Axiocam 512

Microscopic assessment of the testes was performed following standard dehydration and paraffin embedding method^{[39 ]}, and haematoxylin and eosin stained tissue slides prepared. The prepared slides were coded and examined under a Carl Zeiss microscope (Germany). On inspection, images were taken using a Zeiss Axiocam 512 camera by a pathologist unaware of the various treatment groups from which the slides were prepared.

Based on the block-surface method (Spoendlin and Schrott, 1987 (link)), it is feasible to assess the cochlea quantitatively at a light and electron microscopic level within the osseous spiral lamina and the cochlear nerve. ANFs of all specimens were divided into equal and comparable segmented regions, from basal to apical. Sections were digitized using a Zeiss Axio Imager.M2 equipped with a Zeiss Axiocam 512. Full-resolution images were acquired with a Plan Apochromat 63 × 1.4 lens and used to evaluate DD, MT, and G-ratio of the ANFs. Data processing was done with ImageJ as well as MATLAB (version R2018b)¹, using a toolbox (Zaimi et al., 2016 (link)) for semi-automatic segmentation to investigate the dendrite of the ANFs. For a systematic comparison, an equal number of ANFs were counted in all three regions.
Statistical analyses were conducted using MATLAB and Python programming language (version 2.7)². All data shown in Table 1 were collected based on the degree of hearing loss at three regions of the basal, middle, and apical with 450, 520, and 150 fiber numbers, respectively. The data were compared with the healthy specimen as control. The Kruskal–Wallis test was used to determine if there were significant differences between groups in each region, separately. Pairwise Conover test was applied to compare the medians of all groups.

Leaf samples were stained for iron using Perls' reagent as previously described (Meguro et al., 2007 (link)). Samples were mounted in glycerol (50% v/v) and imaged on an Axio Zoom.V16 stereo microscope with an Axiocam 512 color camera (Zeiss, Cambourne, UK). For measuring iron concentrations, dried leaf samples were digested in 0.25 ml nitric acid (69% w/v) and 0.25 ml hydrogen peroxide (30% w/v) at 90°C. After neutralizing with 1 ml ammonium acetate (15% w/v), samples were reduced with 0.1 ml ascorbic acid (4% w/v). Fe²⁺ was quantified using the colorimetric iron chelator ferene (3‐(2‐pyridyl)‐5,6‐bis‐[2‐(5‐furyl‐sulfonic acid)]‐1,2,4‐triazine, 0.1% w/v) and absorbance was measurement at 593 nm. Iron and other elements in pea seeds were quantified by Inductively Coupled Plasma‐Optical Emission Spectroscopy following digestion of ground samples in nitric acid (55% w/v) and hydrogen peroxide (6% w/w) at 95°C for 16 h.

Conidia, mycelium, and biofilm treated with PLGA-coumarin6-NPs were observed, and images were acquired using an Axio Imager M2 fluorescence microscope (Zeiss, Wetzlar, Germany) motorized on the 3 axes by using a FITC filter (λ excitation BP 455-495 nm; λ emission BP 05-555 nm). The thickness of the sample provided a Z-stack image scan performed with an Axiocam 512 (Zeiss) monochromatic camera and ApoTome 2 (Zeiss) as a fringe projection module to eliminate the out-of-focus signal. Zen 2.5 (Zeiss) image analysis software was used to obtain single-plane images as Z-stack maximum projection.

For observation of cleared samples, FAA-fixed samples were washed with water and cleared with chloral hydrate solution (8 g chloral hydrate, 1ml glycerol, and 2ml water) for at least 1 day. The cleared samples were observed using an Axio Imager A2 microscope (Zeiss) and photographed with an Axiocam 512 color camera (Zeiss). Fluorescence observation was performed using the ClearSee method (Kurihara et al., 2015 (link)) with a confocal laser microscope LSM800 or LSM900 (Zeiss). YFP and GFP were excited at 488 nm, and fluorescent images were acquired with the detection range from 490 to 546 nm. Differential interference contrast (DIC) images were taken by a T-PMT detector. Image adjustments were performed using the ImageJ software.

Fluorescence live-cell imaging was performed using a Zeiss Axio Observer microscope equipped with a Zeiss Axiocam 512 monocamera and a set of objectives and filters, which could capture the right size pictures and separate signals from different channels, and captured images were then analyzed and exported by ZEN imaging software (Carl Zeiss Vision GmbH, München, Germany).