Dcf300 fx

Adult fish were anaesthetised with 0.004% MS-222 (Sigma-Aldrich, Germany) and photographed with a Canon EOS 5D MarkII camera and a Macro 100 objective. Metamorphic fish (Figure 4) were anaesthetised, embedded in 1.5% low melting point agarose with 4.5 mg/ml ± epinephrine (Sigma-Aldrich) for melanosome contraction, and photographed under a Leica M205 FA stereomicroscope with a Leica DCF300 FX camera using the software LAS V4.1 to allow multifocus images.

Micrographs were acquired using either a Leica DCF300FX digital camera coupled to a Leica MZ16F dissecting microscope (436/20 nm excitation 480/40 nm emission, 470/40 nm excitation, 510 nm long pass emission and 525/50 nm emission, 10x zoom) and controlled with Leica FireCam Software or a Leica DM2500 compound microscope coupled with a Q-Imaging Micropublisher 3.3 camera, controlled with Q-Capture software. All prepared samples were imaged prior to analysis by flow cytometry. Selected sorted samples which were to be subjected to SEM imaging were placed onto a Nuclepore^TM polycarbonate filter and washed three times with 10 ml milli-Q H₂O to remove any residue of PBS that would cause salt crystals to form on the Nuclepore^TM filter. The resultant filter paper was placed onto an SEM stub and sputter coated (Q150T-ES, Quorum Technologies, UK). The prepared sample was visualised by SEM (JSM-6390LV SEM, JEOL, Japan) and images captured.