TY - JOUR T1 - Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy JF - bioRxiv DO - 10.1101/2021.12.22.473713 SP - 2021.12.22.473713 AU - Lixue Shi AU - Aleksandra Klimas AU - Brendan Gallagher AU - Zhangyu Cheng AU - Feifei Fu AU - Piyumi Wijesekara AU - Yupeng Miao AU - Xi Ren AU - Yongxin Zhao AU - Wei Min Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/12/23/2021.12.22.473713.abstract N2 - Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously due to spectral overlap. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, we describe a new technique called Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS), that integrates SRS microscopy with expansion microscopy (ExM). ExM is a powerful strategy providing significant improvement in imaging resolution by physical magnification of hydrogel-embedded preserved biological specimens. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. We used MAGNIFIERS to visualize nanoscale features in a label-free manner with C-H vibration of proteins, lipids and DNA in a broad range of biological specimens, from mouse brain, liver and kidney to human lung organoid. In addition, we applied MAGNIFIERS to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, we used MAGNIFIERS to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.Competing Interest StatementThe authors declare the following competing financial interest(s): YZ, AK and FF are inventors on several inventions related to ExM methods. ER -