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.

G-Quadruplex (G4) is a noncanonical nucleic acid secondary structure with multiple biofunctions. Identifying G4-related proteins (G4RPs) is important for understanding the roles of G4 in biology. Current methods to identify G4RPs include discovery from specific biological processes or in vitro pull-down assays with specific G4 sequences. Here, we report an in vivo strategy used to identify G4RPs with extensive sequence tolerance based on G4 ligand-mediated cross-linking. Applying this method, we identified 114 and 281 G4RPs in SV589 and MM231 cells, respectively. The results successfully overlapped with all the pull-down assay literature. Through the electrophoretic mobility shift assay (EMSA), we identified some new G4binding proteins. Moreover, enhanced cross-linking and immunoprecipitation (eCLIP) confirmed that one newly identified G4-binding protein, SERBP1, interacts with G4 in the cellular environment. The method we developed provides a new strategy for identifying proteins that interact with nucleic secondary structures in cells and benefit the study of their biological roles.