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.