原子力显微技术在观测微生物表面形态中的应用

原子力显微技术作为一门新发展起来的显微成像技术,不仅具有在近生理条件下对样本实时、高分辨率三维成像等特点,而且能通过力矩测量探知样本物理性状。即给人们认识微生物的表面结构提供又一平台,也为揭示微生物表面结构与功能之间的关系提供一种新方法。介绍了对微生物表面形态观测中常用测量模式和某些样品固定方法:多孔膜技术、凹陷技术,概括近年来原子力显微技术在微生物学中的应用情况。     

Effects of surface-active chemicals on microbial adhesion

Summary A simple, continuously circulating fed-batch culture system of microorganisms was designed and used to study the adhesion of mixed microbial cultures to surfaces of 316 stainless steel, Admiralty brass, and wood. The adhesion of the microbes to the surfaces was monitored by scanning electron microscope analysis. Eighteen non-toxic, non-ionic, or anionic surface-active compounds were tested for efficacy as inhibitors of microbial adhesion to stainless steel and wood surfaces. A rating system was devised to correlate efficacy with the degree of biomass adhered to 316 stainless steel, although correlation could not be made with wood. A correlation was also found between the ability of a compound to lower surface tension and its ability to prevent microbial adhesion.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

High-speed atomic force microscopy: Imaging and force spectroscopy

Atomic force microscopy (AFM) is the type of scanning probe microscopy that is probably best adapted for imaging biological samples in physiological conditions with submolecular lateral and vertical resolution. In addition, AFM is a method of choice to study the mechanical unfolding of proteins or for cellular force spectroscopy. In spite of 28 years of successful use in biological sciences, AFM is far from enjoying the same popularity as electron and fluorescence microscopy. The advent of high-speed atomic force microscopy (HS-AFM), about 10 years ago, has provided unprecedented insights into the dynamics of membrane proteins and molecular machines from the single-molecule to the cellular level. HS-AFM imaging at nanometer-resolution and sub-second frame rate may open novel research fields depicting dynamic events at the single bio-molecule level. As such, HS-AFM is complementary to other structural and cellular biology techniques, and hopefully will gain acceptance from researchers from various fields. In this review we describe some of the most recent reports of dynamic bio-molecular imaging by HS-AFM, as well as the advent of high-speed force spectroscopy (HS-FS) for single protein unfolding.     

AFM-based dual nano-mechanical phenotypes for cancer metastasis

An enhanced mechanical compliance is considered to be a mechanical indicator for metastatic cancer cells. Our study using atomic force microscopy (AFM) revealed that breast cancer cells agreed well with this hypothesis. However, prostate cancer cells displayed a reverse correlation; less metastatic prostate cancer cells were more mechanically compliant. Two-dimensional AFM force spectroscopy was performed to characterize dual mechanical properties—the cell–substrate adhesion as well as the mechanical compliance. Interestingly, prostate cancer cells displayed a strong positive correlation between the cell–substrate adhesion and metastatic potential. However, there was no clearly observable correlation between the cell–substrate adhesion and the metastatic potential despite variations in mechanical compliance of breast cancer cells. These results suggest that the correlation between the dual mechanical signatures and metastatic potential be uniquely identified for cancer cells originating from different organs. We postulate that this correlation could reveal which step of cancer progression is favorable in terms of physical interaction between cancer cells and micro-environments. We expect that based on the “seed and soil hypothesis”, the identification of the dual mechanical phenotypes, could provide a new insight for understanding how a dominant metastatic site is determined for cancer cells originating from specific organs.     

The Detection of Microorganisms and Organic Material on Stainless Steel Food Contact Surfaces

Few methods are available for the differentiation of microorganism and organic material on surfaces, although such mixtures are commonplace, particularly in the food industry, where food debris (soil) and microorganisms frequently foul food contact surfaces and pose challenges in terms of hygiene and cleanability. It would be of value to discern any differences in removal or persistence on surfaces. This review considers some methods which are available. Direct epifluorescence microscopy (DEM) enables visual differentiation, but traditional microbiological culture methods cannot detect organic soil. Atomic force microscopy (AFM) provides images of the surface on the nanometer scale, with minimal preparation, and is able to visualise both cellular and acellular components of the mixture, particularly prior to cleaning. Confocal laser scanning microscopy (CLSM) also has potential in this area. Surface sensitive methods such as X-Ray photoelectron spectroscopy (XPS) and Time-of-flight secondary ion mass spectrometry (ToFSIMS) provide information on chemical species present on a surface. Those chemical species more likely on microbial cells may be differentiated from those more likely in a specified organic soil, thus comparisons may be made as to differential removal of the organic soil and microbial cells. These methods may be of value in studies on the fouling and cleanability of surfaces.     

Ultrastable atomic force microscopy: Improved force and positional stability

Atomic force microscopy (AFM) is an exciting technique for biophysical studies of single molecules, but its usefulness is limited by instrumental drift. We dramatically reduced positional drift by adding two lasers to track and thereby actively stabilize the tip and the surface. These lasers also enabled label-free optical images that were spatially aligned to the tip position. Finally, sub-pN force stability over 100 s was achieved by removing the gold coating from soft cantilevers. These enhancements to AFM instrumentation can immediately benefit research in biophysics and nanoscience.     

