A newly developed frequency domain technique for photoacoustic (PA) image formation, developed by a team at Ryerson University and St. Michael’s Hospital, subdivides PA signals into different frequency bands. Called F-Mode, the new technique leverages information contained in the frequency content of PA signals to generate images with scale-specific contrast. Most existing PA imaging techniques measure amplitude (loudness), displaying areas emitting louder sounds with brighter pixels. What the Ryerson-led team set out to develop was a technique that would measure the frequency (pitch) of sounds emitted from biological structures. “Depending on the size of a biological structure, the pitch of the sound waves it emits will be higher or lower,” said Dr. Michael Moore. “If we could filter incoming sounds by frequency, we could create images that focus on structures of a particular size, which would help to reveal features that might otherwise be hidden or less prominent.” The team demonstrated F-Mode on features of different sizes in samples ranging from biological cells to live zebrafish larvae — all without the use of contrast dyes that would typically be required by other imaging techniques. Images generated with the F-Mode technique surpassed the capabilities of standard maximum amplitude projection (MAP) reconstruction when differentiating between objects of different scale within phantoms. When used in conjunction with label-free photoacoustic microscopy (PAM), F-Mode showed that it could provide a dynamic way to selectively enhance the visualization of organelles in single cells and vessels of different sizes in small, in vivo animal models. In the future, the team plans to apply F-Mode analysis to PA datasets acquired with functional PA to further enhance the selectivity of visualizing arteries and veins in situ. It also plans to apply F-Mode analysis to data sets acquired with ultrahigh-frequency ultrasound techniques, such as those used for single-cell imaging, to provide a dynamic source of contrast for samples with little deviation in local mechanical properties. Dr. Michael Kolios (left) and Dr. Michael Moore (right). Courtesy of Ryerson University. The team is now taking steps toward translating F-Mode into clinical applications, where it could be of widespread benefit. For example, the ability to segment and enhance features of different scales has potential in areas such as ophthalmology, neurosurgery, and the detection of various conditions such as hypertension. In photoacoustic imaging, lightwaves are projected into biological structures, which heat up slightly when they absorb the light. This triggers a tiny expansion in the volume of the structure, which in turn generates sound. The research was published in Communications Physics (https://doi.org/10.1038/s42005-019-0131-y).