Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope
Introduction
Since the first images of dried bacterial cells were obtained with the Atomic Force Microscope (AFM) [1], this technique has significantly contributed to the understanding of the nanoscale structural and physical properties of single bacterial cells [2], [3], [4], [5], [6]. Examples include the high resolution imaging of the dynamics of bacterial membrane proteins [7], [8], the molecular recognition of cellular membrane proteins [9], [10], the visualization of the effects of antibiotics on the cell surface [11], [12], and imaging of the extrusion of bacteriophages [13]. In this way, the AFM has decisively contributed to the emerging field of Nanomicrobiology [5].
Imaging living bacterial cells with the Atomic Force Microscope still poses a major challenge. This limitation arises from the relatively reduced adsorption forces of most living bacteria to the standard substrates used for AFM (such as glass or mica). In contraposition, the non-living bacterial cells (i.e dried bacteria) show stronger adhesion forces, making imaging easier and extensively used [14], [15].
Two different approaches have been reported to overcome the difficulty of imaging living bacteria. The first approach relies on increasing the strength of the forces that immobilize the bacteria to the substrates. The second approach is focused to reduce the shear forces exerted by the AFM tip on the bacteria and which are responsible for cell detachment during imaging. Among the first approach, we can find the physical entrapment of bacterial cells into polycarbonate filters [8], [16] or microwells [17], or the use of specific substrate coatings (such as APTES [11], PEI [18], poly-L-Lysine [19], [20], polyphenolic proteins [21] or gelatine [21], [22], [23]) or surface chemical binding groups (e.g. cross-linking of NH2 groups via glutaraldehyde [24]). Concerning AFM imaging modes, conventional modes such as contact mode or dynamic mode can only be used when bacteria are relatively strongly attached to the substrates [25]. For weakly attached bacteria (for most coated planar substrates) the use of the intermittent contact mode with magnetically excited probes seems to offer the best performance [17], [19], [22]. This has been attributed to the fine tuning of the dynamic oscillation in liquid conditions.
Despite these developments, relatively little progress has been made in the nanoscale imaging of living bacterial processes, such as bacterial growth and division [16], [17], specially for bacterial cells on planar substrates [19], [26]. The use of planar substrates provides a more natural condition to study these bacterial processes. They offer a less constrained space (compared to physical entrapment methods) for bacterial growth and division, together with weak electrostatic adsorption forces. In this way, it mimics the bacterial natural way of adhesion onto several types of substrates, including those present in biofilm formation on natural and synthetic surfaces [27], [28]. In this paper, we present the use of an alternative AFM imaging mode to study living bacterial cells, the so called dynamic jumping mode. With this method, we have been able to image living bacterial cells weakly absorbed onto planar substrates, following its growth and division. When using dynamic jumping mode, the probe is oscillated at its resonance frequency and approached to the sample until a prefixed oscillation amplitude set point is reached. At this point, the probe is retracted a given distance and laterally displaced out of contact from the sample until the next point. This out of contact lateral displacement, together with the use of the intermittent contact mode and of soft probes, drastically reduces the shear forces exerted onto the weakly absorbed bacterial cells. It should be noted that dynamic jumping mode offers a better performance than its static version [29], which has already been widely used in the imaging of viruses on planar substrates in physiological conditions [30], [31].
With the use of the dynamic jumping mode we have been able to image living single bacterial cells belonging to two different Escherichia coli strains, the MG1655 and the enteroaggregative (EAEC) 042, both being weakly adsorbed onto planar gelatine coated substrates. In addition, we have been able to monitor the growth and division of E. coli 042 in its native state over long periods of time.
Section snippets
Cell types and cultures
E. coli strain MG1655 is well known to be the common non-pathogenic laboratory E. coli strain for biological research [32], while strain 042 is the archetype of the EAEC pathotype [33], [34], [35]. EAEC strains display a characteristic aggregative or ‘‘stacked-brick’’ pattern of adherence to intestinal epithelial cells [36]. When grown at initial stages of biofilm, bacteria secrete less extracellular polymeric substance (EPS) [37].
Stock samples of the common laboratory strain E. coli MG1655 and
Imaging bacterial cells on planar substrates in buffer solution
For further reference, we started the analysis by analyzing the E. coli 042 strain grown according to protocol 1 in both dry and re-hydrated conditions. Fig. 1A shows an image obtained under nitrogen ambient flow (~0% Relative Humidity) of a dried (and hence dead) bacterial cell on a gelatinized gold substrate. Dried cells presented a rod-shaped structure ~2 μm long and ~1 μm wide and with a maximum height ~261±6 nm (N=13), as obtained from cross-sectional profiles taken along the main bacterial
Discussion
We have shown that the dynamic jumping mode implemented with soft cantilevers enables the nanoscale AFM imaging of viable and metabolically active bacteria on planar substrates. The use of weak forces (lower than 0.2 nN), together with the lateral displacement of the probe far away from the sample (which drastically reduces lateral shear forces) are at the basis of this capability. Based on the results obtained, this mode can be considered as an alternative to other existing AFM imaging modes
Conclusions
We have shown that dynamic jumping mode AFM constitutes a powerful technique for the observation of physiological processes of viable bacteria that are weakly attached to biocompatible gelatinous coated planar substrates. Images of intact and viable bacterial cells have been obtained for cells suspended in buffer solution for two different E. coli bacterial strains on different substrates, thus predicting a wide applicability of this imaging method. We have observed that when imaging in
Acknowledgments
This research has been financially supported by the Spanish Ministry of Education and Science under Grant no. TEC2010-16844, and by the European Commission under Grant no. NMP-280516. We acknowledge T. Wiegand for useful suggestions.
