Fluorescence spectral jumps

Figure Cl.5.8. Spectral jumping of a single molecule of terrylene in polyethylene at 1.5 K. The upper trace displays fluorescence excitation spectra of tire same single molecule taken over two different 20 s time intervals, showing tire same molecule absorbing at two distinctly different frequencies. The lower panel plots tire peak frequency in tire fluorescence excitation spectmm as a function of time over a 40 min trajectory. The molecule undergoes discrete jumps among four (briefly five) different resonant frequencies during tliis time period. Arrows represent scans during which tire molecule had jumped entirely outside tire 10 GHz scan window. Adapted from...

Once a single molecule line has been isolated with sufficient signal/noise ratio in a fluorescence excitation spectrum (Chapter 1.1), it may or may not exhibit instability due to spectral jumps [40]. Large irreversible jumps move the molecular line outside... 

The intensity auto-correlation method has long been in use at high temperature to study dynamics in solutions [88]. Intensity fluctuations can arise from spectral jumps or changes, from rotational or translational diffusion with respect to the exciting beam, and from any process which can modulate the emitted intensity. Since correlation of single molecule fluorescence works at liquid helium temperature as well as at room temperature (see Section 2.1), it probably can cover the whole intermediate... 

In the second type of experiment that measures single molecule spectral dynamics one performs repeated fluorescence excitation scans of the same molecule. In each scan the line shape is described as above, but now there is the possibility that the center frequency of the line will change from scan to scan because of slow fluctuations. Thus one can measure the center frequency as a function of time, producing what has been called a spectral diffusion trajectory. This trajectory can, in principle, be characterized completely by the spectral diffusion kernel of Eqs. (16) and (19), but of course it must be understood that only the slow Kj < 1 /t) TLSs contribute. In fact, the experimental trajectories are really too short to be analyzed with this spectral diffusion kernel. Instead, it is useful [11, 12] to consider three simpler characterizations of the spectral diffusion trajectories the frequency-frequency correlation function in Eq. (14), the distribution of frequencies from Eq. (15), and the distribution of spectral jumps from Eq. (21). For this application of the theoretical results, in all three of these formulas j should be replaced by s, the labels for the slow TLSs. 

In general, the correlation technique as well as the quantum jump technique are powerful tools to unravel complicated molecular photophysical dynamics for a single absorber. This statement is exemplified by the investigation of the chromophore terrylene, for which no kinetical parameters of the triplet state were known from ensemble measurements. The ISC rates presented here were determined solely by experiments on single molecules. Actually, it would be quite difficult to measure absolute rates of photophysical ISC parameters by other techniques when the triplet quant yield is smaller than 10 . Recently, fluorescence correlation spectroscopy was also proposed as an appropriate method for the determination of triplet parameters of fluorophores in solution [75]. Additionally, it is a helpful tool to investigate spectral diffusion of single absorbers as discussed in Sections 1.4 and 1.5. 

The simplest case of spectral diffusion is that of a single molecule coupled to a single TLS in its neighborhood. Upon interaction with acoustical phonons, the TLS will jump from one configuration to the other, thereby changing the molecular optical transition frequency. The fluorescence intensity will therefore fluctuate according to the jumps, giving rise to two lines in the excitation spectrum and to a correlation fimction ... 

Figure 13. Stochastic simulation of the fluorescence intensity (top) and frequency (middle) correlation functions for a model of spectral diffusion in a glass. The molecule is coupled to a three dimensional distribution of tunneling systems with distributed microscopic parameters. The bottom panel shows the timescale (horizontal) and amplitude of the frequency jumps (relative to the lifetime limited linewidth, vertical scale). The inset in the top panel shows the line-shape and the lifetime limited linewidth as a small bar (from Ref. 77).

To study rates of antibody-hapten reactions the principal methods employed have involved stopped flow or temperature jump techniques. The former was first used by Sturtevant et al. (64) and Day et al. (65). The temperature jump method has been employed by Froese, Sehon, and their collaborators (66-68) and more recently by Pecht et al. (69). Both methods are utilized in conjunction with very rapid optical measurements (in the millisecond range). For example, Sturtevant et al. took advantage of a spectral shift which occurs upon combination of anti-Dnp antibody with the dye, 2-(Dnp-azo)-I-naphthol-3,6-disulfonic acid (64). With the same hapten, and with e-Dnp-L-lysine and e-Dnp-6-aminocaproate. Day et al. (65) used the method of fluorescence quenching (Section VI,D) with a stopped flow apparatus. In the temperature jump technique the components are first equilibrated, a temperature increment is rapidly induced (up to 10°C in 0.1 isecond), and the rate of reequilibration at the new temperature is measured. Velocity constants can be estimated from the data the mathematical approaches required are described in the references cited. 

Fig. 18.15 Spectroscopy of single pentacene molecules in p-terphenyl crystal (W. P. Ambrose, Th. Basche and W. E. Moemer, 1. Chem. Phys. 95, 7150 (1991). (a) Fluorescence excitation spectrum of a single molecule at 1.5 K (0 MHz detuning = 592.407 nm, at the wing of the inhomogeneous hneshape) (b) Fluorescence excitation spectrum of the full inhomogeneous line at 1.5 K. (c) The dependence of the single molecule homogeneous hnewidth on temperature (the solid line is a fit to the data), (d) Two views of spectral diffusion The upper panel shows a time sequence of excitation spectra (each taken over a period of 1 s). The lower panel shows the jumps in the peak frequency as a function of time.

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