*3.5. Confocal Laser-Scanning Fluorescence Microscopy*

Cells treated with Red Nile stained nanospermidine were analyzed by confocal laserscanning fluorescence microscopy. The images (Figure 14A) showed that nanospermidine can interact with the cells after short periods of time. The images reveal a uniform distribution of fluorescence in BR6 and a spotted-like dispersion in NLF. The quantitative analysis of fluorescence intensity (Figure 14B) showed that nanospermidine interaction was higher in BR6 than in NLF. WS1 did not provide fluorescent images after treatment with Red Nile stained nanospermidine for the same time period as the tumor cells.

**Figure 14.** Confocal microscopy of BR6 (**A**) and NLF (**B**) after 15 min treatment with Nanospermidine (NS) stained with Nile Red and controls treated with Nile Red solution at the same concentration used with NS. Cell nuclei were evidenced by 1 µg/mL Hoechst 33348 staining. (**C**) Fluorescence intensities of the treated cells and controls. Photographs were taken at 40× magnification, bar = 20 µm. The images were analyzed by Image J Software and reported as fluorescence intensity per unit area. (mean ± SD, *n* = 3) (\*\* *p* < 0.01, \*\*\* *p* < 0.001).
