Quanta 200 electron microscope

Disrupted Malpighian tubules were smeared on microscope slides, air-dried, fixed in methanol (5 min), and stained with Giemsa solution (Accustain, Sigma-Aldrich; 45 min in a 1:10 dilution). Dried smears were embedded in Entellan (Merck). For scanning electron microscopy, cover glasses with smears of infected Malpighian tubules were air-dried and coated with gold particles using a Baltec SCD 040 sputter device. Micrographs were taken with a Quanta 200 electron microscope (FEI Company). For transmission electron microscopy, samples were fixed for several days with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C, rinsed with cacodylate buffer, and post-fixed for 1.5 h with reduced OsO₄ at room temperature without darkening (fresh 1:1 mixture of 2% OsO₄ and 3% K₄[Fe(CN)₆]). The samples were then rinsed with distilled water, and after dehydration in a series of ethanol (15 min each in 30%, 50%, 70%, 90%, 100%, 3 × 100% water free), they were embedded in Spurr’s resin^{55 (link)}. Ultrathin sections were stained with saturated aqueous uranyl acetate for 30 min in the dark, followed by Reynolds’ lead citrate^{56 (link)} for 5 min. During the latter, sodium hydroxide pellets were put next to the grids in order to remove CO₂ from the atmosphere and thus prevent precipitation of lead carbonate. The sections were investigated using a Philips CM 120 BioTwin electron microscope.

The cross-section SEM micrographs of the samples were obtained with a Quanta 200 electron microscope (from FEI Company, Hillsboro, OR, USA), working at 20 kV in low vacuum mode, without any coating. The samples were fixed on aluminum stubs with double-adhesive carbon tape. The average pore size was calculated using ImageJ software.

Square pyramidal microneedle arrays measuring 1000 μm tall and 450 μm in width, spaced at 450 μm between needles (edge to edge) were designed using Solidworks 2016. The array was 12 × 12 microneedles, for a total of 144 microneedles per patch. The margin from the last microneedle row to the edge of the array was 450 μm in all directions (Additional file 1: Figure S1). This computer aided design file was exported in a Standard Triangle Language (.STL) format, imported into Autodesk’s Print Studio software, centered on the build area with microneedles oriented along the z axis and exported as a .tar.gz file without introducing additional supports. Layer thicknesses of 10 μm, 25 μm, and 50 μm were investigated. The file was uploaded to the Autodesk Ember and fabricated using Autodesk’s Standard Clear PR48 Resin (formulation given in Additional file 2: Figure S2). All microneedle arrays were imaged without sputter coating using an FEI Quanta 200 electron microscope (Hillsborough, Oregon, United States) in low vacuum mode at 0.028 Torr, 20.0 kV, and 3.0 spot size. Microneedle physical dimensions (height, width, tip radius of curvature) were measured using Image J (National Institutes of Health, Bethesda, Maryland, USA). Measurements were taken from three needles at random locations on two separate arrays.

For scanning electron microscopy (SEM) analysis, AOA cells were harvested using 0.22 μm polycarbonate GTTP membrane (Millipore, USA), and then fixed in 2.5% (vol/vol) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 24 h at 4 °C, and dehydrated using a graded ethanol series (70 to 100%) and finally 100% tertiary butanol. The samples were examined with a Quanta 200 electron microscope (FEI, the Netherlands). For transmission electron microscopy (TEM) analysis, cells were collected from 500 ml of culture by centrifugation at 6000 g for 10 min, and then negatively stained with 1% (wt/vol) phosphotungstic acid. Ultrathin sections were produced with an EM UC6 ultramicrotome (Leica, Germany), stained with uranyl acetate and lead citrate, and examined with an H-7650B electron microscope (Hitachi, Japan).

Eight MP fragments were randomly selected from samples of each site and preserved for SEM. The fragments were washed with fresh ddH₂O and dried for 12 h at 70 °C. Dry samples were sputter-coated using a S150A Sputter Coater (Edwards, Irvine, USA) with a gold layer of 2 nm. The coated plastic fragments were visualised and imaged using a Quanta 200 electron microscope (FEI, Hillsboro, United States) with a 10 kV electron beam and varying magnifications.