, which is connected with their routine use. We would like to point out that the fluorescence spectroscopy is an indirect technique and that the interpretation of results needs great care and precaution. It almost always requires additional information on the system. 

Steady-state and time-resolved fluorescence spectroscopy Absorption and fluorescence spectra were measured with a Hitachi 557 spectrophotometer and a Hitachi 850 spectrofluorometer, respectively. The time-resolved fluorescence spectra were measured with the apparatus reported previously [4,6] in principle, the time-correlated single photon counting system under a low excitation condition. The pulse intensity (540 nm, 6 ps (fwhm)) was in a range of 10 to 10 photons/cm. The time resolution of our optical set-up was 6 ps. Correction of spectral sensitivity and data treatment were carried out as reported previously [4,6]. 

Time resolved fluorescence measurements were carried out using a single photon counting apparatus. Transient absorption data were taken with a flash photolysis system as described by Durrant et al. 1989a. 

For fluorescence measurements, by far the most versatile and widely used time-resolved emission technique involves time-correlated single-photon counting [8] in conjunction with mode-locked lasers, a typical mo m apparatus being shown in Figure 15.8. The instrument response time of such an apparatus with microchannel plate detectors is of the order of 70 ps, giving an ultimate capability of measurement of decay times in the region of 7 ps. However, it is the phenomenal sensitivity and accuracy which are the main attractive features of the technique, which is widely used for time-resolved fluorescence decay, time-resolved emission spectra, and time-resolved anisotropy measurements. Below ate described three applkations of such time-resolved measurements on synthetic polymers, derived from recent work by the author s group. 

The wavelength of fluorescence emission is different from its excitation wavelength, and a single fiber is sufficient for transport of both excitation and emission radiation. A single fiber is also used to measure fluorescence quenching, which occurs when a compound that absorbs in the same spectral region as the emission is present. In the future it may be possible to make time-resolved fluorescence measurements. However, fluorescent lifetimes are between 1 and 10 nanoseconds, and such measurements would require complex and costly apparatus. 

NADH can also be determined in vivo with an optical fiber using time-resolved fluorescence measurements [209]. This new technique exploits the rapid decrease in fluorescence over a few nanoseconds, and also eliminates the reduction in fluorescence by the optical fiber. The apparatus is shown in Figure 4.34. The excitation beam is provided by a sub-nanosecond nitrogen laser (337 nm). This excitation beam and die emitted fluorescence spectrum are transmitted by a single optical fiber with a diameter of 200 p.m. If NADH concentrations can be directly measured in vivo then a wide variety of biosensors can be envisaged that exploit the enzymatic redox reactions involving this cofactor. 

Figure 4.34 Apparatus for time-resolved fluorescence measurements using a single fiber configuration (M - mirror L - lens PM -photomultiplier).

Brune et have described a new tandem axis laser magnetic resonance (LMR), resonance fluorescence, and resonance absorption fast-flow apparatus for the study of the kinetics of elementary gas reactions. They placed emphasis on the simultaneous time-resolved detection of reactants and products, crosscalibration of detection axes, the use of multiple sources of free radicals and computer simulation of the time-dependence of the reactant and product concentrations in experiments designed to determine elementary reaction rate constants. They provided data for the reactions N + OH — H+NO, N-1-H02— products, O+OH— -H+02, and 0 + H02- OH+O2. 

Some degree of temporal resolution of emission may be obtained by incorporating a phosphoroscope attachment in the simple apparatus described above. A mechanical or electronic device is used to allow periodic and out-of-phase excitation and detection of luminescence. In the simplest case a mechanical shutter interrupts the excitation beam periodically and the detection system is gated so that emission is observed only after a fixed interval of time has elapsed after excitation. Under these conditions short-lived processes such as prompt fluorescence will have decayed to zero intensity and only longer-lived emission will be recorded. For mechanical devices the limit of measurable lifetime is of the order of 1 ms, thus allowing time resolved studies to be made of certain phosphorescence and delayed emission procesres (see ... 

In the present paper we describe an apparatus for recording transient emission spectra that yields data which approach the ideal multidimensional case. We emphasize in the discussion the advantages of multichannel detection for transient emission data. We also briefly compare our approach to alternative methods for recording time and wavelength resolved fluorescence data on the picosecond time scale. 

The apparatus shown in Figure 7.14 was developed to achieve high sensitivity measurements of both the fluorescence intensity and time-resolved lifetime of the... 

Time-resolved techniques (time-resolved fluoroimmunoassay (TRFIA)) make use of the fact that some compounds have long decay times resulting in phosphorescence or delayed fluorescence (e.g., lanthanide chelate complexes) (Figure 1). Apparatus for time-resolved measurements use normally a pulsed excitation source and electronically gated detectors. By this way background fluorescence can be eliminated. 

Towrie, M., Grills, D.C., Dyer, L., Weinstein, J.A., Matousek, R, Barton, R., Bailey, R.D., Subramaniam, N., Kwok, W.M., Ma, C., Phillips, D., Parker, A.W. and George, M.W. (2003) Development of a broadband picosecond infrared spectrometer and its incorporation into an existing ultrafast time-resolved resonance Raman, UV/visible, and fluorescence spectroscopic apparatus. Appl. Spectrosc., 57, 367-380. 

---