Tecnai bio twin g transmission electron microscope

Previously reported procedures were employed for the O-GNR synthesis from multi walled carbon nanotubes (Sigma-Aldrich,St Louis, MO) and preparation of O-GNR-DSPE-PEG dispersions.^9,10 For atomic force microscopy (AFM) and TEM characterization, O-GNR-PEG-DSPE samples diluted to 5 µg/ml using a 1:1 ethanol water mixture were probe sonicated, (Cole Parmer Ultrasonicator LPX 750) and centrifuged at 2000 rpm for 30 minutes. The supernatant was collected and drop cast onto silicon wafers (AFM samples) or copper grids (Ted Pella) (TEM samples) and dried overnight. AFM was performed using a Nano Surf Easy Scan 2 AF microscope (NanoScience Instruments Inc, Phoenix, AZ), operating in tapping mode with a V-shaped cantilever. TEM was performed on a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR), at 80 kV. Digital images were acquired using an XR-60 CCD digital camera system (AMT, Woburn, MA).

O-GNRs were synthesized from multi-walled carbon nanotubes (MWCNTs)(Sigma-Aldrich, Length=5–9 μm) using the oxidative longitudinal unzipping method [17 (link)]. Briefly, MWCNTs (150 mg) were suspended in 30 ml concentrated (96%) H₂SO₄. After 4 h, 4.75 mM KMnO₄ was added slowly and stirred for an h followed by further stirring for another h at 55–70 °C in an oil bath. This solution was poured on ice (400 ml) containing 5mL 30% H₂O₂ and the ice-H₂O₂ slurry was allowed to melt. The solution obtained was centrifuged at 3000 rpm for 30 minutes, after which the supernatant was discarded. The pellet obtained was then washed with 36% HCl. Ethanol and ether washes were used for flocculation and the final product (O-GNR) was obtained as pellet after centrifugation (30 minutes, 3000 rpm). This product was dried overnight in a vacuum oven at 60°C.
O-GNRs were characterized using atomic force microscopy (AFM) and Transmission Electron Microscopy (TEM). AFM images were obtained using a Nano Surf Easy Scan 2 AFM (NanoScience Instruments Inc, Phoenix, AZ) operating in tapping mode using a V-shaped cantilever and TEM images were obtained using a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR), at 80 kV.

Six well plates with surfaces covered with ACLAR® film (Electron Microscopy Sciences, Hatford, PA) were plated with CMV/U251 and MCF-7 cells at a density of 5× 10⁵ cells per plate, and exposed to O-GNR-PEG-DSPE for 3 h. At the end of 3 h, cells were fixed with 2.5% electron microscopy grade glutaraldehyde (Electron Microscopy Sciences, Hatford, PA) in 1X PBS. After fixation, the films containing fixed cells were placed in 2% osmium tetroxide in 1X PBS, dehydrated through graded ethanol washes, and embedded in durcupan resin (Sigma-aldrich, St. Louis, USA). Areas with high cell densities were blocked, cut into 80 nm ultra-thin sections using an Ultracut E microtome (Reichert-Jung, Cambridge, UK), and placed on formvar-coated copper grids. The sections were then viewed with a Tecnai Bio Twin G transmission electron microscope (FEI, Hillsboro, OR), at 80 kV. Digital images were acquired using an XR-60 CCD digital camera system (AMT, Woburn, MA).