Near-Atomic Three-Dimensional Mapping for Site-Specific Chemistry of ‘Superbugs’

Abstract

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.

Graphene‐Enhanced 3D Chemical Mapping of Biological Specimens at Near‐Atomic Resolution

“…Knowledge acquired by various high‐resolution imaging techniques including (cryo‐)electron microscopy, confocal imaging, and secondary ion mass spectrometry (SIMS) has contributed to a fundamental understanding of cell biology as well as the engineered development of biomimetic materials with new physical and chemical properties . 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 and bacterial cells …”

“…Organic materials, however, are more sensitive to the application of a laser due to the possibility of thermally induced phase transition of the constituent molecules during the field evaporation process . In previous studies, it has been demonstrated that a thin layer of conductive metal coated on an APT specimen can help fulfil the required voltage distribution for field evaporation of nonconductive materials . This physical vapor deposition (PVD) based approach, however, also demonstrated limitations to achieve ultrathin coating of high‐aspect ratio geometries, i.e., tip specimens for APT .…”

“…In previous studies, it has been demonstrated that a thin layer of conductive metal coated on an APT specimen can help fulfil the required voltage distribution for field evaporation of nonconductive materials . This physical vapor deposition (PVD) based approach, however, also demonstrated limitations to achieve ultrathin coating of high‐aspect ratio geometries, i.e., tip specimens for APT . Figure S1a,b (Section S1, Supporting Information) presents an example of APT specimen prepared from resin‐embedded bacterial cells with the previously devised PVD protocol.…”

Atom Probe Tomography for 3D Structural and Chemical Analysis of Individual Proteins

“…Specimen fracture and field-induced deformation has therefore, to date, largely precluded routine atom probe analysis of soft materials. [15] Notable exceptions include studies of ferritin clusters [16] and molecules, [17,18] mammalian and bacterial cells, [19,20] and organic fibers in the chiton tooth. [21] In the present work, we used a sol-gel method to embed rabbit immunoglobulin G (IgG) [22] in a solid silica matrix.…”

An atomic-scale view at the composition of amyloid-beta fibrils by atom probe tomography

“…Previous studies have demonstrated near-atomic resolution of chemically-resolved data of bulk bio-mineralized hard biological matter such as teeth and bone [45][46][47][48][49][50][51] . Progress was also made on more soft materials such as cells 52,53 and also with ferritin as example for proteins [54][55][56] . In early designs of the atom probe microscope, single amino acids, nucleic acids and other polymers were successfully analyzed [57][58][59][60] .…”