Single-photon fluorescence anisotropy

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. 

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

Fluorescent chemical sensors occupy nowadays a prominent place among the optical devices due to its superb sensitivity (just a single photon sometimes suffices for quantifying luminescence compared to detecting the intensity difference between two beams of light in absorption techniques), combined with the required selectivity that photo- or chemi-luminescence impart to the electronic excitation. This is due to the fact that the excitation and emission wavelengths can be selected from those of the absorption and luminescence bands of the luminophore molecule in addition, the emission kinetics and anisotropy features of the latter add specificity to luminescent measurements8 10. 

D. J. S. Birch, A. S. Holmes, J. R. Gilchrist, R. E. Imhof, S. M. A1 Alawi and B. Nadolski, A multiplexed single-photon instrument for routine measurement of time-resolved fluorescence anisotropy, J. Phys. E Sci. Instrum. 20, 471-473 (1987). 

Sample preparation was given elsewhere [2]. Femtosecond fluorescence upconversion and picosecond time-correlated single-photon-counting set-ups were employed for the measurement of the fluorescence transients. The system response (FWHM) of the femtosecond fluorescence up-conversion and time-correlated single-photon-counting setups are 280 fs and 16 ps, respectively [3] The measured transients were fitted to multiexponential functions convoluted with the system response function. After deconvolution the time resolution was 100 fs. In the upconversion experiments, excitation was at 350 nm, the transients were measured from 420 nm upto 680 nm. Experiments were performed under magic angle conditions (to remove the fluorescence intensity effects of rotational motions of the probed molecules), as well as under polarization conditions in order to obtain the time evolution of the fluorescence anisotropy. 

The sum of the [1/ for m-C 1P3 and m-C 1P4 is substantially smaller than the limiting anisotropy. This strongly suggests that, at a time shorter than the resolution of single-photon timing, there is already a process leading to loss of fluorescence polarization in the meta-substituted dendritic systems. 

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 11.17 Single and two photon excited fluorescence anisotropy decays recorded for rhodamine 6G in ethylene glycol, the initial anisotropies for both processes are close to the theoretical maxima (dashed lines) for excitation from an isotropic ground state and (in the case of the two-photon excited population) a diagonal transition tensor dominated by Sxx...

A synchronously pumped DCM dye laser was used as the picosecond light source in the pump-probe experiments. A Soleil-Babinet compensator in the excitation beam was set so that the polarization of the excitation and probe beams were parallel or perpendicular. Fluorescence anisotropies were measured with a Spex fluorolog spectrometer. The time resolved fluorescence was measured by single photon counting. 

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. 

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