Tecnai biotwin

Freshly glow-discharged 200 mesh Formvar/carbon-coated copper grids (Electron Microscopy Services, CG200-Cu) were inverted on drops of gradient-purified PsV diluted 1:9 in phosphate-buffered saline (PBS), and virus allowed to adsorb for five minutes. The grids were washed twice in deionized water and stained by two one-minute incubations on drops of Nano-W (Nanoprobes, Nephank, NY) before removing excess stain by gentle blotting with Whatman #1 filter paper. The grids were air-dried before viewing on a FEI Tecnai Biotwin transmission electron microscope at 80Kv. Images were taken using a Morada CCD camera and iTEM (Olympus) software, and ImageJ was used to provide sizing information based on a scale bar embedded in the images.

We applied our approach to three thoracic aorta specimens excised from adult (8–14 weeks of age) male mice having a C57BL/6 genetic background to demonstrate and validate our TEM image segmentation and analysis pipeline in the specific context of vascular applications. To test our methodology against substantially different collagen fibril architectures, we show results for one wild-type control ascending thoracic aorta (ATA), one descending thoracic aorta (DTA) with disrupted TGF-β signaling (denoted as Tgfbr1r2; see Kawamura et al. [13 (link)] for details), and one Fbn1^C1041G/+ ATA representative of Marfan syndrome (denoted as MFS; see Weiss et al. [23 (link)] for details). Specimen excision and preparation as well as image acquisition methods have been described previously [13 (link), 23 (link)]. Briefly, segments were fixed in 2.5% glutaraldehyde/2% paraformaldehyde in sodium cacodylate buffer at room temperature for 2 h at 4°C. Samples were rinsed in sodium cadodylate buffer before post-fixation in 1% osmium tetraoxide for 1 h and subsequent staining using 2% uranyl acetate for 1 h. Afterward, samples were washed and embedded in resin and sections of the adventitial layer were imaged using a FEI Tecnai BioTwin transmission electron microscope.

Fe NWs were coated with APTES and BSA through the formation of covalent bonds between the coating agent and the iron oxide (Fe₂O₃) interphase that surrounds the NWs formed after releasing the NWs from the alumina. The morphology of BSA and APTES coated NWs was analyzed by TEM (TecnaiBioTWIN; FEI Company).
NWs were also coated with thiol-polyethylene glycol (PEG-SH) (50 μmol PEG/g Fe). In this case, the NWs had to be first coated with APTES and further activated with 2-Iminothiolane (2-IT) and aldrithiol as previously described53 , to finally react with thiol-PEG (2 kD).

The Center for Cellular and Molecular Imaging Electron Microscopy Facility at the Yale School of Medicine prepared the samples. Cells were fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium cacodylate buffer plus 2% (wt/vol) sucrose, pH 7.4, for 30 min at RT and 30 min at 4 °C. After rinsing, cells were scraped in 1% (wt/vol) gelatin and centrifuged in a 2% (wt/vol) agar solution. Chilled cell blocks were processed with osmium and thiocarbohydrazide-osmium liganding. Samples were incubated overnight at 60 °C for polymerization. The blocks were then cut into 60-nm sections using a Leica UltraCut UC7 and stained with 2% (wt/vol) uranyl acetate and lead citrate on Formvar/carbon-coated grids. Samples were imaged using a FEI Tecnai BioTwin at 80 kV, equipped with a Morada CCD and iTEM (Olympus) software for image acquisition.