Microbubbles ranging from 10–150 μm in diameter were clearly visualized, as were air sacs and numerous tracheae, including a main longitudinal tracheal trunk that runs adjacent to the heart (Fig. 1C and Additional files 1, 2, 3, 4, 5, 6 and 7). The buoyant bubbles accumulated along the ventral edge of the dorsal diaphragm, which partially compartmentalizes the dorsal heart region. Within the heart, the buoyant bubbles demarcated the dorsal edge of the heart lumen. The ventral edge of the heart lumen could not be clearly identified, but the paths of microbubbles during flow within the heart suggested its ventral extent. Microbubble flow, compression of the pericardial sinus and changes in the tracheal diameters were all clearly visualized (Additional files 2, 3, 4, 5, 6 and 7).
Additional file 1: Synchrotron x-ray phase-contrast video of heartbeat in the grasshopper Schistocerca americana. Movie depicts the grasshopper's 8th abdominal segment in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm. The dorsal edge of the abdomen can be seen at the top. The diagonal lines running from upper left to lower right are margins of the x-ray transparent Kapton tube. In this sequence, pulsatile movements in the dorsoventral axis associated with heartbeat can been seen, but flow within the heart is not apparent due to lack of contrast between hemolymph and the surrounding tissue. Large collapsing circular structures are air sacs. Tracheal tubes are also visible, particularly the two main tubes running horizontally at the bottom of the frame. (MOV 7 MB)
Additional file 2: Typical hemolymph flow in the heart of the grasshopper Schistocerca americana I. Movie depicts the grasshopper's 3rd abdominal segment in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm. The dorsal edge of the abdomen can be seen at the top. The diagonal line in the lower left corner is a margin of the x-ray transparent Kapton tube. In this video, bubbles can be seen in three locations: within the perivisceral sinus, collected en masse and abutting the dorsal diaphragm (lower third of image); within the heart, moving rapidly left and right; and surrounding the heart in the pericardial sinus, either static or moving less rapidly than within the heart. The dorsal diaphragm moves dorsoventrally in association with heartbeat. A main tracheal trunk running antero-posteriorly is also compressed in association with these movements, but not strictly so. Within the heart, flow is complex and non-uniform, as evidenced by the trajectories of the bubbles. There is net transport of the hemolymph anteriorly, but the bubbles can be seen moving posteriorly as well. Additionally, vortical trajectories of bubbles can be seen. Due to bubble buoyancy, most bubble trajectories in the heart occur in dorsal margin. (MOV 19 MB)
Additional file 3: Typical view of hemolymph flow in the heart of the grasshopper Schistocerca americana II. Movie depicts the grasshopper's abdomen in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm, mostly of the 5th abdominal segment, although the start of the 4th can also be seen on the right side of the image. The dorsal edge of the abdomen can be seen near the top, and the abdominal segment boundary appears on the right. The diagonal lines running from lower left to upper right are margins of the x-ray transparent Kapton tube, whose outer boundary can be seen at the top. This video shows net transport of hemolymph in the heart toward the right (anterograde flow), but intermittent backflow to the left can be seen as well. Note that unlike in Additional file 2, the main tracheal trunks are not compressed along with the pulsatile movements. (MOV 10 MB)
Additional file 4: Oscillatory hemolymph flow in the heart of the grasshopper Schistocerca americana I. Movie depicts the grasshopper's 3rd abdominal segment in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm. The dorsal edge of the abdomen can be seen near the top. The diagonal lines running from upper left to lower right are margins of the x-ray transparent Kapton tube; the spotting in the upper right corner is due to residual material on the tube wall. In this video, the large bubbles in the heart enter the frame from the left, reverse direction, and exit to the left, demonstrating oscillatory flow with no net transport. Also note the slower, steadier movement of much smaller particles near the dorsal abdominal wall. These are the injected Definity microbubbles, which appear to be located within the pericardial sinus that surrounds the heart. (MOV 13 MB)
Additional file 5: Oscillatory hemolymph flow in the heart of the grasshopper Schistocerca americana II. Movie depicts the grasshopper's 6th abdominal segment in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm. The dorsal edge of the abdomen can be seen near the top. The diagonal lines running from upper left to lower right are margins of the x-ray transparent Kapton tube. This video demonstrates a second sequence in which the local heart flow is oscillatory, with no net transport anteriorly or posteriorly. Additionally, the primary bubble movement in the dorsal heart follows a diagonal trajectory (left side), evidencing a bend in the heart tube. (MOV 7 MB)
Additional file 6: Possible entrance of a bubble through an incurrent ostium in the heart of the grasshopper Schistocerca americana I. Movie depicts the grasshopper's 5th abdominal segment in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm. The dorsal edge of the abdomen can be seen near the top. The diagonal lines in the upper left corner are margins of the x-ray transparent Kapton tube. In this sequence, the bubble of interest appears to begin its trajectory outside the heart in the pericardial sinus; it moves dorsally and then anteriorly to the ostium opening; it then enters the heart and is swept anteriorly along with the flow. Although we interpret this trajectory as a heart entrance event, other possibilities remain. (MOV 9 MB)
