Figure 5.

Images of fixed (a–e) and live (f–i) human umbilical vein endothelial cells (EC) after induced co-internalization of 2 μm latex microspheres and PtP-C343. 87 ± 3 % of the total number of microspheres were internalized, of which 86 ±7 % contained PtP-C343. 2P excitation was the same as in the experiments (e, f) in Figure 4. a) Overlaid phase-contrasted and conventional fluorescence images show several cells with nuclei stained by a blue fluorescent dye (DAPI). The cell marked by the square contains several vesicles, where microspheres (white) are surrounded by the probe solution (red). b) Magnification (3.75-fold) of the perinuclear region of a selected cell. Small vesicles containing the probe, but no microspheres, appear as reddish dots around the nucleus. c) The same cell viewed through the eye-piece of the two-photon imaging microscope. The scanned area (square) and the nucleus (dashed line) are marked. d) Integrated intensity image of phosphorescence (50 × 50 pixels²). e) Phosphorescence lifetime image. f) Nucleus of a cell surrounded by internalized micro-spheres. g) Integrated intensity image of phosphorescence and the emission spectra (to the left) collected from the areas 1–3 (marked by the squares). h) Phosphorescence lifetime image. i) pO₂ image calculated from the lifetime image (h).