Figure 3.

Absolute intensity thresholding for region-specific RICS analysis. (A) One image frame from a 100-frame confocal image series of a HeLa cell coexpressing cytosolic Venus fluorescent protein and membrane-targeted MyrPalm-YFP is shown. Scale bar represents 1 μm. (B) Intensity distribution histogram is provided of (black) all pixels in the frame, (red) an arbitrary ROI with ${\langle I\rangle }_{5\mathrm{\times }5}$ > 300 kHz, and (green) an arbitrary ROI with ${\langle I\rangle }_{5\mathrm{\times }5}$ < 300 kHz. For comparison, both the absolute photon count and the count rate per pixel are displayed. (C) The data from (A), were preprocessed using Eq. 9, and intensity-thresholded to select (top image) the membrane or (bottom image) the cytosol. (D) SACFs were calculated from the preprocessed ROI in (C). The inset is a top view of the SACFs. (E) The average arbitrary-ROI SACF was fitted using Eq. 14 and color-coded using the weighted residuals goodness-of-fit parameter r_w. Fig. S2 contains further details on the experiment. Results for a representative cell are shown. The average error on the SACF data (% error on a data point relative to the SACF amplitude) was 3–4%, the error on D was 10–25%, and the reduced χ² fit quality parameter was 1.9–2.4. To see this figure in color, go online.