Development of High-Speed Atomic Force Microscope-Infrared Microscopy in the Aqueous Phase

About This Grant

This project proposes the development of a high-speed atomic force microscopy-infrared (HS-AFM-IR) instrument that will enable real-time, label-free imaging of the dynamics of biomacromolecules with high spatial resolution in the aqueous phase. Current imaging technologies such as cryogenic electron microscopy (cryo-EM), fluorescence microscopy, and atomic force microscopy (AFM) each offer distinct advantages but also corresponding limitations. For example, cryo-EM provides high-resolution static images but requires flash- frozen samples, limiting its use in observing dynamic molecular processes in real-time. Fluorescence microscopy can track dynamic events but relies on extrinsic labels, while conventional AFM primarily reveals surface topography and lacks chemical sensitivity. These gaps hinder studying certain biomacromolecule interactions, such as protein folding, enzyme catalysis, and drug-target binding, under physiologically relevant conditions. The proposed HS-AFM-IR system integrates the strengths of AFM and infrared (IR) imaging and spectroscopy to overcome these limitations. This novel instrument will achieve ~5 nm spatial resolution and real-time dual- composition chemical imaging capability in fluid environments by combining high-speed AFM with photothermal action-based AFM-IR technique. The system’s unique ability to capture dynamic biomacromolecules in real-time, without the need for extrinsic labels, will make it a necessary tool for studying biomolecular processes inaccessible by current microscopy tools. It will allow researchers to track the co-localization of multiple biomacromolecules and observe real-time antibody-antigen kinetics at the molecular level. The system will also provide insights into how external factors such as small drug molecules, ions, or environmental changes affect biomolecular conformations and interactions. In our project, we will demonstrate the time-resolved label-free IR imaging ability of HS-AFM-IR on standard system of streptavidin–biotin system of direct binding and G protein–coupled receptor system of competitive binding. This technology has the potential to advance the foundation of biomedical research, particularly in fields related to drug discovery, protein chemistry, and molecular biophysics. By enabling the direct observation of biomolecular dynamics in their native environments, the HS-AFM-IR system could help accelerate the development of new therapies for diseases related to protein misfolding and aggregation, such as amyloid disease, and improve the understanding of molecular mechanisms. The outcome of this project will be a widely applicable tool that fills a gap in the current microscopy techniques, advancing our ability to study biomacromolecules’ interactions and dynamics in situ and in real-time.