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  • Faster confocal technique explores heart’s pumping mechanism in vivo

BioPhotonics
Jun 2006
Gwynne D. Koch

The cardiovascular system is the first functional organ system to develop, and the embryonic heart begins pumping blood before features such as chambers and valves are discernible. Until recently, scientists held that the valveless embryonic heart circulates blood by peristaltic contractions. Now, a study by researchers at the California Institute of Technology in Pasadena, the University of Cincinnati and National Taiwan University suggests that another mechanism may be responsible. Their findings could lead to new treatments of heart diseases that arise from congenital defects.

Speed and resolution limitations of conventional imaging methods have made in vivo examination of the structure and function of the embryonic heart tube difficult. Although point-scanning confocal microscopy provides higher structural resolution than other imaging techniques, it has an inherent speed limitation because the sample is scanned point by point. Conversely, high-speed bright-field imaging offers inadequate structural resolution.


Captured using line-scanning confocal microscopy, images of a fluorescently labeled embryonic zebra fish heart tube enabled scientists to examine the pumping mechanisms at work in the developing heart. Transparent zebra fish embryos were used because they enable easy viewing and develop completely in only a few days.


By employing line-scanning confocal microscopy, Morteza Gharib, a professor of aeronautics and bioengineering at the university, and his research team examined blood cell and heart wall movements in GFP-labeled embryonic zebra fish hearts. With this technique, the sample is scanned line by line rather than point by point, enabling capture rates of about 100 times faster. Using a Zeiss LSM 510 confocal microscope and a linear array CCD camera, they captured time-lapse images of the hearts before valve formation.

Bidirectional confocal scans (256 x 256 pixels) were captured at 151 fps. Time series were triggered at a random time in the cardiac cycle and taken for 300 to 500 frames. Upon completion of a time series at one Z-section, the optical plane was moved 3 to 5 μm and the acquisition process was repeated. Four-dimensional data sets were reconstructed from 15 to 25 optical sections and analyzed using Imaris software from Bitplane AG of Zurich, Switzerland. Blood cell velocities were computed from image sequences of five to 10 cardiac cycles.

The researchers identified several biomechanical properties of the embryonic heart tube that contradicted the theory of peristalsis as the main pumping mechanism. Their observations of wave propagations, pressure gradients, and blood cell trajectories and velocities supported what they refer to as the “hydroelastic impedance pumping” model, in which pumping results from dynamic suction caused by elastic wave propagation and reflection in the heart tube.

In this model, contraction of a small collection of cells situated near the entrance of the heart tube initiates a series of forward-traveling elastic waves that reflect back after impinging on the end of the tube. At a specific range of contraction frequencies, the waves constructively interact with the preceding reflected waves to generate a dynamic suction action.

Abnormalities in the suction action in adult hearts are common in patients with congestive heart failure. According to Gharib, the study provides evidence of an embryonic root for the observed suction action and enables scientists to reconsider how embryonic cardiac mechanics may produce anomalies in the mature heart. The team is exploring this pumping mechanism in other vertebrates.

Science, May 5, 2006, pp. 751-753.


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