Dfc295 camera

A Leica S8Apo binocular (Leica, Wetzlar, Germany) was used for harvesting of sporophytes and gametangia. An upright DM6000 equipped with a DFC295 camera (Leica) was used for taking microscopic pictures. Both devices ran under the control of the Leica Application suite version 4.4. Microsoft PowerPoint was used for processing (brightness and contrast adjustment) the images.

Printability was characterized by the filament fusion and filament collapse test, described by Ribeiro et al. (2017) (link). Briefly, the filament fusion test was performed by printing three layers of a meandering pattern with increasing strand spacing using the three-axial BioScaffolder plotter (BioScaffolder 3.1, GeSiM, Radeberg, Germany). The hydrogel was extruded through a nozzle with an inner diameter of 840 µm (Globaco, Rödermark, Germany). After plotting, the structures were imaged with a stereo light microscope (Leica M205 C with DFC295 camera, Wetzlar, Germany) and analyzed with the image software Fiji (ImageJ 1.53f51, Schindelin et al., 2012 (link)). Filament segment length and filament thickness were measured and the quotient of these measurements was evaluated as a function of the corresponding filament spacing. The measurements were performed with a sample size of n = 5.
The filament collapse test was performed by printing a single filament on a platform with pillars with the bioprinter BioScaffolder 3.1, with the gap distance increasing with every pillar (1, 2, 4, 8, 16 mm). Immediately after printing, a photo was taken and analyzed with the software Fiji. For the analysis, the angle of filament deflection was measured and plotted against the corresponding gap distance. The measurements were performed in triplicates.

The mass flow of mc6, mc8, and mc10 was conducted through a conical needle with an inner diameter of 250 μm (Globaco, Rödermark, Germany); needle and plate were at room temperature. The inks (n = 3) were allowed to flow through the needle for 50 s at different air pressures (100, 150, 200, 250 kPa), then the mass of the extruded volume was measured. Resulting mass flow $\dot{m}$ was calculated: $\dot{m}=\frac{m}{50 s}$
Filament fusion tests of the mc inks (n = 3) were performed following the protocol of Ribeiro and co-workers [29 (link)]. Shortly, three layers of the inks were plotted on top of each other. Each layer consisted of meandering strands with increasing strand distances. Immediately after plotting, the scaffolds were imaged by a stereo light microscope (Leica M205 C equipped with DFC295 camera, Wetzlar, Germany). Resulting images were analyzed by Fiji Image software [56 (link)] and actual strand distance, fused segment length and strand width were obtained from the data. The quotient of segment length to strand width was evaluated as function of the strand distance.

Pressed polyvinyl chloride clear, non-curling plastic coverslips, 0.17 to 0.25 mm thick, 22 mm width (Fisher Scientific, UK) were cut into 10 mm² width and sterilized in UV light were used to induce biofilm on the wound. The bacterial pathogen; Methicillin-resistant Staphylococcus aureus (ATCC 43300) available in the department was used in the study. Mupirocin (2% w/w) ointment was from Avabon Ointment, Avalon Pharma, Riyadh, KSA, and Luria Bertani broth was purchased from SRL Pvt. Ltd., Mumbai, India. Stains and other chemicals were purchased from different chemical suppliers and were of analytical grade. A microscope (Leica DM 2500 LED connected to a DFC 295 camera) was used to capture images.

The methodology used to identify and count trichomes was fully described by [31 (link)]. The fifth leaf of each plant was dissected into 15 × 3 mm strips of the leaf blade for the analysis. The samples were affixed to the edge of microscope slides using transparent nail polish. A support made of Styrofoam with a slit was used to secure the slides horizontally but elevated from the stage to keep the trichomes perpendicular to the objective-stage direction. Photographs were taken using a stereomicroscope Leica S8AP0 (Wetzlar, Germany) at 50× magnification coupled to a Leica DFC295 camera (Wetzlar, Germany). The lateral views of the trichomes allowed the correct measurement, classification, and counting of each type. Eight individual plants per genotype were sampled, and four different strips were analyzed per plant for each leaf side (abaxial/adaxial). From the pictures, trichome counting was performed for density estimation. Trichome length measurements were performed using the manufacturer’s analytical software (Leica Application Suite 4.0, Wetzlar, Germany).

The H and E staining was conducted using standard procedure [21 (link)]. The modified Gram’s staining was performed as follows [18 (link)]. The paraffin blocks were deparaffinized and hydrated with distilled water. The slides were placed on a staining rack and a drop of crystal violet stain was added onto the tissue section for 1 min followed by washing in tap water. The section was flooded with Lugol’s iodine for 1 min followed by washing with tap water. The sections were blotted dry and acetone was poured over the section until no color came out, followed by washing. The slides were then stained with basic fuchsin for 3 min followed by washing and drying. The slide was quickly dipped into acetone (two dips) and then directly into the picric acid-acetone mixture until a ‘salmon’ color developed. The slide was dipped quickly into two changes of acetone, air-dried, and dipped into xylene. Slides were dried and observed under the microscope attached to a camera (Leica DM 2500 LED connected to a DFC 295 camera).
A diagrammatic representation of the methodology used in the study is given in Figure 1.