PNNL

The Science

DOE researchers have developed an infrared nano-imager that may be used to visualize and fingerprint biological molecules in their native liquid environments. The novel imager boasts nanometer spatial resolution, chemical selectivity, and ultrahigh sensitivity. The approach combines in-liquid atomic force microscopy with evanescent infrared illumination of the samples using a total internal reflection geometry. This geometry increases sensitivity through directional signal emission, while minimizing signal loss as a result of infrared light absorption. The initial study investigated the structures of solvated biomimetic peptoid sheets as well as proteins.

The Impact

Visualizing biological systems in their native environments and natural states is a grand challenge in biology. Although fluorescence and electron microscopy are the tools of choice in this context, they both suffer from inherent deficiencies, including the requirement of tagging biomolecules with fluorophores or easily induced damage in soft matter. Vibrational spectroscopic methods are non-destructive, but they have inherent sensitivity limits and limited spatial resolution in their standard implementations. In the case of infrared spectroscopy, strong absorption from the liquid is also restrictive. This work builds on early demonstrations of infrared nano-spectroscopy and extends the approach to enable such measurements in liquids. The new method of in situ infrared nano-spectroscopy minimizes background signals and enhances sensitivity via infrared antenna enhancement and directional signal emission. It could be used to study pH-induced protein folding or formation of a passivation layer on battery electrodes, as well as other types of biomolecular, cellular, catalytic, or electrochemical systems in liquid.

Summary

Researchers at the U.S. Department of Energy’s Pacific Northwest National Laboratory, led by Scott Lea, in collaboration with the Raschke group at CU-Boulder have developed a method of infrared scattering-type scanning near-field optical microscopy (IR s-SNOM) for nanoscale chemical and biological imaging directly in liquid. In this setup, mid-infrared light focused onto a sharp tip of an atomic force microscope (AFM) through a zinc selenide prism generates a light field concentrated above the prism around the region of the AFM tip and sample. The local IR properties of the sample are then broadcasted with nanoscale spatial resolution via enhanced directional signal emission.

Previous methods of using IR s­-SNOM to make nanoscale measurements in liquid used a membrane to separate the AFM tip and liquid. However, this barrier prevented direct access to the sample with the AFM tip, had limited applicability due to stringent sample requirements, and suffered from large background signals.Our approach is more broadly applicable to a wide range of sample systems and improves signal quality and sensitivity through directional signal emission and efficient background noise suppression.

The researchers used their setup to image nanocrystals of catalase in liquid, as well as to detect the structure and chemical composition of a peptoid monolayer with sensitivity down to a few zeptomoles. Because the AFM is fully immersed in liquid, this imaging approach could accommodate changes in pH, solvent composition, and temperature for studies of protein folding and ion transport.

Funding

This work was supported by the Department of Energy, Office of Science, Biological and Environmental Research Bioimaging Technology. The preparation of peptoid nanosheets was supported by the DOE Office of Basic Energy Sciences, and Biomolecular Materials Program at PNNL. This work was performed in the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science User Facility sponsored by BER and located at Pacific Northwest National Laboratory.