Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-L-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane

Influence of Fermi surface topology on the quasiparticle spectrum in the vortex state

We study the influence of Fermi surface topology on the quasiparticle density of states in the vortex state of type II superconductors. We observe that the field dependence and the shape of the momentum and spatially averaged density of states is affected significantly by the topology of the Fermi surface. We show that this behavior can be understood in terms of characteristic Fermi surface functions and that an important role is played by the number of points on the Fermi surface at which the Fermi velocity is directed parallel to the magnetic field. A critical comparison is made with a broadened BCS type density of states, that has been used frequently in analysis of tunneling data. We suggest a new formula as a replacement for the broadened BCS model for the special case of a cylindrical Fermi surface. We apply our results to the two gap superconductor MgB$_2$ and show that in this particular case the field dependence of the partial densities of states of the two gaps behaves very differently due to the different topologies of the corresponding Fermi surfaces, in qualitative agreement with recent tunneling experiments.Comment: 12 pages 12 figure

TopoStats – a program for automated tracing of biomolecules from AFM images

We present TopoStats, a Python toolkit for automated editing and analysis of Atomic Force Microscopy images. The program automates identification and tracing of individual molecules in circular and linear conformations without user input. TopoStats was able to identify and trace a range of molecules within AFM images, finding, on average, ~90% of all individual molecules and molecular assemblies within a wide field of view, and without the need for prior processing. DNA minicircles of varying size, DNA origami rings and pore forming proteins were identified and accurately traced with contour lengths of traces typically within 10 nm of the predicted contour length. TopoStats was also able to reliably identify and trace linear and enclosed circular molecules within a mixed population. The program is freely available via GitHub (https://github.com/afm-spm/TopoStats) and is intended to be modified and adapted for use if required

Tuneable poration : host defense peptides as sequence probes for antimicrobial mechanisms

The spread of antimicrobial resistance stimulates discovery strategies that place emphasis on mechanisms circumventing the drawbacks of traditional antibiotics and on agents that hit multiple targets. Host defense peptides (HDPs) are promising candidates in this regard. Here we demonstrate that a given HDP sequence intrinsically encodes for tuneable mechanisms of membrane disruption. Using an archetypal HDP (cecropin B) we show that subtle structural alterations convert antimicrobial mechanisms from native carpet-like scenarios to poration and non-porating membrane exfoliation. Such distinct mechanisms, studied using low- and high-resolution spectroscopy, nanoscale imaging and molecular dynamics simulations, all maintain strong antimicrobial effects, albeit with diminished activity against pathogens resistant to HDPs. The strategy offers an effective search paradigm for the sequence probing of discrete antimicrobial mechanisms within a single HDP

Charge transfer and doping-dependent hybridization of C-60 on noble metals

K-doped C-60 monolayers on polycrystalline Cu, Ag, and Au are studied by means of ultraviolet photoelectron spectroscopy. The polycrystalline character of the substrate allows an accurate subtraction procedure to isolate the photoemission signal of the monolayer. Charge transfers of 1.0-1.8 electrons per molecule are found for the undoped monolayer. On high K doping the hybridization with the substrate is shown to practically disappear, and the overlayer almost totally suppresses the substrate signal. [S0163-1829(98)02120-1]

PEGylated surfaces for the study of DNA–protein interactions by atomic force microscopy

DNA–protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA–protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000–2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show – through the use of biotinylated DNA and streptavidin – that this selective adsorption extends to DNA–protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM

Cooperative amyloid fibre binding and disassembly by the Hsp70 disaggregase

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure