In the mammalian nervous system, myelin provides electrical insulation for the neural circuit by forming a highly organized, multilayered thin film around the axon fibers. Here, we investigate the spectral reflectance from this subcellular nanostructure and devise a new label-free technique based on a spectroscopic analysis of reflected light, enabling nanoscale imaging of myelinated axons in their natural living state. Using this technique, we demonstrate three-dimensional mapping of the axon diameter and sensing of dynamic changes in the substructure of myelin at nanoscale. We further reveal the prevalence of axon bulging in the brain cortex in vivo after mild compressive trauma. Our novel tool opens new avenues of investigation by creating unprecedented access to the nanostructural dynamics of live myelinated axons in health and disease.

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Fluorescent optical probes have rapidly transformed our understanding of complex biological systems by providing specific information on biological targets in the natural living state. However, their utility is often limited by insufficient brightness, photostability, and multiplexing capacity. Here, we report a conceptually new optical probe, termed ‘reflectophore’, which is based on the spectral interference from a dielectric microsphere. Reflectophores are orders-of-magnitudes brighter than conventional fluorophores and are free from photobleaching, enabling practically unlimited readout at high fidelity. They also offer high-degree multiplexing, encoded in their optical size, which can be readily decoded through interferometric detection with nanoscale accuracy, even in turbid biological media. Furthermore, we showcase their biological applications in cellular barcoding and microenvironmental sensing of a target protein and local electric field.

Compensation of sample-induced optical aberrations is crucial for visualizing microscopic structures deep within biological tissues. However, strong multiple scattering poses a fundamental limitation for identifying and correcting the tissue-induced aberrations. Here, we introduce a label-free deep-tissue imaging technique termed dimensionality reduction adaptive-optical microscopy (DReAM) to selectively attenuate multiple scattering. We established a theoretical framework in which dimensionality reduction of a time-gated reflection matrix can attenuate uncorrelated multiple scattering while retaining a single-scattering signal with a strong wave correlation, irrespective of sample-induced aberrations. We performed mouse brain imaging in vivo through the intact skull with the probe beam at visible wavelengths. Despite the strong scattering and aberrations, DReAM offered a 17-fold enhancement of single scattering–to–multiple scattering ratio and provided high-contrast images of neural fibers in the brain cortex with the diffraction-limited spatial resolution of 412 nanometers and a 33-fold enhanced Strehl ratio.