Cryo‐FIB Lift‐Out and Electron Tomography Workflow for Bacteria–Nanopillar Interface Imaging Under Native Conditions: Investigating Dragonfly Inspired Bactericidal Titanium Surfaces

Dragonfly- and cicada-wing-inspired titanium nanostructured surfaces exhibit promising bactericidal properties. However, direct visualization of the bacteria-material interface under hydrated conditions remains limited, restricting experimental interrogation of how bacteria interact with nanostructured surfaces. This limitation reflects a long-standing methodological gap, as visualization of bacteria-nanostructure interfaces has largely relied upon indirect or dehydrated imaging approaches. Cryo-electron tomography (cryo-ET) enables 3D visualization of cellular ultrastructure in a native hydrated environment, but imaging bacteria attached to metallic nanostructures by cryo-ET requires preparation of thin electron-transparent lamellae. Obtaining such lamellae from vitrified bacteria on titanium substrates is technically challenging because cryo-focused ion beam (cryo-FIB) milling must simultaneously section soft biological material and hard metal whilst bacteria embedded in vitreous ice are not directly visible. Here, we establish a correlative cryogenic workflow enabling targeted extraction and cryo-ET imaging of defined bacterium-nanopillar interfaces. Individual bacteria interacting with titanium nanopillars are identified by correlative cryo-fluorescence microscopy, followed by targeted cryo-FIB lift-out, transfer to a receiver grid, and thinning into electron-transparent lamellae. The data presented demonstrate that bacteria-nanostructure interfaces can be targeted, extracted, and structurally analysed in situ under fully hydrated conditions. This workflow provides a methodological framework for future cryo-ET studies of bacteria-nanotopography interactions.