SpH bleedthrough into the pHuji channel is linearly correlated and therefore can be corrected. (A) Pixel-by-pixel fluorescence correlation plots between SpH and pHuji values fitted with simple linear regressions generated by analyzing TIR-FM images (256 × 256 pixels) of cells expressing SpH-cargos alone, simultaneously acquired in both SpH and pHuji channels under identical conditions as in Fig. 2. This panel shows the first frame (fr1) of a movie of TfR-SpH expressing cell post-background subtraction (post-bs) without Gaussian blur (pre-gb). (B) Similar analysis post-Gaussian blur (post-gb, σ = 2). (C) Frame 500 (fr500) of the same movie as in A and B post-bs and post-gb (σ = 2).