S3000n vpsem

Fiber alignment across acellular tubular lamellar constructs was imaged using SEM (Hitachi S3000N VPSEM, Berkshire, UK) as described previously in section in vitro Cell Orientation on Bilayer Fiber Scaffold. Similarly, fiber alignment (Rhodamine stained) and cell orientation (Hoechst 33342 nuclear stain and molecular calcein stain for live cells) across cellular tubular lamellar constructs were imaged using laser confocal microscopy (Leica TCS SP5, Leica Biosystem UK Ltd, UK) as described previously in sections in vitro Cell Orientation on Bilayer Fiber Scaffold and Cell Distribution.

In order to evaluate the structural changes in chronically implanted microelectrode arrays, all arrays were imaged before implantation and after explantation using a variable pressure scanning electron microscope (Hitachi S-3000N VP-SEM). The environmental mode in the VP-SEM was chosen for imaging the arrays to enable direct placement of the samples into the SEM chamber without the use of carbon or conductive coating. This is very critical especially for pristine electrodes as they will be implanted into an animal after the SEM imaging procedure. Such a method was used so as to cause no damage to the arrays from the imaging process for both pre-implant as well as explanted arrays. When the pre-implant images were taken, electrode arrays were handled with care to prevent any damage to the microwires. The post-explant images were taken on the electrode arrays extracted from the implanted tissue. The post-explant arrays were carefully placed on a holder positioned on to the SEM stage. The following parameters were used to take the pre- and post-implant images: (1) environmental secondary electron detector (ESED) mode with an acceleration potential of 12 kV, (2) working distance range was set between 18 and 40 mm, and (3) magnification was varied according to need and the minimum magnification was set at 20X.

Cell morphology was assessed at 1 and 7 days using SEM and confocal microscopy. For SEM (Hitachi S3000N VPSEM), samples (n = 2) were washed in PBS and fixed in 2.5% v/v glutaraldehyde in PBS at 4 °C for 2 h. As previously described, samples were dehydrated through increasing concentrations of ethanol in distilled water (50%–100% v/v), chemically dried in hexamethyldisilazane, mounted on carbon-tabbed stubs, and gold-sputter coated. For confocal microscopy (Leica SP8), samples were fixed with 10% neutral buffered formalin for 30 min, washed with PBS, permeabilized for 1 h with 0.1% Triton X-100 in PBS (Sigma) then blocked with 2% bovine serum albumin (Sigma-Aldrich) at 37 °C for 1 h. F-actin stain (Alexa-Fluor488 phalloidin 1:300, Life Technologies) was applied and incubated for a further 2 h at 37 °C. Samples were subsequently washed with PBS and placed on glass coverslips for imaging.