While the thickness of this silicate film may be tuned by altering the number of PAH/silicate layers, we chose to add only one layer of silicate for a final thickness of ca. 2 nm, as this would still allow for plasmonic sensing to occur beyond the thin layer of silicate glass.
After cooling down to room temperature on this monolayer (henceforth denoted as monolayer 1), a multilayer was grown at a deposition rate of 5 Å/min to a final thickness of 300 Å.
The sections were then reduced to a final thickness of ~30 μm by means of a series of silicon carbide (SiC) abrasive papers (280, 400, 800, 1200, and 2500; Buehler Ltd., Lake Bluff, IL) in a grinding/polishing machine (Metaserv 3000, Buehler Ltd., Lake Bluff, USA) under water irrigation.
The sections, taken from the central level of the lumbar vertebral body and from the femur mid-diaphysis and distal metaphysis (for this last considering the more proximal section of the patellar grove), were glued to a glass slide and ground to a final thickness of about 40 μm.
The sections were mounted on acrylic slides (Maertin, Freiburg, Germany) and ground to a final thickness of approximately 60 μm on a rotating grinding plate (Struers, Ballerup, Denmark).
The sections were glued onto opalescent acrylic glass, ground to a final thickness of ~80 mm and surface stained with toluidine blue and basic fuchsin.
The sample was thinned to a final thickness of ∼100 nm and imaged using a JEOL 2010F.
Subsequently, all specimens were glued with acrylic cement (Technovit 7210 VLC, Heraeus Kulzer, Wehrheim, Germany) to opaque Plexiglas and ground to a final thickness of approximately 40 μm.
The central section from each hybrid layer was reduced to a final thickness of 50 μm by microgrinding and polishing with a cutting and grinding device (Exakt, Apparatebau GmbH, Norderstedt, Germany).
The film growth was confirmed by transmission electron microcopy and was found to increase as a function of the number of deposited layers with a final thickness of 18 nm.
