Figure 7.

Confocal Microscopy Qualitative Assessment of pH Nanosensor Function and Lysosome Colocalization

(A) Endosomes 3 hr post-transfection containing bPEI w/w 2 or PLL w/w 2 nanoparticles formed with the pH nanosensor show qualitative functioning of the nanosensor for live cell microscopy using GB319 cells, whereby the pH-insensitive fluorophore intensity was not dependent on pH, whereas the pH-sensitive fluorophores were quenched when DNA was present in lysosomes. Images were thresholded for analysis of colocalization of DNA via the pH-insensitive channel with lysosomal tracking dye. Compartments containing DNA non-colocalized with lysosomes are shown in pink, whereas lysosomes not containing DNA are shown in red, and compartments containing DNA colocalized with lysosomes are shown in yellow. Scale bars, 20 μm. (B) Scattergrams show the analysis of the representative microscopy images with distribution of pixels with DNA fluorescence intensity shown on the x axis, lysosome fluorescence intensity shown on the y axis, and region 3 showing thresholded colocalized pixels. (C) Quantification of Pearson’s correlation coefficient M1 for colocalization of DNA and lysosomes showed PLL nanoparticles colocalized with lysosomes at both 3 and 24 hr post-transfection, whereas bPEI nanoparticles partially avoided an initial lysosomal fate at 3 hr, but not at 24 hr. Quantified results were tested using multiple t tests corrected for multiple comparisons using the Holm-Sidak method. Errors bars show mean ± SEM of five images taken with the same microscope settings. *p < 0.05.