Motion-based structure separation for label-free, high-speed, 3D cardiac microscopy.

Capturing the dynamics of individual structures in the embryonic heart is an essential step for studying its function and development. Label-free brightfield (BF) microscopy allows for higher acquisition frame-rates than techniques requiring molecular labeling, without interfering with embryo viability or needing complex equipment. However, since different structures contribute similarly to image contrast, label-free microscopy lacks specificity. Here we mitigate this problem by separating a single-channel image series into multiple channels specific to different cardio-vascular structures, based only on their motion patterns. The technique combines images from multiple cardiac cycles and z-sections after non-uniform temporal registration to produce 3D+time image volumes of one full cardiac cycle with separate channels for static, transient and periodically moving structures. The resulting data is well-suited for velocity analysis and 3D-visualization. We characterize the separating capabilities of our technique on a synthetic cardiac dataset and demonstrate its practical applicability, by reconstructing three-channel views of the beating embryonic zebrafish heart with an effective frame rate of 1000 volumes (256×256×20 voxels each) per second. This technique enables quantitative characterization of dynamic heart function during cardiogenesis.

Characterization of HCN and cardiac function in a colonial ascidian.

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the rhythmic beating of mammalian hearts. We identified an HCN homolog in the colonial ascidian Botryllus schlosseri, a nonvertebrate chordate which possesses a tubular heart that beats bidirectionally. Contractions initiate at one end of the heart and travel across the length of the organ, and these periodically reverse, suggesting the presence of two pacemakers, one on each side. We find that HCN expression is highly enriched in cells scattered throughout the myocardium. We functionally analyzed the role of HCN channels in heartbeat using the antagonists Cilobradine and Zatebradine, which decreased the heartbeat in a reversible manner. We also assessed the role of ß-adrenoreceptors in regulating HCN function using the antagonist Metoprolol, which lowered heartbeat rate (HR), as well as the agonist Isoproterenol, which did not alter HR, but caused simultaneous beating, analogous to a fibrillation. Measurements of direction and velocity of blood flow by making use of a novel system to study heart function in model systems amenable to live imaging revealed a significant correlation between heartbeat arrhythmia and drug treatment, similar to that observed with the same drugs in vertebrates. These results suggest that the heart pacemaker in tunicates may be homologous to that in their vertebrate counterparts in both development and function.

High-speed multicolor microscopy of repeating dynamic processes.

Images of multiply labeled fluorescent samples provide unique insights into the localization of molecules, cells, and tissues. The ability to image multiple channels simultaneously at high speed without cross talk is limited to a few colors and requires dedicated multichannel or multispectral detection procedures. Simpler microscopes, in which each color is imaged sequentially, produce a much lower frame rate. Here, we describe a technique to image, at high frame rate, multiply labeled samples that have a repeating motion. We capture images in a single channel at a time over one full occurrence of the motion then repeat acquisition for other channels over subsequent occurrences. We finally build a high-speed multichannel image sequence by combining the images after applying a normalized mutual information-based time registration procedure. We show that this technique is amenable to image the beating heart of a double-labeled embryonic quail in three channels (brightfield, yellow, and mCherry fluorescent proteins) using a fluorescence wide-field microscope equipped with a single monochrome camera and without fast channel switching optics. We experimentally evaluate the accuracy of our method on image series from a two-channel confocal microscope.

Joint dynamic imaging of morphogenesis and function in the developing heart.

In the developing heart, time-lapse imaging is particularly challenging. Changes in heart morphology due to tissue growth or long-term reorganization are difficult to follow because they are much subtler than the rapid shape changes induced by the heartbeat. Therefore, imaging heart development usually requires slowing or stopping the heart. This, however, leads to information loss about the unperturbed heart shape and the dynamics of heart function. To overcome this limitation, we have developed a non-invasive heart imaging technique to jointly document heart function (at fixed stages of development) as well as its morphogenesis (at any fixed phase in the heartbeat) that does not require stopping or slowing the heart. We review the challenges for imaging heart development and our methodology, which is based on computationally combining and analyzing multiple high-speed image sequences acquired throughout the course of development. We present results obtained in the developing zebrafish heart. Image analysis of the acquired data yielded blood flow velocity maps and made it possible to follow the relative movement of individual cells over several hours.