Longpass dichroic mirror

First, a Leica SP5 confocal laser scanning microscope (CLSM, Leica Microsystems) equipped with hybrid detectors, a 40× immersion objective lens (HCX PL APO lambda blue), a motorized stage, and a tuneable laser (470–670 nm) was used. QDs, Alexa Fluor 546, and DRAQ5 were excited at 405, 561, and 647 nm, respectively, and their individual emissions were collected at 560–580, 560–620, and 665–795 nm. Second, a custom-made two-photon laser scanning microscope (2P-LSM) equipped with a femtosecond-pulsed titanium–sapphire laser (Chameleon Ultra II; Coherent) and a Zeiss W Plan Apochromat 20× (NA 1.0) water immersion objective lens was used. For excitation of the QDs, Hoechst and Alexa 546 the laser was respectively set at 850 nm and at 830 nm. The emitted light was split by two longpass dichroic mirror (Semrock) one 560 nm, and another 405 nm, and collected by photo-multiplier tubes (Hamamatsu). For the z-stacks acquisitions no laser power depth compensation was performed. All images were processed with the FIJI (an image-processing package based on ImageJ), Imaris 8.30 (Bitplane), Graph Pad Prism 6 (Graph Pad Software, Inc.) and Matlab (version 7, MathWorks) software. For 3D imaging of microscopy z-stacks as volumes, the Java-based ImageJ 3D Viewer plugin developed by Benjamin Schmid (Biozentrum Universität Würzburg) was used.