Single-photon fluorescence principles

The principle of the determination of time-resolved fluorescence spectra is described in Section 6.2.8. For solvent relaxation in the nanosecond time range, the single-photon timing technique can be used. The first investigation using this technique was reported by Ware and coworkers (1971). Figure 7.3 shows the reconstructed spectra of 4-aminophthalimide (4-AP) at various times after excitation. 

Two techniques, phase and pulse fluorometry, are used for the direct measurement of fluorescence decay rates, and their principles are described by Birks and Munro (1967), Parker (1968), and Birks (1970). The photon sampling method has proved useful and versatile. This is an iterative technique in which single photons are counted as a function of the time at which they appear after excitation and a complete decay curve is built up. (For recent references see e.g. Zimmerman et al., 1973, 1974). Wider use of the photon sampling technique will increase the precision of lifetimes obtained and extend the range of compounds studied to those with shorter lifetimes or very low fluorescence yields. 

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. 

Figure 11.13 Representation of fluorescence decay and of the principle of the measurement. On the graph, each point represents the contents of a memory channel (coupling time/numher of photons). The exponential curve of decay appears here in the form of a straight line. This is due to the choice of a logarithmic scale for the ordinate. On the right, a representation of the TCSPC (time-correlated single-photon counting).

The bimolecular reaction dynamics of geminate recombination or acid-base neutralization have until recently been studied with time-resolved techniques probing electronic transitions. Time-resolved fluorescence using time-correlated single photon counting detection is limited to a time resolution of a few picoseconds. UV/vis pump-probe experiments, in principle, may have a time resolution of a few tens of femtoseconds, but may be hampered by overlapping contributions of... 

Perhaps the most significant contrihution that two-photon excited fluorescence has made in biological research to date is in the area of fluorescent imaging. Prior to the invention of two-photon microscopy in 1990, depth resolution in fluorescence microscopy was achieved using the confocal microscope, the principles of which are illustrated in Figure 11.9. Fluorescence is excited via single-photon excitation... 

This relationship is modified by two constants the molecular shape factor/ (a function of the molecular dimensions) and the boundary coefficient C, which takes into account the interaction between the solvent and the solute. In principle, two-photon fluorescence anisotropy decays in isotropic media should yield the same diffusion times as for single photon excitation, but with significantly increased initial fluorescence anisotropy this can be seen in Figure 11.17, which compares single- and two-photon anisotropy decays for the fluorescent probe rhodamine 6G in ethylene glycol. Rotational drflusion times for small molecular probes vary from nanoseconds to hundreds of picoseconds for isotropic rotational drflusion in low viscosity solvents. 

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]. 

Figure 3.7 The principles of one- and two-photon excitation. In one-photon excitation (left) a single photon, whose energy matches the electronic transition between the ground state and first singlet state causes an electron to be excited. Subsequent relaxation of the electron results in fluorescence emission. In two-photon excitation, two photons, each with approximately half the energy of the electronic transition, are absorbed simultaneously resulting in excitation of an electron and subsequent fluorescence.

---