High-speed atomic force-Raman microscopy

Abstract

A comprehensive understanding of the mechanisms underlying biological systems requires correlative analysis of multiple complementary molecular properties through multidimensional measurements. High-speed atomic force microscopy (HS-AFM) is a powerful tool for elucidating biomolecular structural dynamics at the single-molecule level with high spatiotemporal resolution. However, structural information alone is often insufficient for fully understanding the biological function mechanisms. Here, we report high-speed atomic force-Raman microscopy (HS-AFRM), which enables multimodal measurements combining video-rate structural imaging with chemical-bond analysis. Raman spectroscopy is a powerful, non-invasive technique that probes molecular vibrations to provide chemical information. We achieved several key technical developments that facilitated the seamless integration of HS-AFM and micro-Raman spectroscopy, allowing reliable correlative measurements of structural and chemical information. We validated the versatility of the developed system using representative samples, including two-dimensional materials and a protein. Furthermore, we demonstrate probing of changes in the surrounding environment, which are inaccessible by HS-AFM alone. Multimodal measurements incorporating fluorescence spectroscopy were also demonstrated as an additional practical extension. This multimodal approach substantially enhances the analytical capability of HS-AFM, providing a powerful platform for revealing correlated structural and chemical properties across diverse research fields.