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 decade1. 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.
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This work was supported principally by the Agence Nationale de la Recherche (ANR), within the project ANR MUSLI. We thank the Fondation Pierre-Gilles de Gennes for funding C.E. The laboratory was also supported by LABEX WIFI (Laboratory of Excellence ANR-10-LABX-24) within the French Program “Investments for the Future” under reference ANR-10-IDEX-0001-02 PSL*.
M.T. is a co-founder, shareholder and scientific advisor of Supersonic Imagine. All other authors declare no competing financial interests.
Extended data figures and tables
a, Stack of B-mode images. The region of interest corresponds to a region of 2 mm × 1.1 mm within the cortex. b, Spatiotemporal filtering of the B-mode images shows the presence of decorrelating microbubbles in each frame (1–4). c, The four representative frames are separated by 44 ms (1–4). d, Computed two-dimensional PSF of the rescaled and filtered ultrafast acquisitions. These echoes are then interpolated and the Cartesian coordinates of their centre is obtained (1–4). The summit of each two-dimensional Gaussian profile identifies the centroid of each separable source.
Extended Data Figure 2 uULM coronal scan (anterior–posterior) of the entire rat brain through a thinned skull window.
a–i, The ultrasound probe was driven by a micro-step motor to perform uULM on different imaging planes separated by 500 μm. We reconstructed the vascularization of the rat brain at the following coordinates: Bregma −0.5 mm (a), −1 mm (b), −1.5 mm (c), −2 mm (d), −2.5 mm (e), −3 mm (f), −3.5 mm (g), −4 mm (h), −4.5 mm (i).
Extended Data Figure 3 Anterior–posterior scan of in-plane velocity maps of the rat forebrain through a thinned skull window.
a–i, Velocity maps for the different coronal planes presented in Extended Data Fig. 2.
Individual microbubble tracks within the cortex. (AVI 5333 kb)
Ultrafast ultrasound localization microscopy over multiple coronal plane of the cortex. (AVI 3137 kb)
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Errico, C., Pierre, J., Pezet, S. et al. Ultrafast ultrasound localization microscopy for deep super-resolution vascular imaging. Nature 527, 499–502 (2015). https://doi.org/10.1038/nature16066
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