Use ROONS-EGFP as a model to determine the relationship between macroscopic fluorescence intensity and the total number of channels on the membrane patch. (A, top) Two single-channel traces of ROONS-EGFP channel recorded at the potential of 80 mV. Transitions between opening and closing were recorded in the presence of 3.5 µM cGMP, a subsaturating concentration for ROONS-EGFP. (bottom left) Maximal channel opening recorded with 35 µM cGMP. The current level corresponding to the closed state is indicated by a gray line. (bottom right) The current trace of the same patch recorded in the absence of cGMP. The gray line represents the current level of the open state. (B) Histogram of the top recording trace from A. Averaged amplitude of single-channel currents was 4.29 ± 0.57 pA (n = 7). (C, left) Macroscopic current recorded under 35 µM cGMP and a voltage step from 0 to 40 mV. (right) The corresponding brightfield and fluorescence images. (D, left) Histogram of the pixel intensities of the whole fluorescence image collected using four different exposure durations. The Cascade 1K is a 16-bit camera so that the pixel intensity ranges from 0 to 65,535. (right) Total fluorescence intensity from the ROI surrounding the patch of membrane patch (green) and the background (blue), which was separately recorded after moving the recording pipette out of the view. (E) Cross-plots of fluorescence intensity versus current amplitude. Two x axes show the macroscopic current (bottom) and the corresponding number of channels (top). The amplitude of single-channel current, 4.29 pA, was used in the conversion. Linear fit statistics: Pearson correlation coefficient, 0.823. Green curves show the confidence bands (95%).