Atomic force microscopy: a powerful tool for studying bacterial swarming motility

Swarming motility is a fascinating phenomenon by which some bacteria use flagella to move over solid surfaces. Understanding the molecular mechanisms underlying swarming motility requires studying the factors that induce and control flagella expression in swarming cells. Traditionally, flagella are observed by optical or electron microscopy, but none of these techniques combine versatility and easiness, with quantitative and high-resolution information. We report an atomic force microscopy (AFM)-based approach for the fast imaging of bacterial phenotypes (cell shape, flagella expression) in swarming motility studies. Cells from the gram-positive bacterium Bacillus thuringiensis sv. israelensis were inoculated on energy-rich media containing increasing agar concentrations. Following swarming assays (2 days), the cell morphology and the amount of flagella were directly observed by AFM imaging in air. Consistent with the macroscopic swarming behavior, cells harvested from the rim of colonies spreading on soft agar were hyperflagellated, elongated and arranged in chains. Increasing the agar concentration led to much lower amounts of flagella and to shorter rod-shaped cells, a finding consistent with the slower swarming motility of the cells. Cells taken from colony centers on soft and hard agar surfaces were generally non-flagellated, rod-shaped, rarely arranged in chains, and exhibited lysis and sporulation. This study shows that AFM imaging can readily discriminate between swarming and non-swarming cells, and quantify their morphological details, thus offering an important tool to study the dynamics of bacterial populations.     

Differential dynamic microscopy: probing wave vector dependent dynamics with a microscope

We demonstrate the use of an ordinary white-light microscope for the study of the q-dependent dynamics of colloidal dispersions. Time series of digital video images are acquired in bright field with a fast camera, and image differences are Fourier analyzed as a function of the time delay between them. This allows for the characterization of the particle dynamics independent of whether or not they can be resolved individually. The characteristic times are measured in a wide range of wave vectors and the results are found to be in good agreement with the theoretically expected values for Brownian motion in a viscous medium.     

Polymer confinement and bacterial gliding motility

Cyanobacteria and myxobacteria use slime secretion for gliding motility over surfaces. The slime is produced by the nozzle-like pores located on the bacteria surface. To understand the mechanism of gliding motion and its relation to slime polymerization, we have performed molecular dynamics simulations of a molecular nozzle with growing inside polymer chains. These simulations show that the compression of polymer chains inside the nozzle is a driving force for propulsion. There is a linear relationship between the average nozzle velocity and the chain polymerization rate with a proportionality coefficient dependent on the geometric characteristics of the nozzle such as its length and friction coefficient. This minimal model of the molecular engine was used to explain the gliding motion of bacteria over surfaces.     

Ultrasonic radiation in dynamic force microscopy

In dynamic force microscopy the cantilever of an atomic force microscope is vibrated at ultrasonic frequencies in the range of several 10 kHz up to several MHz while scanning a sample surface. The amplitude and phase of the cantilever vibration as well as the shift of the cantilever resonance frequencies provide information about local sample surface properties. In several operation modes of dynamic force microscopy, for example force modulation microscopy, tapping mode or atomic force acoustic microscopy, the sensor tip is in contact with the sample at least during a fraction of its vibration cycle. The periodic indentation of the tip with the sample surface generates ultrasonic waves. In this paper, the ultrasonic radiation of a vibrating cantilever into a sample and its contribution to the damping of the cantilever vibration are calculated. The theoretical results are compared to experiments.     

Damping mechanism in dynamic force microscopy

A general theory is presented which describes the damping in dynamic force microscopy due to the proximity of the surface, consistently with resonant frequency shift effects. Orders of magnitude for the experimentally measured "dissipation" and image corrugation are reproduced. It is suggested that the damping does not mainly result from energy dissipation, but arises because not all solutions of the microlever equation of motion are accessible. The damping is related to the multivalued nature of the analytical resonance curve, which appears at some critical tip-surface separation.     

Use of laser scattering for quantitative determinations of bacterial motility

Laser light intensity correlation spectroscopy is used to measure bacterial motility. The angular dependence of the scattering spectra from motile E. Coli K₁₂ bacteria is in agreement with theoretical predictions. By taking appropriate integrations of the spectra, the swimming speed distribution of the bacteria is determined.     

