Figure 3.

Evaluation of cytoplasmic nanoparticle loading in live cells by osmotic lysis of pinosomes. COS-7 cells were incubated in hypertonic solution (10 minutes, 37°C, Invitrogen I-14402) for pinocytic loading of QD565ITK nanocrystals (NCs, Quantum dot corporation). Shifting to hypotonic culture medium caused the osmotic lysis of the internalized pinosomes and release of NCs into the cytoplasm. (a) Bright-field image at × 100 magnification. Scale bar for (a) to (c): 4 $\mu$m. (b)–(c) Epifluorescence images from a time-resolved image stack of the same cell. Green circles identify individual NCs that intermittently changed from ON to OFF state (blinking) between frame 250 (b) and 253 (c). Cell viability following loading was tested using the trypan blue exclusion assay at low magnification, × 10. Osmotic shock without (d) and with 1 nM QD565ITK nanocrystals in the extracellular fluid (e) did not compromise cell viability. (f) In contrast, adding ethanol reliably killed cells as reported by the dark trypan blue labelling. Scale bar for (d) to (f): 40 $\mu$m.