Fabrication of focused electron beam induced deposition tips for high-speed atomic force microscopy using benchtop scanning electron microscopy.

High-speed atomic force microscopy (HS-AFM) movies have contributed significantly to our understanding of biomolecular dynamics, because these experiments have resolved conformational changes at submolecular resolution in aqueous environments. However, the resolution of HS-AFM images and AFM images in general critically depend on the sharpness of the tip. Focused electron beam-induced deposition (FEBID) in a scanning electron microscope (SEM) yields AFM tips with high aspect ratio and small tip radius, formed by deposition of metal-organic precursors such as ferrocene, producing amorphous carbon deposits, permissive for the reproducible acquisition of high-resolution images. Although FEBID tips are essential for HS-AFM image quality, it is common to rely on commercial cantilevers, because of limited SEM access or perceived technical barriers. Here, we showcase the use of a commercial benchtop SEM for the fabrication of FEBID tips for high-resolution AFM. To facilitate the fabrication process, we developed a cantilever and sample holder that minimize drift and mechanical vibrations during SEM operation and enable efficient and reproducible fabrication of sharp AFM tips. The shape of the FEBID tips was evaluated as a function of beam defocus, deposition time, acceleration voltage and beam current. We describe an optimized workflow for FEBID tip fabrication (eight tips can be fabricated within 1-2 h) and demonstrate high-resolution and long-term stable HS-AFM imaging of annexin V and plasmid DNA using the FEBID tips fabricated in this study. We anticipate that this protocol may be useful for a wide range of AFM practitioners and facilitate the reproducible acquisition of high-resolution AFM data.