Scale-invariant correlations in dynamic bacterial clusters

In Bacillus subtilis colonies, motile bacteria move collectively, spontaneously forming dynamic clusters. These bacterial clusters share similarities with other systems exhibiting polarized collective motion, such as bird flocks or fish schools. Here we study experimentally how velocity and orientation fluctuations within clusters are spatially correlated. For a range of cell density and cluster size, the correlation length is shown to be 30% of the spatial size of clusters, and the correlation functions collapse onto a master curve after rescaling the separation with correlation length. Our results demonstrate that correlations of velocity and orientation fluctuations are scale invariant in dynamic bacterial clusters.     

Adhesion hysteresis in dynamic atomic force microscopy

The effects of adhesion hysteresis in the dynamic‐dissipation curves measured in amplitude‐modulation atomic force microscopy are discussed. Hysteresis in the interaction forces is shown to modify the dynamics of the cantilever leading to different power dissipation curves in the repulsive and attractive regimes. Experimental results together with numerical simulations show that power dissipation, as measured in force microscopy, is not always proportional to the energy dissipated in the tip–sample interaction process. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

扫描隧道显微术中的微分谱学及其应用

文章介绍了扫描隧道显微术中微分谱学的原理及其在实验中的诸多应用。微分谱（dI／dV谱）和dI／dV成像可用来研究电子局域态密度在能量和空间的分布，即微分谱固定空间一点，反映电子态密度以能量为变量的分布；而dI／dV图像则反映某给定能量的电子局域态密度以空间为变量的分布，二次微分谱（d^2I／dV^2谱）和二次微分成像可以用来反映分子的非弹性隧穿过程，从而研究分子的振动态。     

扫描隧道显微术中的微分谱学及其应用

文章介绍了扫描隧道显微术中微分谱学的原理及其在实验中的诸多应用.微分谱(dI/dV谱)和dI/dV成像可用来研究电子局域态密度在能量和空间的分布,即微分谱固定空间一点,反映电子态密度以能量为变量的分布;而dI/dV图像则反映某给定能量的电子局域态密度以空间为变量的分布.二次微分谱(d2I/dV2谱)和二次微分成像可以用来反映分子的非弹性隧穿过程,从而研究分子的振动态.     

Velocity and diffusion imaging in dynamic NMR microscopy

The imposition of resolution gradients in a pulsed-gradient spin-echo (PGSE) NMR sequence induces motionally dependent phase and amplitude modulation in the image, a technique which we have termed dynamic NMR microscopy. Fourier analysis of this modulation gives a dynamic displacement profile for each pixel which can then be analyzed to obtain velocity and diffusion maps. The application of this method at high spatial resolution is motivated by a desire to measure vascular flow in living plants and variations in molecular self-diffusion under the influence of velocity shear in narrow capillaries. The theory of dynamic NMR microscopy is presented and potential artifacts discussed, including the effect of slice selection gradients, PGSE gradient nonuniformity, and specific problems associated with the measurement of self-diffusion in the presence of velocity gradients. It is demonstrated that a double-echo PGSE pulse sequence can be used to restore coherent phase shifts associated with steady-state flow, and examples of self-diffusion maps and signed velocity maps from sequences of phase-encoded images obtained by projection reconstruction are given. This method has been applied at 20,um transverse resolution in laminar capillary flow.     

Measuring viscous dissipative interactions in dynamic force microscopy

The periodic motion of a harmonic pendulum in an arbitrary force field including viscous damping is studied as it pertains to dynamic force microscopy. It is shown that the damping constant as a function of tip-sample distance and thus the dissipative force can be obtained unambiguously by measuring the driving-force amplitude versus displacement of the force sensor. This methodology provides the basis for quantitative force spectroscopy of dissipative interactions.     

Kinetics of colloidal fractal aggregation by differential dynamic microscopy

We study the kinetics of an aggregation process induced by adding salt to a stable colloidal suspension of 73 nm (diameter) particles. Despite the subdiffraction size of the colloidal particles, the process is monitored via optical microscopy, which is used here to obtain time-resolved scattering information about the colloidal aggregates. The radius of the aggregates is determined as a function of time and their fractal dimension is extracted. Our results are compatible with a diffusion limited aggregation process, as independently confirmed by spectral turbidimetry measurements on the same sample.     

Investigations of dynamic photorefractivity regime by optical polarizing microscopy

We present a technique, based on optical polarizing microscopy, and results of direct observation of the optical interference field effect on the transient domains excited by ac electric field in a nematic planar cell with photosensitive aligning layers. The light source used in a microscope operated in DC mode as well as in triggered pulse one. Obtained microscopic snapshots of transient domain structure confirmed our assumption of the transient domains reordering (trapping) by the low intensity optical interference field.     

