Emergence of multi-drug resistant Gram-negative bacteria has caused a global health crisis and last-line class of antibiotics such as polymyxins are increasingly used. The chemical composition at the cell surface plays a key role in antibiotic resistance. Unlike imaging the cellular ultrastructure with well-developed electron microscopy, acquisition of a high-resolution chemical map of the bacterial surface still remains a technological challenge. In this study, we developed an atom probe tomography (APT) analysis approach to acquire mass spectra in the pulsed-voltage mode and reconstructed the 3D chemical distribution of atoms and molecules in the subcellular domain at the near-atomic scale. Using focused ion beam (FIB) milling together with micromanipulation, site-specific samples were retrieved from a single cell of Acinetobacter baumannii prepared as needle-shaped tips with end radii less than 60 nm, followed by a nano-scale coating of silver in the order of 10 nm. The significantly elevated conductivity provided by the metallic coating enabled successful and routine field evaporation of the biological material, with all the benefits of pulsed-voltage APT. In parallel with conventional cryo-TEM imaging, our novel approach was applied to investigate polymyxin-susceptible and -resistant strains of A. baumannii after treatment of polymyxin B. Acquired atom probe mass spectra from the cell envelope revealed characteristic fragments of phosphocholine from the polymyxin-susceptible strain, but limited signals from this molecule were detected in the polymyxin-resistant strain. This study promises unprecedented capacity for 3D nanoscale imaging and chemical mapping of bacterial cells at the ultimate 3D spatial resolution using APT.
The direct imaging of individual atoms within the cellular context holds great potential for understanding the fundamental physical and chemical processes in organisms. Here, a novel approach for imaging of electrically insulated biological cells by introducing a graphene encapsulation approach to "disguise" the low-conductivity barrier is reported. Upon successful coating using a water-membrane-based protocol, the electrical properties of the graphene enable voltage pulsing field evaporation for atom probe tomography (APT). Low conductive specimens prepared from both Au nanoparticles and antibiotic-resistant bacterial cells have been tested. For the first time, a significant graphene-enhanced APT mass resolving power is also observed confirming the improved compositional accuracy of the 3D data. The introduction of 2D materials encapsulation lays the foundation for a breakthrough direction in specimen preparation from nanomembrane and nanoscale biological architectures for subsequent 3D near-atomic characterization.physical and chemical properties. [1][2][3][4][5][6][7][8] Atom probe tomography (APT), the only technique offering 3D chemical measurements with near atomic resolution, has recently demonstrated its unique capability as a biological imaging tool to mammalian [9] and bacterial cells. [10] However, the broader application of APT to the field is largely limited by the challenges in the sample preparation and the nonconductivity of biological specimens. A requirement of APT experiments is that the specimen material must be shaped into a sharp needle, typically with a tip radius less than 75 nm. By applying a positive voltage to the specimen under ultrahigh vacuum and cryogenic conditions, this tip geometry allows the generation of the sufficient electric field intensity to cause field ionization and evaporation of surface atoms. Controlled voltage pulsing allows the evaporation of individual and molecular ions that eventually reach a position sensitive detector. The impact positions and sequence of hits are employed to reconstruct their original position within the specimen and the time of flight is utilized to determine the atomic species. [11,12] In order to maintain a field density Cellular ImagingThe ORCID identification number(s) for the author(s) of this article can be found under https://doi.
Goethite (α-FeOOH) is dispersed throughout the earth's surface, and its propensity to recrystallize in aqueous solutions determines whether this mineral is a source or sink for critical trace elements in the environment. Under reducing conditions, goethite commonly coexists with aqueous Fe(II) (Fe(II) aq ), which accelerates recrystallization by coupled electron transfer and atom exchange. Quantifying the amount of the mineral phase that exchanges its structural Fe(III) atoms with Fe(II) aq is complicated by recrystallization models with untested assumptions of whether, and to what extent, the recrystallized portion of the mineral continues to interact with the solution. Here, we reacted nanoparticulate goethite with 57 Fe-enriched Fe(II) aq and used atom probe tomography (APT) to resolve the threedimensional distribution of Fe isotopes in goethite at the sub nm scale. We found that the 57 Fe tracer isotope is enriched in the bulk structure (tens of nanometers deep), with some samples having 57 Fe penetration throughout at a level that is similar to the isotopic composition of Fe(II) aq . This suggests that some particles undergo near-complete recrystallization. In other cases, however, the distribution of 57 Fe is more heterogeneous and generally concentrates near the particle periphery. Nanoparticle encapsulation and subsequent APT can hence capture hidden recrystallization mechanisms which are critical to predicting mineral reactivity in aqueous solutions.
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