Live-cell Raman imaging based on bioorthogonal Raman probes with distinct signals in the cellular Raman-silent region (1800–2800 cm⁻¹) has attracted great interest in recent years. We report here a class of water-soluble and biocompatible polydiacetylenes with intrinsic ultrastrong alkyne Raman signals that locate in this region for organelle-targeting live-cell Raman imaging. Using a host-guest topochemical polymerization strategy, we have synthesized a water-soluble and functionalizable master polydiacetylene, namely poly(deca-4,6-diynedioic acid) (PDDA), which possesses significantly enhanced (up to ~10⁴ fold) alkyne vibration compared to conventional alkyne Raman probes. In addition, PDDA can be used as a general platform for multi-functional ultrastrong Raman probes. We achieve high quality live-cell stimulated Raman scattering imaging on the basis of modified PDDA. The polydiacetylene-based Raman probes represent ultrastrong intrinsic Raman imaging agents in the Raman-silent region (without any Raman enhancer), and the flexible functionalization of this material holds great promise for its potential diverse applications.

High-resolution optical microscopes that can break 180 nm in spatial resolution set to conventional microscopies are much-needed tools. However, current optical microscopes have to rely on exogenous fluorescent labels to achieve high resolution in biological imaging. Herein, we report near-resonance enhanced label-free stimulated Raman scattering (SRS) microscopy with a lateral resolution near 130 nm, in which the high-resolution image contrast originates directly from a low concentration of endogenous biomolecules, with sensitivity gains of approximately 23 times. Moreover, by using a 0.3-m-long optical fiber, we developed hyperspectral SRS microscopy based on spectral focusing technology. Attributed to enhancements in spatial resolution and sensitivity, we demonstrated high-resolution imaging of three-dimensional structures in single cells and high-resolution mapping of large-scale intact mouse brain tissues in situ. By using enhanced high-resolution hyperspectral SRS, we chemically observed sphingomyelin distributed in the myelin sheath that insulates single axons. Our concept opens the door to biomedical imaging with ~130 nm resolution.