Eos 550d

After 28 days from the grafting, mice were sacrificed in a CO₂ chamber. The scaffolds were harvested, observed by a circular lens (Canon EOS 550 D), and images captured (Figure 1B).

Photographs were taken using a digital camera Canon EOS 550D or EOS 5D Mark III, coupled to a Canon 50 mm macro lens, or to a MP-E 65 mm macro lens, both equipped with polarizing filters. Resulting photographs were optimized using Adobe Photoshop CS6. Unless specified, reproduced photographs of fossil specimens are dry-ethanol composites.

To observe nuclei present in the growing hyphal tips, an agar block excised from the actively growing margin of the P. noxius colony was put on a sterilized slide in a sterile moist chamber and incubated for 2 days. After removal of the agar block, the mycelium present on the slide was mounted in 20 μl of a DAPI (4',6-diamidino-2-phenylindole) solution (10 μg/ml in ddH₂O) for 10 min and then sealed with a coverslip. DAPI staining was also conducted to observe the conidia from a naturally occurring basidiocarp and the arthospores dislodged from the P. noxius colony. Material was observed under a Leica DMLB microscope (Buffalo Grove, IL, USA) equipped with filter cube A (BP 340–380 nm, LP 425 nm). Images were captured by using a Canon (Ohta-ku, Tokyo, Japan) digital camera EOS 550D.

Larva at 3 dpf was mounted in 3% methylcellulose and video-recorded for the blood flow in caudal vein (60frames/second) under a transmitted-light stereomicroscope (Leica, MDG28) equipped with a digital single-lens reflex camera (Canon, EOS 550D). The velocity of blood flow was estimated from the traveling distance of a same red blood cell captured in a serial video stills with the software Celltracker [43 (link)] on MATLAB R2015a system. The cardiac stroke area, ejection fraction and cardiac output of larvae were calculated with the following equations: Stroke area = end diastolic area − end systolic area; Ejection fraction = stroke area / end diastolic area; Cardiac output = stroke area × heart rate. End-systolic area and end-diastolic area of heart were measured from the bright field time lapse image sequence analysis on a serial video stills with the software ImageJ, an open platform for scientific image analysis (https://imagej.net/ImageJ).

Larvae at 1 dpf were placed on a concave slide with embryo water under a transmitted-light stereomicroscope (Leica, MDG28) equipped with a digital single-lens reflex camera (Canon, EOS 550D). Larval spontaneous contraction was video-recorded in 60 frames per second and analyzed with EthoVision XT (Noldus, Wageningen, The Netherlands).

Three specimens were used to investigate and illustrate the thoracic skeleton. One complete and undamaged specimen was dehydrated in a graded ethanol series and critical-point dried (Balzer CPD 030) to visualize the outer lateral and dorsal view. Another specimen was sagitally cut and macerated in 5% KOH (1 h in a heating cabinet with 60 °C) and likewise dried at critical point. Critical-point drying was applied to improve the contrast of the thoracic sclerites against the membranous areas and to visualize the sclerites in more detail. One specimen was fixed in a ventrally overstretched position to expose the neck region and subsequently dried using the HMDS (Hexamethyldisilazane, Carl Roth GmbH & Co KG, item number 3840.2) procedure [35 (link)]. Photographs of the HMDS-dried specimen were taken using a digital camera (OLYMPUS Pen E-P2) mounted on a stereomicroscope ZEISS Stemi SV11. The critical-point dried specimens were photographed with a CANON EOS 550D equipped with a macro lens (100 mm) and a ring flash (METZ 15 MS-1). The overall sharp images are composed of image stacks edited in Helicon Focus® (Helicon Soft) and Adobe Photoshop® CS3.