Calculation of the frequency shift in dynamic force microscopy

A theoretical study of the quality and the range of validity of different numerical and analytical methods to calculate the frequency shift in dynamic force microscopy is presented. By comparison with exact results obtained by the numerical solution of the equation of motion, it is demonstrated that the commonly used interpretation of the frequency shift as a measure for the force gradient of the tip–sample interaction force is only valid for very small oscillation amplitudes and leads to misinterpretations in most practical cases. Perturbation theory, however, allows the derivation of useful analytic approximations.     

Probing bacterial interactions: integrated approaches combining atomic force microscopy, electron microscopy and biophysical techniques

Recent developments in the application of Atomic Force Microscopy (AFM) and other biophysical techniques for the study of bacterial interactions and adhesion are discussed in the light of established biological and microscopic approaches. Whereas molecular-biological techniques combined with electron microscopy allow the identification and localization of surface constituents mediating bacterial interactions, with AFM it has become possible to actually measure the forces involved in bacterial interactions. Combined with the flexibility of AFM in probing various types of physical interactions, such as electrostatic interactions, specific ligand-receptor interactions and the elastic forces of deformation and extension of bacterial surface polymers and cell wall, this provides prospects for the elucidation of the biophysical mechanism of bacterial interaction. However, because of the biochemical and a biophysical complexity of the bacterial cell wall, integrated approaches combining AFM with electron microscopy and biophysical techniques are needed to elucidate the mechanism by which a bacterium interacts with a host or material surface. The literature on electron microscopy of the bacterial cell wall is reviewed, with particular emphasis on the staining of specific classes of cell-wall constituents. The application of AFM in the analysis of bacterial surfaces is discussed, including AFM operating modes, sample preparation methods and results obtained on various strains. For various bacterial strains, the integration of EM and AFM data is discussed. Various biophysical aspects of the analysis of bacterial surface structure and interactions are discussed, including the theory of colloidal interactions and Bell's theory of cell-to-cell adhesion. An overview is given of biophysical techniques used in the analysis of the properties of bacterial surfaces and bacterial surface constituents and their integration with AFM. Finally, we discuss recent progress in the understanding of the role of bacterial interactions in medicine within the framework of the techniques and concepts discussed in the paper.     

Surface plasmon interferometric microscopy for three-dimensional imaging of dynamic processes

A method, which we named surface plasmon interferometric microscopy, for real-time displaying of the dynamic evolution of the refractive index (RI) of a sample in three-dimensions is demonstrated experimentally. The Fourier fringe analysis technique is employed to get the phase variations of the samples by demodulating the interference patterns captured by a CCD camera, and the 3D RI distribution can be obtained through numerical interpolation from the relation between the phase and the RI of the samples. Our method may provide an interesting way to monitor fast dynamics of physical, biological, and chemical processes in real time.     

Exact solution of the frequency shift in dynamic force microscopy

The exact frequency shift of an AFM non-uniform probe with an elastically restrained root, subjected to van der Waals force, is derived. The original distributed system is considered and then its exact fundamental solutions and the general frequency equation are derived. Results are compared with those by the force gradient method and the perturbation method. The effects of several parameters on the sensitivity of measurement are investigated. Results show that the interpretation of frequency shift by using the force gradient method is unsatisfactory. The smaller the amplitude of oscillation and the tip–surface distance are, the larger the frequency shift. The design of a taper beam is recommended for increasing the sensitivity of measurement.     

Laplace field microscopy for label-free imaging of dynamic biological structures

We present Laplace field microscopy as a method for generating intrinsic contrast of transparent specimens. This technique uses a spatial light modulator to perform the Laplacian of the field in the Fourier plane of a microscope image. The resulting image incorporates phase information and thus renders high contrast images from phase objects. We demonstrate the potential of the method by imaging index-matched beads, unlabeled tissue slices, and dynamic live cells.     

Identification of nanoscale dissipation processes by dynamic atomic force microscopy

Identification of energy-dissipation processes at the nanoscale is demonstrated by using amplitude-modulation atomic force microscopy. The variation of the energy dissipated on a surface by a vibrating tip as a function of its oscillation amplitude has a shape that singles out the dissipative process occurring at the surface. The method is illustrated by calculating the energy-dissipation curves for surface energy hysteresis, long-range interfacial interactions and viscoelasticity. The method remains valid with independency of the amount of dissipated energy per cycle, from 0.1 to 50 eV. The agreement obtained between theory and experiments performed on silicon and polystyrene validates the method.