Q150t es sputter

SEM images were collected using a Field Emission (FE) SEM Zeiss Gemini 500. Each sample was mounted on the stub with conductive carbon tape and a thin film (5 nm) of chromium was deposited on the sample surface using a Quorum Q 150 T ES sputter to make it conductive for measurement purposes.

To prepare the sample for SEM, a drop of diluted CNCs in water was deposited onto a thin glass slide. Then, the glass slide was attached to the SEM sample holder with conductive double sided tape. After the water was evaporated under ambient air, the sample was sputter coated with gold for 60 seconds at 20 mA current with a Quorum Q-150T ES Sputter to help prevent charging. Planar cross-sections from CNC aerogel structures were also obtained through cryofracture using liquid N₂; then cross-sections were again attached to SEM sample holders with conductive double sided tape and sputter coated with gold before SEM was performed at 10 kV accelerating voltage.

In order to acquire an enhanced Raman signal during intracellular imaging (see below), a 50 nm layer of 99.95% pure platinum was deposited on glass bottom culture dishes by sputter coating technique using a Q150T ES sputter (Quorum Technologies, Ltd). Afterwards, the platinum-coated dishes were sterilized by 20 minutes UV exposure in a laminar flow hood. Subsequently, fibroblast NIH-3T3 cells were seeded on the dishes at the density of 50·10³ cells per cm² and grown in DMEM medium supplemented with 1% Pen/Strep and 10% FBS. Cells were incubated at 37 °C and 5% CO₂ concentration for 24 h before replacing the medium with a fresh one supplemented with 1% Pen/Strep and 2% FBS previously mixed with a freshly synthesized solution of clusters at final medium:clusters volume ratio of 2:1. Under these conditions, the reduced concentration of serum does not let the formation of a protein corona that could inhibit the cellular uptake of nanoparticles by cells^{58 (link)}. After 24 h incubation at 37 °C and 5% CO₂ concentration, cells have been washed three times with phosphate buffer 1X to remove extracellular probes and directly analyzed by means of Raman spectroscopy (see below).