Med020

After chip-TIRFM imaging, both the coverslip and the PDMS frame were removed and the samples were fixed with 0.1% glutaraldehyde. Thereafter, the samples were incubated with methylcellulose followed by centrifugation at 4700 rpm (Heraeus Megafuge 40 R, Thermo Scientific) in a falcon tube. After drying (2 × 10 min) at 40 °C on a heating plate, the photonic chips were transferred to an electron beam evaporator (MED 020, Leica Microsystems). The specimen was then coated with platinum/carbon (Pt/C, 10 nm) by rotary shadowing at an angle of 8°^{57 (link)}. Thereafter, the photonic chips were mounted on a 25 mm Pin Mount SEMclip (#16144-9-30, Ted Pella) and imaged at 4 nm pixel size with a scanning electron microscope (Auriga 40 CrossBeam, Carl Zeiss Microscopy) at a low-accelerating voltage (1.5 keV). Supplementary Information S14 illustrates various steps of SEM imaging on a photonic chip.

HGFs were cultured on 10 mm tissue culture plastic coverslips (Sarstedt, Newton, NC, USA) in 24-well plates for four weeks, fixed with 2.5% glutaraldehyde (EM grade, Electron Microscopy Sciences (EMS), Hatfield, PA, USA), postfixed with 1% osmium tetroxide (EMS) and dehydrated in a graded series of ethanol followed by critical point drying (Samdri-795; Tousimis Research Corporation, Rockville, MD, USA). The coverslips were mounted on stubs and coated with 20-nm carbon (Med020, Leica Microsystems Inc, Concord, ON, Canada). Cells were visualized by SEM (Helios 650 Nanolab dual beam, FEI, Hillsboro, OR, USA) using the backscattered electrons (BSE) imaging mode (atomic number contrast) and electron energy sectioning^{73 (link)}. Once a suitable area was identified, a cross section was created with the Focused Ion Beam (FIB), exposing the interior of the cells. The BSE imaging mode was used to distinguish the cell and intracellular calcified vesicles based on atomic number contrast. To confirm the presence of Ca (L alpha of 3.9 keV) and P (K alpha 2.013) areas of high density were analyzed using EDX point analysis (Octane Pro Detector, EDAX, Mahwah, NJ, USA).

Once LM imaging was performed the wafers were “lifted” up by application of buffer solution close to the edges of the wafer. After few washes in PBS (3 × 10 min) the wafers were quickly washed with 2% methylcellulose (Sigma-Aldrich) (2 × 5 sec) and left in a drop of methylcellulose at 4 °C for 1–2 min. The wafer was then placed, as fast as possible, in an Eppendorf tube and centrifuged at 14100 × g for 90 sec, mounted on a SEM aluminum stub (Agar Scientific) using an adhesive carbon pad, left drying at room temperature for 5 min and transferred to an electron beam evaporator (MED 020, Leica Microsystems). The specimen was then coated with Platinum/carbon (Pt/C, 2 nm) by unidirectional or rotary shadowing at an angle of 8 degrees (Supplementary Fig. 4).

Osteoclasts plated on bone slices for 3 days were unroofed as described above and fixed for 10 min in a 0.2 M sodium cacodylate buffer (pH 7.4) containing 2% paraformaldehyde (Electron Microscopy Sciences 15710) and 2.5% glutaraldehyde (Electron Microscopy Sciences 16220), then washed with distilled water. The samples were then prepared for observation following the protocol: they were dehydrated through a graded series (25–100%) of ethanol, transferred in acetone and subjected to critical point drying with CO₂ in a Leica EM CPD300, then sputter-coated with 3 nm platinum with a Leica EM MED020 evaporator, and were examined and photographed with an FEI Quanta FEG250.