High-throughput fabrication of geometrically complex nanoenvironments for single-molecule fluorescence microscopy

Nanoenvironments with complex geometries mimic biological interfaces such as the synovial joint, making them valuable tools for investigating single-molecule behavior in biomimetic systems. Current environments that replicate both the interfacial gap (0.01 μm) and roughness (10 μm characteristic wavelength) of the native synovial joint are typically fabricated using focused ion beam (FIB) milling. However, FIB requires serial fabrication of nanoenvironments, making it unfavorable for high-throughput fabrication. In this study, we fabricate multiple 3D nanoenvironments simultaneously by reactive ion etching (RIE) of optically transparent borosilicate glass substrates. Here, we use grayscale photolithography to define 3D photoresist patterns and RIE to transfer photoresist patterns to borosilicate substrates. We characterize etched surface roughnesses using atomic force microscopy and validate etched geometries using laser scanning confocal microscopy. We demonstrate that the range of nanofabricated feature depths and pitches is sufficient to create biomimetic synovial joint environments. These environments will be used to characterize the dynamic behavior of biopolymers using single-molecule fluorescence microscopy.