Fei xl30

Lipid-free samples were made using TEST medium with no egg yolk or lipid added (control). Additionally, samples of a cryoprotectant-free medium (freezing medium buffer without lipid and glycerol: FMB) were included in this analysis. Thus, fresh sperm cells and FMB, TEST, TEST-PC, TEST-Y, and TEST-Y-PC cryopreserved samples were fixed for at least 24 hours in 3% glutaraldehyde at 4 C. Representative samples (n ¼ 5) of each treatment were washed four times by centrifugation at 16,000 Â g for 5 seconds and resuspended in 0.1 M sodium phosphate buffer (pH 7.2). This buffer was then replaced with buffered osmium tetroxide (1%) and left for 1 hour, and the samples were washed with sodium phosphate buffer twice by centrifugation as described. Samples were deposited onto 0.2-mm filters, dehydrated in a graded series of ethanol up to 100%, and then critical point dried (Samdri PVT-3; Tousimis). The dried samples were mounted on aluminum stubs and sputter-coated with gold. Imaging was performed with an FEI XL30 scanning electron microscope (FEI Company) at 25 kV accelerating voltage.

The media was size graded by dry sieving through a series of 10" stainless steel sieves with mesh sizes ranging between 0.18 mm and 1.18 mm. To fully characterise the performance of the media, it was first un-hybridised by passing 12 M HCl through a fixed bed of Layne RT , which resulted in dissolution of the iron nanoparticles and their subsequent removal from the media. This left the base ion exchange resin, which was split into two sub samples and put into the chloride and sulphate form respectively using strong solutions of each as a regeneration step. The distribution of key elements within the media in its native and adapted forms (iron, chloride, sulphur, phosphorus) were measured using an FEI XL30 environmental scanning electron microscope (FEI UK Ltd, Cambridge, United Kingdom) in low-vac mode, and then subjected to elemental spectroscopic analysis using an Oxford Instruments EDX with INCA software (Oxford Instruments, Abingdon, Oxfordshire, United Kingdom). Prior to analysis, the beads were frozen in liquid nitrogen and cut into cross sections to enable imaging of the internal surfaces. Target points for spectroscopy were measured firstly at an area 15 µm from the outer surface of the beads, and then across the diameter of each bead at intervals of 50 μm (Figure 1).