*2.12. Confocal Fluorescence Microscopy*

Fluorescence microscopy was performed with a Zeiss Observer SD spinning disk confocal microscope using a Yokogawa CSU-X1 spinning disc unit and an oil objective (63× magnification) and BP 525/50 (WGA488) and LP 690/50 (Atto633) emission filters. A 488 nm and a 639 nm laser were used for excitation. At 24 h prior to transfection, 3500 T24 cells per well were seeded in 8-well plates in 280 μL growth medium. A total of 20 μL MSN-454-GE11 covalently labeled with Atto-633 carboxy (200 μg mL−<sup>1</sup> MSN in HEPES buffer) were added to each well and incubated for 45 min or 6 h, respectively, at 37 ◦C followed by addition of WGA-488 to the medium for cell membrane staining. Cells were washed with PBS and after addition of 300 μL fresh medium, cells were directly imaged.

The cellular uptake of nanoparticles was quantified using the ImageJ macro "Particle\_in\_Cell-3D" developed by Adriano A. Torrano and Julia Blechinger, Department of Chemistry and Center for NanoScience (CeNS, University of Munich (LMU), Munich, Germany. http://imagejdocu.tudor.lu/ doku.php?id=macro:particle\_in\_cell-3d)
