Faster confocal technique explores heart’s pumping mechanism in vivo
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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