References (47)
- et al.
Physico-mechanical characterisation of cells using atomic force microscopy: current research and methodologies
J. Microbiol. Methods
(2011) - et al.
Recent progress in cell surface nanoscopy: light and force in the near-field
Nano Today
(2012) - et al.
Single-molecule imaging on living bacterial cell surface by high-speed AFM
J. Mol. Biol.
(2012) - et al.
Antibiotic-induced modifications of the stiffness of bacterial membranes
J. Microbiol. Methods
(2013) - et al.
Comparative studies of bacteria with an atomic force microscopy operating in different modes
Ultramicroscopy
(2001) - et al.
Imaging living cells surface and quantifying its properties at high resolution using AFM in QITM mode
Micron
(2013) - et al.
Immobilizing live Escherichia coli for AFM studies of surface dynamics
Ultramicroscopy
(2014) - et al.
Immobilisation of living bacteria for AFM imaging under physiological conditions
Ultramicroscopy
(2010) - et al.
AFM imaging of bacteria in liquid media immobilized on gelatin coated mica surfaces
Ultramicroscopy
(2003) - et al.
Comparison of the indentation and elasticity of E. coli and its spheroplasts by AFM
Ultramicroscopy
(2007)
Nanoscale effects of antibiotics on P. aeruginosa
Nanomedicine
Bacterial biofilms and biofouling
Curr. Opin. Biotechnol.
Minimizing tip-sample forces in jumping mode atomic force microscopy in liquid
Ultramicroscopy
The role of capsid maturation on adenovirus priming for sequential uncoating
J. Biol. Chem.
Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM
Ultramicroscopy
Characterization of curli A production on living bacterial surfaces by scanning probe microscopy
Biophys. J.
Quartz tuning fork studies on the surface properties of Pseudomonas aeruginosa during early stages of biofilm formation
Colloids Surf. B: Biointerfaces
Jumping mode scanning force microscopy: a suitable technique for imaging DNA in liquids
Appl. Surf. Sci.
From atoms to integrated circuit chips, blood cells, and bacteria with the atomic force microscope
J. Vac. Sci. Technol. Vac. Surf. Film
Frontiers in microbial nanoscopy
Nanomedicine
Towards nanomicrobiology using atomic force microscopy
Nat. Rev. Microbiol.
Using nanotechniques to explore microbial surfaces
Nat. Rev. Microbiol.
Imaging the nanoscale organization of peptidoglycan in living Lactococcus lactis cells
Nat. Commun.
Cited by (17)
Molecular dynamics model for the antibactericity of textured surfaces
2021, Colloids and Surfaces B: BiointerfacesCitation Excerpt :In the case of a spherocylindrical bacterium, cellular geometry was instead described by the sectional eccentricity e, defined as the ratio between the vertical and horizontal diameters, d1 and d2, of the elliptical section estimated from AFM topographical profiles. Using data from the literature [52,53], values of Δh/h0 ∼ 0.9 μm and e ∼ 0.7 were obtained, where h0 is the undeformed cell diameter. The simulation routine was divided in two distinct phases.
Microbial adhesion and ultrastructure from the single-molecule to the single-cell levels by Atomic Force Microscopy
2019, Cell SurfaceCitation Excerpt :However, all these techniques present limitations and should be wisely chosen depending on the applications. Chemical substrate modifications (i or ii) are often selected when one wants to observe bacterial growth for instance (Van Der Hofstadt et al., 2015). However, gelatin substrates are not recommended as they can cause AFM tip contamination.
Atomic force microscopy study of morphological modifications induced by different decontamination treatments on Escherichia coli
2017, UltramicroscopyCitation Excerpt :At the same time, the treatment reduces growth rate, inhibits cell division, and determines cell death [28]. In the case of ethanol solution 6.25% treatment, images reveal (Fig. 4) piled bacterial cells with reduced dimensions and very smooth surface appearance, forming structures similar to biofilms [26], despite the growing condition (200 rpm) and sample preparation process. It is known that the modification of shapes and dimensions help E. coli cells cope with and adapt to external conditions [29].
Image-guided Quality Control of Biomanufacturing Process
2017, Procedia CIRPAtomic force microscopy for biomedical nanotechnology
2023, Analytical Techniques for Biomedical Nanotechnology