Intravital imaging and extracellular domain antibodies to peer into the heart
Studying the heart is a bit like trying to photograph a hummingbird mid-flight: it’s a fast-moving and dynamic organ that just won’t stay still. On top of that, a lot of imaging techniques fail to capture some of the finer cellular details in action. Until now.
In a new paper, researchers have developed and optimized an intravital imaging technique to observe cardiac tissue at the cellular level in a mouse model. This became achievable with a little help from fluorescently-labeled primary antibodies targeting the extracellular domain of a membrane protein (1).
Stabilization, Fluorescence, and Precision Imaging
The researchers looked to improve the classic intravital imaging method and built their protocol around a suction-based stabilization system. They utilized LysM-eGFP transgenic mice which express GFP in immune cells in the cardiac tissue. After surgically exposing the heart, they applied a vacuum to secure the tissue against a glass coverslip. This step reduced the jarring effects of cardiac and respiratory movements without significantly affecting the tissue’s physiological state. Combining this stabilization with dual-mode confocal and two-photon microscopy allowed them to capture high-resolution images of live cardiac tissue.
To visualize the cardiac microvasculature, they injected a FSD647-conjugated anti-CD31 antibody to the mouse’s tail vein. This antibody binds to an extracellular epitope of CD31 on endothelial cells, which lights up the vasculature in impressive detail. With these labeled extracellular antibodies, some DiD-labelling and GFP expression, the team was able to track simultaneously the flow of red blood cells and neutrophils in cardiac vasculatures. AI-driven motion correction then processed these real-time images to compensate for any remaining movement – the results were sharp, quantifiable data!
Cellular Dynamics in Real Time
Their setup provided a front-row seat to cellular behavior in the heart, where immune cells crawled along blood vessel walls and red blood cells flowed through arterioles at high velocities (Figure 1). The team also used second-harmonic generation imaging to visualize the heart’s structural proteins, like sarcomeres and fibrillar collagens, without any additional labelling.
Intravital imaging isn’t new but doing it in the heart has always been a bit of a challenge – the whole organ is in constant motion, and the resulting artefacts have typically made meaningful in vivo data hard to come by. But this protocol solves that and opens the door to some incredible experiments. Immune cell tracking? Vascular dynamics in disease? Drug testing in a real-time, physiologically relevant system? All of it becomes possible.
Figure 1. In vivo real-time cellular imaging of immune cell and RBC flowing dynamics in the heart. Ultra-fast real-time recording of flowing neutrophil and RBC dynamics in the arteriole of the left ventricle in live heart tissue. Sub-second video-rate cellular trafficking of flowing LysM+ neutrophil and DiD-labeled RBC dynamics in cardiac vessels in LysM-GFP mouse was indicated by arrowheads. Scale bar, 100 µm. Image and legend from Ahn et al. (1).
Conjugated Extracellular Domain Antibodies: Tools to Build on this Success
While the study used a commercially available CD31 antibody to label endothelial cells, its success underscores the value of extracellular domain conjugated antibodies for live imaging. At Alomone Labs we’ve developed a comprehensive collection of extracellular domain fluorescently labeled primary antibodies designed specifically for live-cell and in vivo research. These antibodies target the extracellular epitopes of membrane proteins, which preserves intracellular signaling and lets you make real-time observations. And their utility extends well beyond cardiac imaging: extracellular domain antibodies are ideal for tracking cellular behavior, studying receptor dynamics, and monitoring physiological changes in a whole range of systems.
A Window to the Future
This study shows how even the most challenging tissues, like the heart, can now be studied at a level of detail previously thought impossible. With the right imaging and bio-detection tools you can push the boundaries of what’s observable in vivo.
Alomone’s extracellular domain conjugated antibodies could help bring this technique to a wider range of models, to make dynamic, real-time imaging more accessible and versatile. The beating heart has never been more within reach.
Take a look at our full collection of fluorophore-conjugated extracellular domain antibodies
Reference
1. S. Ahn, J.-Y. Yoon, P. Kim, Intravital imaging of cardiac tissue utilizing tissue-stabilized heart window chamber in live animal model. Eur Heart J Imaging Methods Pract 2, qyae062 (2024). DOI: https://doi.org/10.1093/ehjimp/qyae062.
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