Tm4000 plus tabletop sem

All specimens or samples were fixed in 2.5% glutaraldehyde solution and were deposited by cytocentrifugation on cytospin slides, followed by staining with a 1% phosphotungstic acid aqueous solution (pH = 7) for 3 min. All samples were then sputter coated with a 10-nm-thick layer of platinum to reduce charging of the imaged samples.
For image acquisition, we first used Hitachi’s TM4000Plus tabletop SEM, approximately 60 cm in height and 33 cm wide, to evaluate bacterial structure. We used backscattered-electron imaging for detection. The voltage of acceleration was 10 kV, and magnifications varied from ×250 to ×7,000. Using the same accelerating voltage, we then used Hitachi’s SU5000 SEM for the images with higher resolution and magnifications. Magnifications varied from ×5,000 to ×15,000. The evacuation time after loading specimens into the SEM chamber was less than 2 min. All cocultures of samples/Saccharibacteria cells were acquired using the same acquisition settings regarding magnification, intensity, and voltage mode. Here, each microbial form that presented a coccus shape and a physical size between 100 and 400 nm and was outside or attached to a bacterium was considered a CPR cell.

Bovine tissue strips were imaged using scanning electron microscopy (SEM) to observe any architectural damage caused to the dCELL tissues’ structural matrix following decellularization compared to untreated cellular BTM (n = 3). Bovine tissue strips were fixed in 2.5% v/v Glutaraldehyde (TAAB Laboratories Equipment Ltd, Reading, UK) for 24 h at 4 °C. The strips were dehydrated in ascending concentrations of Ethanol (50–100% v/v; (Decon Laboratories Inc., King of Prussia, PA, USA)), critical point dried with Hexamethyldisilazane (HMDS; Sigma-Aldrich, Gillingham, UK) and mounted onto carbon-tabbed SEM stubs (Agar Scientific Ltd., Essex, UK). Mounted strips were Gold/Palladium (AuPd) sputter-coated to increase electrical conductivity and imaged using a HITACHI TM4000 Plus tabletop SEM at high vacuum with a 15 kV electron beam.