*2.8. Confocal Laser-Scanning Fluorescence Microscopy*

Confocal laser scanning microscopy (CLSM) is a type of high-resolution fluorescence microscopy that generates high-resolution images by superposition of photons emitted from the fluorescence sample and reaching the detector during one exposure period [41].

To image with CLSM, the cells were grown on glass coverslips. After 24 h, the samples were incubated at 37 ◦C for 30 min in the presence of 1 µg/mL Hoechst 33342 to stain cell nuclei, and, for the last 15 min, they were exposed to NS 0.2 mg/mL stained with Nile Red. The staining of NS was obtained by the addition of 1% (*w*/*w*) Nile Red to the semisolid mixture used for the preparation of NS. Reconstitution was done according to the preparation of NS nanomicelles followed by extensive dialysis to remove any residual unloaded dye. After cell exposure to Nile Red stained NS, the cells were washed with PBS 3 times, fixed with 3% formaldehyde for 10 min at room temperature, and washed repeatedly with 0.1 M glycine/PBS and PBS. As controls, the cells were exposed to Nile Red solution at the concentration corresponding to the NS treatment. Specimens were embedded in Mowiol and analyzed using a Nikon C1s confocal laser-scanning microscope, equipped with a Nikon PlanApo 40, 1.4-NA oil immersion lens. Excitation was performed at 405 nm with an argon laser and emission was recorded at 650 nm. The images were analyzed by Image J Software (version 1.53a, U. S. National Institutes of Health, Bethesda, MD, USA).
