Electron beam lithography on biological membrane enabled by ethanol ice resist

Cryogenic electron microscopy is revolutionizing structural biology, yielding increasingly high-resolution data from unwieldy proteinaceous complexes. Yet, despite its power and widespread use, the method is limited to imaging. For delicate biological specimens the surface patterning and deterministic functionalization inherent in electron beam lithography is usually out of reach due to beam-induced damage. Ice Lithography (IL) has the potential to bridge cryogenic electron microscopy and electron beam lithography and achieve direct patterning of fragile, chemically sensitive and biologically active materials. In IL the specimen is cooled inside an electron microscope. Subsequent gas injection leads to a conformal coating of ice (i.e., solid phase condensed gas) over the specimen. This ice acts as a resist layer for lithographic processing. Ices can be stable over long timescales, and the sign of the resist can vary with chosen condensed gas species. In this work we introduce ethanol ice as a negative resist for IL that minimizes specimen damage incurred via electron irradiation. Purple membrane patches from Halobacterium salinarum are employed as test targets. Electron beam exposure at 125 K creates an ethanol-based material that is stable in ambient conditions and does not significantly disturb the ~5 nm thick architecture of purple membrane. An overview of our current understanding of the IL process as well as the materials properties of the ethanol-based material will be provided. Some future directions will also be discussed.