Localization-based techniques for super-resolution imaging of vascular dynamics

The mammalian vascular system is a marvel of biological engineering, comprising an extensive network of vessels that sustains every organ and tissue in the body. Within this network, microvessels smaller than 100 μm—including capillaries, arterioles, and venules—are the principal sites of exchange for oxygen, nutrients, and waste. Yet, their minute scale and structural complexity have long impeded detailed observation, leaving the role of microcirculation unresolved in key pathological processes underlying diseases such as cancer, neurovascular disorders, diabetic microangiopathy, and chronic inflammation.

For decades, our ability to visualize microvascular structures and functions has remained a formidable challenge. Conventional angiographic techniques, including X-ray angiography, Doppler ultrasound, magnetic resonance imaging, fluorescence microscopy, optical coherence angiography, optoacoustic microscopy and tomography, and intravascular methods, provide access to vascular anatomy and dynamics, but their capabilities remain constrained by tissue motion and resolution-depth trade-offs.

Recently, a new toolset of localization-based microangiography methods leveraging fluorescence, ultrasound, and hybrid optoacoustic contrast has emerged, breaking long-standing resolution-depth barriers and offering unprecedented noninvasive quantification of vascular anatomy and blood flow down to the capillary scale. These approaches are inspired by single-molecule localization microscopy (SMLM), a Nobel Prize-awarded method that surpasses the optical diffraction limit (∼200–300 nm). In contrast to SMLM, which relies on stochastically photoactivated yet static fluorescent molecules in superficial or transparent samples, localization-based microangiography exploits flowing exogenous particles or cells in vessels as sparse emitters. Precise localization overcomes intrinsic diffraction- or diffusion-related resolution limits in deep, living tissues while enabling simultaneous extraction of blood flow velocity and direction via particle tracking. By harnessing different contrast mechanisms (fluorescence, acoustic scattering, and optical absorption), these methods feature complementary advantages in resolution, depth, and application domains (Figure 1B). Here, we outline how these emerging methods uncover fundamental mechanisms underlying microvascular dysfunction and lay the groundwork for next-generation diagnostic tools.