Figure 3: Confocal microscopy and permeation statistics. (A)The confocal micrograph confirms permeation of fluo-4-AM into P. pastoris . The cells were imaged using an Olympus FV-1000 laser-scanning confocal microscope under 60x magnification and at excitation and emission wavelengths of 488 and 505 nm, respectively. (B) The permeation is non-uniform. However, 45 minutes incubation is sufficient to stain about 21% of the population and generate a detectable signal.
We do not anticipate these phenomena to occur in the assay since incubation of the cells with the dye occurs in PBS in those tests. In light of this conclusion and the fact that we still observe a detectable signal in the assays despite the dye permeating into a little more than fifth of the population of cells, we decided against optimizing the dye incubation time.