Fesem merlin

Volume size distribution and Z-potential of T22-GFP-H6 protein nanoparticles and T22-GFP-H6-ATTO nanoconjugates was determined in a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) respectively at 633 nm. All samples were measured in triplicate. T22-GFP-H6-S-Cy5 nanoconjugates cannot be analyzed by DLS or ELS, since Sulfo-Cyanine 5 fluorescence (excited at 633 nm) prevents reliable light scattering measurements.
Ultrastructural morphometries of T22-GFP-H6, T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 (size and shape) were determined at nearly native state with field emission scanning electron microscopy (FESEM). Drops of 3 µL of diluted samples at 0.3 mg/mL were directly deposited on silicon wafers (Ted Pella Inc., Redding, CA, USA) for 30 s, excess of liquid blotted, air dried and immediately observed without coating with a FESEM Zeiss Merlin (Zeiss, Oberkochen, Germany) operating at 1 kV and equipped with a high resolution in-lens secondary electron detector. In a qualitative approach, representative images of a general fields and nanostructure details were captured at two high magnifications (150,000× and 400,000×). In a quantitative approach, nanoparticles and nanoconjugate average size were analyzed by Image J software (Image J 1.48v, NIH, Bethesda, MA, USA) from FESEM images.

IB geometry and surfaces were studied in a nearly native state with a Field Emission Scanning Electron Microscope (FESEM). IBs in deionized water were deposited over silicon wafers (Ted Pella, Reading, CA, USA) in microdrops of 10 μL. After air drying, uncoated samples were observed in a FESEM Zeiss Merlin (Zeiss, Oberkochen, Germany). The equipment was operated at 2 kV with a high resolution in-lens secondary electron detector [35 (link)].

Microdrops of JAMF1 IB suspensions were air-dried on silicon wafers and micrographed in a FESEM Zeiss Merlin (Zeiss) running at 1 kV

Ultrastructural characterization of intracellular and isolated IBs was performed with two high-resolution electron microscopy techniques. Imaging of intracellular IBs was performed with standard Transmission Electron Microscopy (TEM) procedures adapted to this type of sample [23 (link), 58 (link), 63 (link), 64 (link)]. Briefly, pellets of bacilli with and without IBs were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.2, postfixed in 1% osmium tetroxide containing 0.8% potassium ferrocyanide in PB, dehydrated in acetone, embedded in Spurr resin and polymerized at 60 °C during 48 h. Ultrathin Sect. (70 nm) obtained with an ultramicrotome UCT7 (Leica Microsystems) were placed in Cu grids (200 mesh) and contrasted following routine protocol of uranyl acetate and lead citrate solutions. Samples were observed in a TEM JEM 1400 (Jeol) equipped with an Erlangshen CCD camera (Gatan) and operating at 80 kV.
Ultrastructural morphometry (size and shape) of nanoparticles was performed and characterized at nearly native state with field emission scanning electron microscopy (FESEM). Drops of 20 µL of IBs sample were directly deposited on silicon wafers (Ted Pella Inc.) for 30 s and immediately observed without coating with a FESEM Merlin (Zeiss) operating at 1 kV and equipped with a high-resolution secondary electron detector.

For TEM imaging of JAWS II ultrastructure, cells were collected, fixed, and processed as described previously (17 (link)). Ultrathin sections (70–90 nm) were cut on a Ultracut E microtome (Reichert-Jung, Austria). The images were acquired using Zeiss Libra 120 transmission electron microscopy (Zeiss, Jena, Germany).
Scanning electron microscopy images of the cells surfaces were obtained by incubating JAWSII with 5 µg/ml of NPs for 3 h, then the cells were fixed with warm 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 for 20 min, as described in Ref. (17 (link)) and acquired using FE-SEM Merlin (Zeiss, Jena, Germany).