W plan apochromat 20 na 1.0

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.

To assess
the distribution of mRNA nanoparticles within a collagen-nHA scaffold,
modRNA was tagged with Cy3 using a Mirus Label IT Cy3 nucleic acid
labeling kit (Medical Supply Company, Ireland) according to the manufacturer’s
instructions. The tagged modRNA (5 μg) was complexed with jetPEI
(2:1 v/w) and soak-loaded onto both sides of the collagen-nHA scaffolds.
Scaffolds were imaged on a Carl Zeiss LSM 710, equipped with a W Plan-Apochromat
20× (NA 1.0) with an interslice Z spacing of
1.2 μm to yield a total image Z depth of 31.2
μm. The scaffold autofluorescence was excited using a 405 nm
laser (detection range of 410–509 nm). Cy3 fluorescence was
excited using a 561 nm laser (detection range of 564–681 nm).
Images were recorded at a resolution of 1024 × 1024 pixels with
a dwell time of 0.79 μs. Z stack images were maximum intensity
projected and prepared in ImageJ. To assess the initial depth of particle
incorporation within the scaffold, the scaffolds were also sectioned
with a scalpel to expose the center of the scaffold, which was then
imaged as described above. ImageJ software was used to determine the
depth of the nanoparticle infiltration into the scaffold.