Mapping the Path to Cryogenic Atom Probe Tomography Analysis of Biomolecules

The understanding of protein structure and interactions remains a fundamental challenge in modern biology. While X-ray and electron-based techniques have provided atomic-level protein configurations, they require numerous molecules for averaged views and lack detailed compositional information crucial for biochemical activity. Atom probe tomography (APT) emerges as a promising tool for biological material analysis, though its capabilities for examining biomolecules in their native, hydrated state remain largely unexplored. We present systematic analyses of amino acids in frozen aqueous solutions using two different nanoporous metal supports across various analysis conditions. Our methodology employs a complete cryogenic workflow, including drop-casting, inert gas glovebox freezing, and specimen transfer via a cryogenically cooled ultra-high vacuum shuttle to both focused ion beam microscopy and atom probes. Using water molecular ion ratios as electrostatic field condition indicators, we investigate amino acid fragmentation and behavior. We evaluate the critical factors for successful biomolecular analysis: support material selection, cryogenic specimen preparation, and optimal data acquisition parameters. This work establishes guidelines for cryogenic APT analysis of biomolecules, advancing the technique's application in biological sciences.