Non-invasive imaging deep into organs at microscopic scales remains an open quest in biomedical imaging. Although optical microscopy is still limited to surface imaging owing to optical wave diffusion and fast decorrelation in tissue, revolutionary approaches such as fluorescence photo-activated localization microscopy led to a striking increase in resolution by more than an order of magnitude in the last decade. In contrast with optics, ultrasonic waves propagate deep into organs without losing their coherence and are much less affected by in vivo decorrelation processes. However, their resolution is impeded by the fundamental limits of diffraction, which impose a long-standing trade-off between resolution and penetration. This limits clinical and preclinical ultrasound imaging to a sub-millimetre scale. Here we demonstrate in vivo that ultrasound imaging at ultrafast frame rates (more than 500 frames per second) provides an analogue to optical localization microscopy by capturing the transient signal decorrelation of contrast agents--inert gas microbubbles. Ultrafast ultrasound localization microscopy allowed both non-invasive sub-wavelength structural imaging and haemodynamic quantification of rodent cerebral microvessels (less than ten micrometres in diameter) more than ten millimetres below the tissue surface, leading to transcranial whole-brain imaging within short acquisition times (tens of seconds). After intravenous injection, single echoes from individual microbubbles were detected through ultrafast imaging. Their localization, not limited by diffraction, was accumulated over 75,000 images, yielding 1,000,000 events per coronal plane and statistically independent pixels of ten micrometres in size. Precise temporal tracking of microbubble positions allowed us to extract accurately in-plane velocities of the blood flow with a large dynamic range (from one millimetre per second to several centimetres per second). These results pave the way for deep non-invasive microscopy in animals and humans using ultrasound. We anticipate that ultrafast ultrasound localization microscopy may become an invaluable tool for the fundamental understanding and diagnostics of various disease processes that modify the microvascular blood flow, such as cancer, stroke and arteriosclerosis.

The majority of exchanges of oxygen and nutrients are performed around vessels smaller than 100 mm, allowing cells to thrive everywhere in the body. Pathologies such as cancer, diabetes and arteriosclerosis can profoundly alter the microvasculature. Unfortunately, medical imaging modalities only provide indirect observation at this scale. Inspired by optical microscopy, ultrasound localization microscopy has bypassed the classic compromise between penetration and resolution in ultrasonic imaging. By localization of individual injected microbubbles and tracking of their displacement with a subwavelength resolution, vascular and velocity maps can be produced at the scale of the micrometer. Super-resolution ultrasound has also been performed through signal fluctuations with the same type of contrast agents, or through switching on and off nano-sized phase-change contrast agents. These techniques are now being applied pre-clinically and clinically for imaging of the microvasculature of the brain, kidney, skin, tumors and lymph nodes.

Medical ultrasound is a widely used diagnostic imaging technique for tissues and blood vessels. However, its spatial resolution is limited to a sub-millimeter scale. Ultrasound Localization Microscopy was recently introduced to overcome this limit and relies on subwavelength localization and tracking of microbubbles injected in the blood circulation. Yet, as microbubbles follow blood flow, long acquisition time are required to detect them in the smallest vessels, leading to long reconstruction of the microvasculature. The objective of this work is to understand how blood flow limits acquisition time. We studied the reconstruction of a coronal slice of a rat’s brain during a continuous microbubble injection close to clinical concentrations. After acquiring 192000 frames over 4 minutes, we find that the biggest vessels can be reconstructed in seconds but that it would take tens of minutes to map the entire capillary network. Moreover, the appropriate characterization of flow profiles based on microbubble velocity within vessels is bound by even more stringent temporal limitations. As we use simple blood flow models to characterize its impact on reconstruction time, we foresee that these results and methods can be adapted to determine adequate microbubble injections and acquisition times in clinical and preclinical practice.