Ultimately, the researchers envisage that the technique could be used to visualize minute details of processes occurring in tissue, such as the growth of tiny blood vessels "Our main goal is to develop a microscope that can see the microvasculature and capillary vessels," said senior author Ori Katz from the Hebrew University of Jerusalem. "It's important to be able to watch these grow with nearby tumours, for example."

Photoacoustic imaging - in which is tissue is irradiated with laser light, leading to localized tissue heating and the generation of acoustic waves - can image far deeper into tissue than a standard optical microscope. Its spatial resolution, however, is fundamentally limited by the acoustic wavelength, which is orders of magnitude longer than the optical diffraction limit.

Working in collaboration with Emmanuel Bossy (now at Université Grenoble Alpes) Katz devised a way to overcome the acoustic diffraction limit by exploiting temporal fluctuations in photoacoustic signals arising from the flow of red blood cells, which provide a strong source of naturally fluctuating absorption.

The tools needed to achieve such "super-resolution" imaging were described nearly a decade ago, in a work on optical microscopy, with the technique super-resolution optical fluctuation imaging (SOFI). The researchers discovered that the mathematics used with SOFI could also be used to improve photoacoustic imaging. "Someone just needed to make the connection," Katz explained. "It's the same equation - the wave equation. Mathematically, you could say it's the same problem."

In previous work, Katz and colleagues demonstrated that the acoustic diffraction limit could be surpassed by exploiting temporal fluctuations in photoacoustic signals induced by dynamic optical speckle illumination. This approach, however, required a long-coherence laser, which is not a standard part of a photoacoustic imaging system, and was also limited by the inherent low amplitude of the photoacoustic fluctuations.

In their latest study, the researchers overcame these limitations by applying an advanced statistical analysis framework to the inherent signal fluctuations caused by red blood cells flowing through the vessels, thus eliminating the need for structured illumination. They validated their approach experimentally using a microfluidic phantom, first with a flowing suspension of absorbing particles in the microfluidic channels and secondly using whole human blood.

The team is now developing the technique with a view to in vivo applications. One challenge here is removal of motion artefacts, arising from heartbeat, changing volume of vessels or microscale movements of the tissue itself. In animal models and humans, absorbers other than blood will be present, and the researchers are also devising ways to suppress background signals from these. They are also working to apply sophisticated reconstruction algorithms that use prior information to further increase resolution and reduce background.

"We have demonstrated that flow-induced fluctuations in photoacoustic signals can be harnessed to provide a resolution beyond the acoustic diffraction limit," the authors concluded. "Our technique can be directly implemented on any conventional photoacoustic imaging system and with any illumination. The technique may be potentially used to image blood microvessels that are otherwise blurred by exploiting the natural flow of endogenous absorbers such as red blood cells."