Atom Probe Tomography of Hydrated Biomacromolecules: Preliminary Results

The folding and structure of biomacromolecules depend on the three-dimensional distributions of their constituents, and this ultimately controls their functionalities and interactions with other biomacromolecules. Atom probe tomography (APT) with its unparalleled compositional sensitivity at nanoscale spatial resolution, could provide complementary information to cryo-electron microscopy, yet routine APT analysis of biomacromolecules in their native state remains challenging. Here, we used a solution containing ferritin as a model biomacromolecule, and following plunge freezing in liquid nitrogen (LN2), performed cryogenic specimen preparation via lift-out, and APT analysis. We measure elements from the ferritin core and shell, yet the proteins themselves appear to have been destroyed. We hence demonstrate the feasibility of preparing and analyzing bulk hydrated biological samples using APT; however, plunge freezing in LN2 is too slow to vitrify the solution into its vitreous state. This caused irrecoverable damage to the protein shell surrounding the ferritin particles due to the ice crystal formation. We report on preliminary data from high-pressure frozen deionized water, demonstrating a proof-of-principle experiments that intact biomacromolecules could be analyzed through a similar workflow in the future. We report on many trials (and errors) on the use of different materials for substrates and different substrate geometries, and provide a perspective on the challenges we faced to facilitate future studies across the community.