Overview of the quantification method used to determine the total volume of phagosomes and PDVs in each cell. (A) A still image from the same video displayed in Fig. 1 D is used to exemplify the quantification method. The main panel shows the red channel in a rainbow scale, where red is the highest intensity level and blue is the lowest intensity level. The smaller panels show the red and DIC channels. The white dashed line indicates the boundary of the cell, and the white boxes indicate the positions of the insets. Arrows within the insets point to phagosomes, while arrowheads point to PDVs. Scale bars: 10 µm (main panels), 2 µm (insets). (B) To quantify the total volume (V) of phagosomes in each cell, we drew a region of interest around the cell and instructed Volocity to select objects within that region, which had a high fluorescence intensity and a volume >1 µm3 (phagosomes are under the masked areas). The volumes of the phagosomes in each region of interest were summed to determine the total volume of phagosomes in each cell. The volumes of the phagosomes displayed in the insets are indicated. (C) To quantify the total volume of PDVs in each cell, we instructed Volocity to select dimmer objects within the region of interest enclosing the cell, which had a volume >0.02 µm3 but <5 µm3 (PDVs are under the masked areas). The volumes of the PDVs in each region of interest were summed to determine the total volume of PDVs in each cell. Phagosomes were excluded from the PDV quantification because lower fluorescence intensity PDVs immediately surrounding the phagosomes were included in the phagosomal volume, which increased the apparent size of the phagosomes above the PDV threshold. (D) To demonstrate that the enlarged phagosomes are excluded from the PDV quantification in C, we modified the PDV settings to select the objects that had a volume >5 µm3. The volumes of these enlarged phagosomes are indicated for comparison with phagosomes quantified in B.