Additional file 7: Possible entrance of a bubble through an incurrent ostium in the heart of the grasshopper Schistocerca americana II. Movie depicts the grasshopper's 6th abdominal segment in lateral view, with dorsal oriented upward and anterior to the right. Field of view is 1.3 × 0.9 mm. The diagonal line in the lower left corner is a margin of the x-ray transparent Kapton tube. This sequence demonstrates a second potential ostial entrance event. Here, the bubble of interest appears to begin its trajectory outside the heart in the pericardial sinus; it moves posteriorly, dorsally, and then anteriorly to the ostium opening; it then enters the heart in a large movement to the upper right. As in Additional file 6, although we interpret this trajectory as a heart entrance event, other possibilities remain. (MOV 11 MB)
As seen within the field of view (1.3 H × 0.9 V mm), the flow patterns were complex and dependent on location. At any particular location along the heart, general flow patterns were repetitive, but not strictly time-periodic, over the course of the measurement (10's of seconds). For most pulsations (e.g., Fig. 2), forward flows coincided with local pericardial compressions (see Methods). Interestingly, back flows, when they occurred, began during pericardial compression, and ended at or after the end of the compression. However, this could be due to a phase lag between the hemolymph and the bubble (see Discussion). The amplitude of pericardial sinus compression varied locally (Additional file 2). Compressions of the longitudinal tracheal trunk were not synchronized with the pericardial sinus compressions (e.g., Fig. 3); however, their frequencies were similar (0.8–1.0 Hz). Air sac compressions also appeared to be independent of pericardial sinus compressions (Additional file 1). Flow within the heart was pulsatile, but did not appear to be bolus-like. Unlike previously observed peristaltic transport in the gut [2] in which the bolus-like transport of food and gut peristaltic waves were readily apparent, the hemolymph transport mechanism within the heart remains unknown from this preliminary study.
The power of this technique is exemplified in the ability to track small-scale flow patterns. For example, bubbles can apparently be seen to enter the heart by moving through incurrent ostia (Fig. 4, Additional files 6 and 7). However, it was not possible to differentiate if the incurrent ostia were opening actively or passively, nor were we able to positively identify movement of bubbles out of the heart through excurrent ostia [22]. The local flow patterns near the ostia, but external to the heart, were complex. In general, bubbles were observed to enter the ostia originating both posteriorly (e.g., Fig. 4a, Additional file 6) and anteriorly (e.g., Fig. 4b, Additional file 7). In either case, once they entered the heart, the bubbles were swept anteriorly with the heartflow. Another example shows the complexity of flow within the heart. In Figure 5, four microbubbles are tracked over the course of 25 frames (~0.83 s) in one section of the heart. This sequence suggests that the bubbles were pulled towards the middle of the image during diaphragm dilation, and upon compression, hemolymph was transported towards the head. The ability to image individual microbubbles allowed us to quantify instantaneous velocities and potentially to map complex flow fields. Such detailed flow information cannot be obtained from any other technique.
Although the main purpose of this paper is to present this new technique, our measurements allow us to make a few preliminary observations on the hemolymph flow in the heart of the grasshopper Schistocerca americana.
1. In general, flow patterns are complex, and are time and location dependent. Furthermore, some flows appear to be three-dimensional. In some observations, there appeared to be no overall transport, but simply a back-and-forth oscillation of the hemolymph (Additional files 4 and 5). This preliminary study suggests that the origin of hemolymph transport may be more complicated than the commonly assumed peristaltic motion [10, 11, 14].
The maximum speed of bubbles that could be tracked was 9.5 mm/s; faster moving bubbles were observed but could not be reliably measured due to motion blur (exposure, 16 ms). Based on this maximum speed, a heart diameter of 0.5 mm, and using the viscosity of water as an estimate of that of hemolymph, we calculate a Reynolds number of 4.8, suggesting that flows are in the laminar regime.
3. For two individuals that were imaged for 20–30 minutes each, there was no evidence of a sustained retrograde fluid transport. The microbubble motion was either pulsatile with overall forward transport, or oscillatory with no net transport.
4. Respiratory structures (tracheae and/or air sacs) were compressed in patterns both synchronous and asynchronous to the local heartbeat.
5. No immediate detrimental effects on the animal were observed, either from the x-rays or from the microbubbles. The animals that were injected with microbubbles and under x-ray irradiation of the heart for up to an hour were alive 24 hours later. This is consistent with our previous results [2] which showed that there were no observable negative effects in insects at similar x-ray intensities under irradiation of the abdomen. Local x-ray damage must have occurred, but effects on heart, tracheal, or abdominal movements and behavior were not apparent within this time frame.