Focused ion beam fabrication of solidified ferritin into nanoscale volumes for compositional analysis using atom probe tomography

Abstract

SummaryThe purpose of this investigation was to conduct atom probe tomography (APT) analyses on ferritin specimens prepared with focused ion beam (FIB) to assess whether this approach can be used to effectively characterize biomaterials. Soft matter is particularly sensitive to ion beam exposure which can induce physical and chemical changes. We employ protective metal films and low-energy ion fluence to mitigate potential problems that may be introduced by FIB. This study had two major objectives: (1) to qualitatively assess the viability of the specimens when subjected to the unique physical conditions of APT analysis, namely ultrahigh vacuum, high electric field, and thermal pulsing using a laser and (2) to quantitatively assess the data from such specimens under various experimental parameters and compare the results with appropriate control specimens. For the first objective, a range of experimental parameters were determined that met the basic criteria necessary to validate that ferritin-based specimens prepared with FIB can retain structural integrity during APT analysis. Initial field evaporation time-of-flight mass spectrometry (TOF-MS) data show that the specimens fabricated with FIB are capable of emitting ions under various laser pulsing conditions with a high electric field applied. For the second objective, the experimental parameter space was narrowed to a range that yielded data quality sufficient to produce meaningful comparison between the ferritin-based specimens and the salt-only controls.

Atom Probe Tomographic Mapping Directly Reveals the Atomic Distribution of Phosphorus in Resin Embedded Ferritin

“…From this information, a unique 3-dimensional reconstruction can be formed revealing the atomic scale composition along with impurity distributions up to a part-per-million sensitivity and subnanometer spatial resolution 6 7 . APT continues to make major contributions in the materials science of structural 8 9 and electronic materials 8 10 , and recently has been demonstrated in geological 11 , bio-mineralogical 12 13 14 , soft organic 15 16 17 and bio-organic 4 18 19 materials. However the regular application of APT to soft biological materials is lacking in large part due to difficulties in specimen preparation.…”

“…The ferritin protein molecule has been the subject of previous Field Ion Microscopy (FIM) based analyses 20 21 and more recent APT-based analyses 18 19 because the iron-rich ferrihydrite core (hydrated iron (III) oxide) can provide an iron signature in the mass spectrum, the detection of which would confirm field evaporation of the protein. Additionally, through an accurate atom-by-atom reconstruction, the mineral core can also serve as a fiducial marker from which the relative composition of the organic protein shell can be tomographically mapped.…”

Cryo-focused ion beam preparation of perovskite based solar cells for atom probe tomography

“…In general, FIB milling with high energy ions (normally Ga) without the application of necessary safeguards induces overall degradation of organic materials from their pristine state [11,[16][17][18][19]. For this reason, one of the approaches used to considerably mitigate damage during FIB preparation is milling under cryogenic conditions, normally below 150 K. It is well established that cryo-FIB preparation modifies the ion sputtering mechanism on the specimen by limiting sample heating during the sputtering process [12,20].…”

Towards Atom Probe Tomography of Hybrid Organic-Inorganic Nanoparticles

“…Greene et al recently used APT to analyse ferritin as a model for characterizing biological materials using APT. In this work, researchers used a solidified organic/salt matrix, which contained numerous ferritin complexes [6]. These APT tips of dense ferritin/salt deposits were able to undergo APT, providing a proof of concept through mass spectrometry results.…”

“…These APT tips of dense ferritin/salt deposits were able to undergo APT, providing a proof of concept through mass spectrometry results. However, they were unable to characterize individual proteins and their experiments suffered from drastic tip bending, preventing meaningful reconstructions [6]. To image individual ferritin protein complexes, we deposited a dilute solution of native equine spleen ferritin onto a silicon post coated with Au-Pd (Figure 1) [7].…”