Steady-State Photon Counting

Steady-state fluorescence spectra, fluorescence quantum yield (F) and lifetimes (tf) of DTT 15 and DTP 23a were estimated as shown in Table 8. F for DTT is higher than DTP. F for DTP is very small and it was difficult to estimate an accurate fluorescence lifetime by the photon counting method due to weak fluorescence. It is noted that the for DTP depends largely on the solvent and is 7.7 x 10-5 in acetonitrile. This low F value has been attributed to an addition reaction with the solvent. 

Every time an excited molecule exits the excited state region by the fluorescence pathway it emits a photon. We can either count the number of photons in a longer time interval (by a steady-state measurement of the fluorescence intensity) or make a time-resolved measurement of the fluorescence decay. These measurements can be done in an ensemble mode or on single molecules—the basic process is the same. The number of photons collected from the donor emission will be depicted by IDA and ID, where we mean the fluorescence intensity of D in the presence (Ida) and absence (ID) of acceptor. All other conditions, other than the presence or absence of acceptor, remain the same. During the same time of the experiment where we have measured the photons emitted by D, many of the excited D molecules have exited from the excited state by a pathway other than fluorescence. Obviously, the number of times a pathway has been chosen as an exit pathway is proportional to the... 

In this final section, we summarize the operation and characteristics of the principal vacuum tube and solid state detectors that are available for red/near-IR fluorescence studies. These include conventional photomultipliers, microchannel plate versions, streak cameras, and various types of photodiodes. Detector applicability to both steady-state and time-resolved studies will be considered. However, emphasis will be placed on photon counting capabilities as this provides the ultimate sensitivity in steady-state fluorescence measurements as well as permitting lifetime studies. 

At the present time, two methods are in common use for the determination of time-resolved anisotropy parameters—the single-photon counting or pulse method 55-56 and the frequency-domain or phase fluorometric methods. 57 59) These are described elsewhere in this series. Recently, both of these techniques have undergone considerable development, and there are a number of commercially available instruments which include analysis software. The question of which technique would be better for the study of membranes is therefore difficult to answer. Certainly, however, the multifrequency phase instruments are now fully comparable with the time-domain instruments, a situation which was not the case only a few years ago. Time-resolved measurements are generally rather more difficult to perform and may take considerably longer than the steady-state anisotropy measurements, and this should be borne in mind when samples are unstable or if information of kinetics is required. It is therefore important to evaluate the need to take such measurements in studies of membranes. Steady-state instruments are of course much less expensive, and considerable information can be extracted, although polarization optics are not usually supplied as standard. 

Instrumentation. The steady-state fluorescence spectra were measured with Perkin-Elmer MPF-44B fluorescence spectrophotometer. The single-photon counting instrument for fluorescence lifetime measurements was assembled in-house from components obtained from EG G ORTEC. A PRA-510B light pulser filled with gas was used as the excitation source. Instrument response function was obtained with DuPont Ludox scatter solution at the excitation wavelength. 

Several ways exist to determine the efficiency by measuring steady-state fluorescence. It is easily observed by inspecting the first equality in Equation 2. The best way to acquire steady-state values of the fluorescence intensity is photon counting. We will not go into the way it is done with hardware and electronics (it is usually done automatically with modem instmmentation), but simply note that by using photon counting one can easily... 

Steady-state emission spectra were recorded with a fluorescence spectrophotometer (Model 850, Hitachi Ltd.). Emission lifetime measurements were carried out using laser excitation pulses and a time-correlated single-photon counting system as described elsewhere. 

Emission spectra and absorption spectra were recorded on a Perkin-Elmer 650-iOS Fluorescence Spectrophotometer and a Perkin-Elmer 320 tn/ Spectrophotometer, respectively. Fluorescence decay data were obtained on a single-photon-counting apparatus from Photochemical Research Associates. The samples were bubbled witli nitrogen for the steady-state fluorescence spectra and the fluorescence decay measurements. In some cases, front face spectra were taken. The data were analyzed by a software package from PRA based on the iterative convolution method. NMR spectra were obtained on a JEOL FX90Q, and FTIR spectra were recorded on a Nicolet 5DX. The elemental analyses were conducted by M-H-W Laboratories of Phoenix, AZ. 

The great sensitivity of fluorescence spectral, intensity, decay and anisotropy measurements has led to their widespread use in synthetic polymer systems, where interpretations of results are based upon order, molecular motion, and electronic energy migration (1). Time-resolved methods down to picosecond time-resolution using a variety of detection methods but principally that of time-correlated single photon counting, can in principle, probe these processes in much finer detail than steady-state techniques, but the complexity of most synthetic polymers poses severe problems in interpretation of results. 

The complexes of 2,3 (sodium salt) and 4 (potassium salt) with P-CD and (2,3,6- tri-0-methyl)-/ -CD were studi using steady-state fluorescence and time-correlated, single-photon counting techniques [52]. The formation of both 1 1 and 2 1 complexes between p-CD and 2,3 was confirmed. Trimethyl- -CD gave evidence only of 1 1 complexes. The fluorescence decay of systems giving exclusively 1 1 complexes was collected at CD concentrations that ensure more than 90% complexation. The analysis performed using a continuous lifetime distribution model... 

In the steady state, = 0, we have tin = Tp, and since Qv has a meaning of absorbed photon current, Fp has the meaning of photon emission current. For the stochastic Bloch equation, a steady photon flux is never reached however, integrating Eq. (4.13) over the counting time interval T,... 

The following estimation illustrates the possible sensitivity of resonant two-photon ionization spectroscopy (Fig. 1.36a). Let Nk be the density of excited molecules in level Ek, Pki the probability per second that a molecule in level Ek is ionized by photons from laser L2 and /la = Nin aikiS.x (1.34) the number of photons absorbed per second on the transition Ei Ek.li Rk is the total relaxation rate of level Ek, besides the ionization rate (spontaneous transitions plus collision-induced deactivation) the signal rate in counts per second for the absorption path length Ax and for incident laser photons per second under steady state conditions is ... 

Steady state absorption and fluorescence measurements were made using a Perkin-Elmer 554 spectrophotometer and a Perkin-Elmer MPF-4 fluorimeter. Fluorescence lifetimes were measured using time-correlated single photon counting [5,7]. Details of the analysis of the decays can be found in [7]. 

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]. 

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