Imaging of live embryos/larvae and measurements of cardiac function

Digital still micrographs of live larvae were obtained with an Olympus SZX-10 stereo microscope equipped with a 5 Mp resolution camera (Infinity 2–5c, from Lumenera) while video recordings where obtained using the same microscope and a Nikon SMZ-800, both with 1.2 Mp resolution video cameras (Unibrain Fire-I 785c). Image magnification was calibrated with a stage micrometer. BTV Pro 5.4.1 (www.bensoftware.com) was used to control the video camera. Video microscopy was performed at 1 and 3 dph (Continuous and Pulse embryo exposures, 60 larvae per treatment), 2 dph (Transient embryo exposure, 30 larvae per treatment) and 3 and 9 dph (Larval exposure, 48 larvae per treatment). Animals were immobilized in a glass petri dish filled with 3% methylcellulose and kept at 8 °C using a temperature controlled microscope stage. Length of ethmoid plate, mandible, area of edema and length, area and circularity of ventricle were measured using ImageJ (ImageJ 1.48r, National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij) with the ObjectJ plugin (https://sils.fnwi.uva.nl/bcb/objectj/index.html) from images as described in Fig. 5. At 0 dph for the embryonic exposure, the area occupied by edema fluid was quantified as the difference between the total area of the yolk sac and the area of the yolk mass, expressed as a percentage of total yolk sac area (Fig. 5). For larval exposure, we quantified edema accumulation at 9 and 18 dph with a similar approach, as the difference between the area occupied by the internal organs and the total pericardial and abdominal area. The assessment of edema at 9 dpf was based on presence/absence, not estimated edema area. ImageJ was also used to measure the ventricular and atrial diastolic (D) and systolic diameter (S) to estimate the fractional shortening (FS = (D − S)/D) (Fig. 5A,B). Atrial beats per minute (BPM) were also counted. Measurements from both images and videos were performed blind.

To quantify oil microdroplets on the eggshell we recorded PAH associated blue fluorescence using a Nikon AZ100 microscope equipped with a DAPI filter set (peak excitation 330–380 nm; emission >425 nm) and a Nikon Intensilight C-HGFI fluorescence light source. Pictures were taken using a 2x objective and 1x zoom and a QICAM monochrome camera (1392 × 1040 pixels, http://www.qimaging.com) resulting in images with a resolution of 0.237 pixels/μm. The microscope UV-light, camera exposure time and gain factor were initially adjusted to the high exposure group eggs so that oversaturation did not occure in the picture. These factors were then held constant (2 sec exposure time, gain 5) for all images. Each picture usually contained 6 eggs. Emission on the eggshell was estimated using Image J; each egg was encircled using the circular ROI (region of interest) tool and mean light intensity measured for each. If the eggshell had artifacts (e.g., small particles) that interfered with the measurement, these were excluded from the ROI before measurement.