ABSTRACT
The number of rhB molecules in bright fluorescent spots (Fig. 16A and B) was determined from the following observations: the digitized histograms, the stairstep photobleaching, and the numerical consistency in the fluorescence photocounts from a single rhB molecule, as described in note 111. The digitized histograms reflect the independent nature of rhB molecules in bright fluorescent spots, within the limits of variability in photocounts around each maximum of
the histograms. In addition to inherent independent nature of each rhB molecule, two technical factors of the current work favor the well-resolved digitized his tograms: the use of the off-z-axis and circularly polarized excitation light and the photon-counting technique in the video camera. This excitation is equivalent to simultaneous irradiation of circularly polarized light whose incident direc tions are along z-and x-axes. Thus, target molecules were equally irradiated using this excitation irrespective of their orientations. Moreover, thanks to the single photon-counting technique, the observed fluorescence intensity is free from fluctuation of the electronics involved in the video microscopy. Thus, the observed distribution of photocounts around each maximum of the histogram is due to the fluctuation of absorbance and fluorescence efficiencies of individ ual rhB molecules; and the shot-noise-determined fluctuation of the observed photocounts themselves.
