A team from Arizona State University (ASU) has introduced evanescent scattering microscopy (ESM), a label-free method for sensitive imaging of biomolecules, including proteins. The single-molecule microscopy technique is based on total internal reflection (TIR), an optical phenomenon that occurs when light passes from a high-refractive medium, like glass, into a low-refractive medium, like water. ESM could allow researchers to visualize proteins and other biomolecules with more clarity and by simpler means than existing microscopy methods. TIR occurs when waves arriving at the interface between one medium and another (e.g., at the interface between glass and water) are not refracted into the second medium (e.g., water), but completely reflected back into the first medium (e.g., glass). The second medium has a lower refractive index than the first, and the waves are incident at a sufficiently oblique angle on the interface. In development, ESM caused a beam of laser light to be directed at a molecular sample at the angle necessary to produce TIR. This created an evanescent field that excited molecules at the glass-liquid interface when the molecules were affixed to a cover glass. Using ESM, the scientists imaged the interference between the evanescent light scattered by the single molecules and by the natural roughness of the cover glass, to produce images of razor-sharp contrast on a plain-glass surface. The experimental setup for ESM. Courtesy of the Biodesign Institute at Arizona State University. The sharpness of the images allowed the researchers to quantify the sizes of single proteins, characterize protein-antibody interactions at the single-molecule level, and analyze the heterogeneity of single-protein binding behaviors. Due to the exponential distribution of evanescent field intensity, ESM also allowed the scientists to track the analyte axial movement with high resolution. The results could be used to analyze DNA conformation changes, providing a way to detect small molecules, such as microRNA. ESM’s use of evanescent wave scattering allows samples, including proteins, to be probed at an extremely shallow depth — typically less than 100 μm. This capability makes it possible to use ESM to create an optical slice, with dimensions comparable to a thin electron microscopy section. Most existing imaging methods that use evanescence require the molecules under investigation to be labeled with fluorescent tags. The use of fluorophores requires significant sample preparation and can interfere with the researcher’s ability to observe molecular interactions. ESM, a label-free imaging method, requires no fluorescent dye or gold coating for sample slides. “The method we report in this study ... is compatible with fluorescence imaging for in situ cross validation, and it reduces the light-induced heating effect that could harm the biological samples,” professor Shaopeng Wang said. The experimental setup for performing ESM, a label-free method for sensitive imaging of biomolecules, including proteins. A beam of laser light is directed at a molecular sample with the proper angle to produce a condition known as total internal reflection. The resulting evanescent wave can excite the molecules at the glass-liquid interface, allowing for exceptionally precise imaging. Courtesy of the Biodesign Institute at Arizona State University. The researchers used ESM to detect four model proteins. They observed protein-protein interactions, including the rapid binding and dissociation of individual proteins and the heterogeneity of single protein binding properties, in a series of experiments. According to the researchers, understanding binding kinetics such as the ones that they observed is essential in designing safer and more effective drugs. Small molecule detection is challenging for label-free optical measurement systems, which are usually considered suitable for detecting biological macromolecules only, the researchers added. They said that ESM could broaden the use of label-free optical microscopy by providing a straightforward method for detecting small biological molecules. The cover glass used for ESM has a lower heating effect, no plasmonic quenching effect, and good optical clearance so that ESM could be integrated easily with fluorescence imaging for multiplexed detection in future applications. ESM could additionally be used to analyze the behavior of single molecules and biological complexes, especially when combined with volumetric fluorescence imaging to understand cell activities systematically. The research was published in Nature Communications (www.doi.org/10.1038/s41467-022-30046-8).