Getting to the heart of the matter

Sam Lee highlights how advances in fluorescence microscopy are aiding in the fight against heart attacks.

Diseases affecting the heart and blood vessels remain the leading cause of death across most of the world. The British Heart Foundation estimates that some 200,000 people in the UK are hospitalised every year because of heart attacks. A heart attack, also called a myocardial infarction, is caused by blockages within the blood vessels that supply the heart muscle.

Heart attacks are commonly caused by fat build-up on the inside of the vessel. If these fat deposits rupture, a blood clot can form, which can detach and lodge itself within a smaller vessel downstream. Deprived of blood, and therefore oxygen, the heart muscle will start to die. Although removing the clot is essential, restoring blood flow can counterintuitively lead to further immediate damage to the muscle tissue, otherwise known as reperfusion injury. Worse still, the smallest blood vessels—just several micrometres wide—may remain blocked, even after the initial clot has been removed.

A blockage of these microvessels is referred to as myocardial infarction with non-obstructive coronary arteries (MINOCA) and cannot currently be visualised in a hospital setting. To understand how reperfusion injury occurs, and to develop treatments that restore flow with minimal tissue damage, researchers have developed cutting-edge microscopy techniques to see inside the microvessels of the heart.

Traditionally, thin slices of heart tissue are stained with antibodies that are designed to attach to, and therefore label, blood vessels and cells. Like microscopic beacons, these antibodies glow (or fluoresce) under laser light; researchers then view this fluorescence with a microscope. This is called fluorescence microscopy and is shown in Figure 1. However, this type of microscopy cannot see through thick tissue.

To see these deeper vessels more clearly, a technique called two-photon microscopy is used, as seen in Figure 2. The physics behind this gets complicated in a hurry, but to put it briefly: light is comprised of photons, and it is these photons that cause fluorescent molecules to glow. In traditional (one-photon) fluorescence microscopy, each fluorescent antibody molecule is excited by a single photon. In two-photon microscopy, each fluorescent molecule absorbs two photons at (practically) the same time. Because two photons are required, and each of these photons has a lower energy and a longer wavelength, they can penetrate deeper into the tissue and cause less damage.

However, both of these techniques can only be used on fixed sections of tissue removed from the heart, not ideal for monitoring the heart function of a living patient. To see inside the microvessels of an intact heart, as it is beating, a technique called intravital microscopy is required.

In animal models, we can access the heart relatively easily: point a microscope at it, introduce fluorescent antibodies, and watch them move through the vessels. But the heart is constantly beating; without external help, the image produced on the microscope will constantly shift out of focus. By using a 3D-printed stabiliser, a small area of the heart can be fixed in place and attached to the microscope to minimise blur. Now, we can watch blood moving through the vessels, observe the inflammatory cells and markers start growing in number after a heart attack, and even see blood clots form in real-time (Figure 3)!

Through these advances in microscopy, biomedical scientists can finally unravel the mechanisms behind reperfusion injury and the blockage of microvessels following a heart attack. Understanding these mechanisms will allow us to develop new ways of treating these conditions and hopefully reduce the catastrophic impact that heart attacks have on patients around the world.

From Issue 21