Orius sc1000 ccd camera

A 3 μL droplet of the aqueous EV dilution was placed onto a holey carbon covered TEM grid (Plano, type S147-4), plotted onto a thin liquid film for 2 s and plunged into a bath of liquid ethane at −165°C using a Gatan CP3 cryoplunger (Pleasanton). The frozen sample was transferred under liquid nitrogen to a Gatan cryo-TEM sample holder (model 914) and investigated at −173°C by low-dose bright-field imaging TEM (JEOL JEM-2100 LaB6). A Gatan Orius SC1000 CCD camera was used for image acquisition.

Mice were sacrificed using CO₂ and perfused intracardially with cold fixative (3% glutaraldehyde, 1% paraformaldehyde, 0.5% acrolein, 4% sucrose, 0.05 M CaCl₂ in 0.1 M cacodylate buffer, pH 7.3). The cerebellum was isolated and postfixed for a minimum of 1 day. For a secondary fixation, the samples were incubated in 2% OsO₄/1% potassium ferrocyanide in 0.1 M cacodylate buffer for 3 hr at 4°C in the dark, followed by dehydration in an ascending water/acetone series – then embedded in epoxy resin ‘Epon’ (glycid ether 100, SERVA, Heidelberg, Germany). The resin was allowed to polymerize for 2 days at 60°C in flat embedding molds. Ultrathin sections (50 nm) were produced using an ultramicrotome (Reichert Ultracut S; Leica, Wetzlar, Germany) and electron micrographs taken on a JEM 1400 electron microscope (JEOL, Akishima, Japan), using an accelerating voltage of 80 kV coupled with Orius SC 1000 CCD-camera (GATAN, Pleasanton, USA).

SEM images were obtained on a FEI Quanta 450 environmental scanning electron microscope (Hillsboro, OR, USA). TEM images were obtained on a computer-controlled TEM JEM-2100F (Jeol Pty Ltd., Peabody, MA, USA), equipped with a field emission gun. SiNWs were scrapped from the etched wafer and were suspended in ethanol. The samples were dried on a 300 lines/mesh copper grid coated with a Formvar film (PST ProSciTech, Queensland, Australia). The instrument was operated at a 200 kV accelerating voltage and images were acquired with a Gatan Orius SC1000 CCD camera (Pleasanton, CA, USA) mounted at the bottom of the column.

Light microscopy of Giemsa and DAPI (4',6'-diamidino-2-phenylindole; Sigma-Aldrich) stained smears was done as described elsewhere [42 (link)]. Standard measurements were performed on Giemsa-stained smears for 50 cells in each biological replicate, and expressed in μm. Methods used for scanning electron microscopy (SEM) and high-pressure freezing transmission electron microscopy (HPF-TEM) were described elsewhere [30 (link)]. HPF-TEM images were captured using Orius SC1000 CCD camera (Gatan, München, Germany).

Sections of the intestine from each treatment group were preserved in 2.5% glutaraldehyde buffered with 0.1M sodium cacodylate trihydrate. The samples were washed twice in 0.1M Na sodium cacodylate trihydrate for 15 minutes. Washed samples were post-fixed in 1% OsO4 in 0.1M Na cacodylate for 1 hour, washed again 0.1M Na cacodylate twice for 10 minutes, then dehydrated in graded ethanol solutions for 15 minutes at each stage [31 ]. Propylene oxide was used to further dehydrate the samples. Dried samples were then infiltrated with a 50:50 solution of propylene oxide: Poly/Bed 812 for 24 hours, thereafter with a 100% mixture of Poly/Bed 812 for 12 hours per manufacturer’s instructions (Polysciences Inc., Warrington, PA, USA). Blocks prepared for TEM were sectioned with a diamond knife and ultramicrotome into 90-150nm sections on 200 mesh grids A JEOL 1400 scope (JEOL Ltd., Akishima, Tokyo, Japan) equipped with a Gatan Orius SC 1000 CCD camera (Gatan Inc. Pleasanton, CA, USA) was used to obtain TEM images. Collected images (magnification x 15000) were analyzed using ImageJ (National Institute of Health, USA) [32 ] and were used to measure microvilli length.