Photobleaching and Intensity Fluctuations

The quantum efficiency for photobleaching, b, may be estimated from the measured single molecule detection rate, Rf, the collection and detection efficiency, D, the fluorescence quantum yield, f, and the bum time, xb/ f = x D x tb). 

Values of b range from 10 to 10 for molecules on surfaces. In general, such a range is indicative of the heterogeneity of the environments, in particular, the different binding geometries, as has been shown for glass [22]. 

Many possible sources can be imagined for the fluctuations including (1) metastable excited states, such as photoionization with transient electron trapping, (2) metastable complex formation, such as association/dissociation with a nearby molecule, (3) isomerization, including small changes in the nuclear coordinates that result in a spectral shift, (4) changes in the molecular configuration of the environment and a concomitant spectral shift, and (5) reorientation of the optically active molecule. 

The latter would result in intensity fluctuations if the excitation were linearly polarized or if the reorientation included an out-of-plane component. In either of these cases, the absorption probability which is proportional to E pp would change 

Other molecules showed intensity variations on time scales less than a minute. For example, (B) has a dark line through the lower lobe and (C) has a noisy appearance. A shear force image obtained concurrently with the data in Fig. 22 had no feedback instability other than a 2-nm peak-to-peak height noise throughout the image. 

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