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. 2016 Oct 18;111(8):1785–1796. doi: 10.1016/j.bpj.2016.09.012

Figure 4.

Figure 4

Molecular mobility near complex cell organelles. (A and B) One image frame from a 200-frame two-color confocal image series of a HeLa cell coexpressing (A) Gag.Venus and (B) the endoplasmic reticulum marker calr-mRFP-KDEL are shown. Scale bars represent 1 μm. (C) The data from (B) was intensity-thresholded (Ithreshold = 500 kHz) to select (left image) regions devoid of ER membranes or (right image) regions with dense ER staining. (D) Selected pixels from (C) were used to create region-specific ROIs in the Gag.Venus imaging channel. (E) SACFs were calculated from the ROIs in (D); y-axes are ×10−3. (F) The average arbitrary-ROI SACF was fitted using Eq. 14 and color-coded using the weighted residuals goodness-of-fit parameter rw; y-axes are ×10−3. Results for a representative cell are shown, but a total of 10 cells were analyzed, using values for Ithreshold that visually separated the cytosol from the ER. Errors on fit parameters are the mean plus SE. The average error on the SACF data (% error on a data point relative to the SACF amplitude) was 1–4%, and the reduced χ2 fit quality parameter was 1.7–3.5. To see this figure in color, go online.