Sem evo 40

Hydromagnesite and aragonite precipitates inhabited by microorganisms were investigated by SEM. The samples were fixed with 5% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7) for 3 h. The samples were rinsed twice for 10 min in 0.1 M sodium cacodylate buffer (~ pH 7) and were frozen at −95 °C until they were placed in an Edwards freeze-dryer for 5–10 h at −60 °C operating at 2 × 10^–2 mbar. The dried samples were sputter-coated with gold and examined using a Zeiss EVO 40 SEM at 10 kV accelerating voltage.

Due to the size of the larval anal papillae and their location, scanning electron microscopy (SEM) was used to measure them. Larvae were fixed in 2% glutaraldehyde, buffered with 0.1 M sodium cacodylate (pH 7.2) for 2 h, and then postfixed in 1% osmium tetroxide and dehydrated through a series of ethanol–water solutions of increasing concentration. Samples were critical-point dried, coated with gold, and observed using a Zeiss Evo 40 SEM (Figure 1).
For larvae, the length of the two anal papillae was measured twice (left and right appendix were distinguished) by two independent people, and the absolute difference in the length of two appendixes generated similar results (Table 1), indicating high repeatability. The FA index was defined as an absolute difference between the right and left appendixes, standardized by the mean. The presence of asymmetry was confirmed using an ANOVA test (Table 2), which tested the differences in length between the longer and shorter appendix in a pair, as previously described [46 (link),47 (link)].

The CGF was fixed in 4% (w/v) paraformaldehyde (PFA) in PBS for 2 h (room temperature), followed by two PBS washings, and final storage in 0.05% (w/v) sodium azide in PBS. After fixation, the CGF was rinsed two times with PBS, dehydrated in scalar ethanol/water solutions (15%, 25%, 50%, 70%, 90%, and 100% ethanol, 10 min each), and then freeze-dried. To observe the inner surface, CGF was cut in the middle with a scalpel, along the transverse plane, and coated with a 7 nm layer of gold and examined under scanning electron microscopy (SEM EVO^® 40, Carl Zeiss AG, Oberkochen, Germany), in variable pressure mode with an accelerating voltage of 20 kV. The sample was placed on the SEM sample holder using double-sided adhesive tape and was observed without any further manipulation at a lower and higher magnification (2 kX and 10 kX). SEM micrographs were then analyzed by ImageJ 1.50c software (NIH, https://rsb.info.nih.gov/ij, accessed on 18 March 2021) to evaluate the average fiber diameter and size distribution in the fibrin matrix (50 measurements for each acquired sample). The diameters are reported as the mean ± standard deviation.

The morphologies of the empty MSs and MS/Ks were examined using an SEM EVO^® 40 (Carl Zeiss AG, Jena, Germany). Approximately 1–2 mg of lyophilized empty MSs and MS/Ks were deposited onto a carbon adhesive disk mounted on an aluminum stub and directly observed at an accelerating voltage of 20 kV.

Scanning electron microscopy (SEM EVO® 40, Carl Zeiss AG) using variable pressure mode and an accelerating voltage of 20 kV was used to observe the surface morphology of chitosan nanofiber mats. Before microscopy analysis, small discs (10 mm diameter) were punched out from each electrospun mat, placed onto the sample holder, and sputter-coated with gold (7 nm) in a vacuum chamber.
SEM micrographs were then processed and analyzed with ImageJ 1.50c. software (NIH, http://rsb.info.nih.gov/ij) to determine the average fibers diameter and size distribution in the electrospun samples (with diameter ϕ in the range <100 nm, 100 < ϕ < 250 nm, 250 < ϕ < 400 nm, 400 < ϕ < 550 nm, and 550 < ϕ < 800 nm), by taking the average values from 200 measurements chosen randomly in the images of each sample. The diameters are reported as average value ± standard deviation.