Chemical force microscopy of cellulosic fibers

Atomic force microscopy with chemically modified cantilever tips (chemical force microscopy) was used to study the pull-off forces (adhesion forces) on cellulose model surfaces and bleached softwood kraft pulp fibers in aqueous media. It was found that for the –COOH terminated tips, the adhesion forces are dependent on pH, whereas for the –CH₃ and –OH terminated tips adhesion is not strongly affected by pH. Comparison between the cellulose model surfaces and cellulosic fibers under our experimental conditions reveal that surface roughness does not affect adhesion strongly. X-ray photoelectron spectroscopy (XPS) and Fourier Transformed Infrared (FTIR) spectroscopy reveal that both substrate surfaces have homogeneous chemical composition. The results show that chemical force microscopy can be used for the chemical characterization of cellulose surfaces at a nano-level.     

Nanostructure and β1-integrin distribution analysis of pig's spermatogonial stem cell by atomic force microscopy

Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis and male fertility. However, spermatogenesis has direct links with some adhesion molecules on SSCs membrane. Β1-integrin (CD29) is such a kind of adhesion molecule and a biomarker of pig's SSCs. Therefore, quantitative characteristics of β1-integrin expression level in a single cell could help us to capture the signal switch and understand the mechanism of spermatogenesis. In this study, atomic force microscopy (AFM) was used to obtain the morphology and ultrastructure of SSCs at nanometer level, and the CD29 Ab-functionalized AFM tip was used to examine β1-integrin distribution on the cell membrane. There were many force-binding spots on about 50% of cell membrane binding to the CD29 Ab-functionalized AFM tip, and the mean bind rupture force was 283.63±12.56PN which was much larger than the non-specific average force 70.75±10.95PN. Meanwhile, β1-integrin on SSCs membrane was distributed non-uniformly, and there were some β1-integrins appeared to be expressed as 150-350 nm nanoclusters on the membrane. Our results discovered the structure of SSCs at nanometer level by AFM. The force between β1-integrin antigen-antibody interactions and the distribution of β1-integrin protein on SSCs membrane were also firstly demonstrated.     

Effect of the covalently linked fatty acid 18-MEA on the nanotribology of hair's outermost surface

Highly ordered lipids adsorbed or grafted on surfaces are known to provide protection and lubrication custom engineered surfaces. We have used atomic force microscopy (AFM) to measure adhesion and frictional properties of the outermost surfaces of a variety of human hairs with the aim of both understanding the role of 18-methyleicosanoic acid (18-MEA), an unusual branched-chain fatty acid covalently bound to the cuticle surface, and investigating how treatments or the ethnic origin affect this layer. Results show that an unmodified silicon nitride AFM tip is able to detect changes at the hair surface that can be related to the absence or presence of this layer due to treatment conditions and in particular that this monolayer has a lubricant effect.     

Probing elasticity and adhesion of live cells by atomic force microscopy indentation

Atomic force microscopy (AFM) indentation has become an important technique for quantifying the mechanical properties of live cells at nanoscale. However, determination of cell elasticity modulus from the force–displacement curves measured in the AFM indentations is not a trivial task. The present work shows that these force–displacement curves are affected by indenter-cell adhesion force, while the use of an appropriate indentation model may provide information on the cell elasticity and the work of adhesion of the cell membrane to the surface of the AFM probes. A recently proposed indentation model (Sirghi, Rossi in Appl Phys Lett 89:243118, 2006), which accounts for the effect of the adhesion force in nanoscale indentation, is applied to the AFM indentation experiments performed on live cells with pyramidal indenters. The model considers that the indentation force equilibrates the elastic force of the cell cytoskeleton and the adhesion force of the cell membrane. It is assumed that the indenter-cell contact area and the adhesion force decrease continuously during the unloading part of the indentation (peeling model). Force–displacement curves measured in indentation experiments performed with silicon nitride AFM probes with pyramidal tips on live cells (mouse fibroblast Balb/c3T3 clone A31-1-1) in physiological medium at 37°C agree well with the theoretical prediction and are used to determine the cell elasticity modulus and indenter-cell work of adhesion. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkin's lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500×500 nm²) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells.     

Comparative studies of bacterial biofilms on steel surfaces using atomic force microscopy and environmental scanning electron microscopy

Environmental scanning electron microscopy (ESEM) and atomic force microscopy (AFM) were compared as tools for the observation of bacterial biofilms developed on carbon steel and AISI 316 stainless steel surfaces under stagnant conditions. Biofilms were generated in batch cultures of two different isolates of marine sulphate reducing bacteria (SRB) and in cultures consisting of mixed populations of acidophilic bacteria, known as "acid streamers";. Imaging of single SRB cells on mica was also carried out to reveal the surface topography of individual bacterial cells at nanometre resolution. Following the removal of biofilms, the stainless steel surfaces were profiled using AFM to determine the degree of steel deterioration. ESEM and AFM studies of bacterial biofilms in-situ, gave both qualitative and quantitative information on biofilm structure at high resolution. The use of AFM image analysis software allowed estimation of the width and height of bacterial cells, the thickness and width of exopolymeric (EPS) capsule and bacterial flagella, as well as characterisation of the surface roughness of the steel, including measurements of depth and diameter of individual pits. Exposure of stainless steel specimens to acid streamers resulted in a significant increase in the surface roughness of the steel, compared to specimens placed in sterile medium.     

ANALYSIS OF LIGAND–RECEPTOR INTERACTIONS IN CELLS BY ATOMIC FORCE MICROSCOPY

ABSTRACT

Atomic force microscopy (AFM) increasingly has been used to analyse “receptor” function, either by using purified proteins (“molecular recognition microscopy”) or, more recently, in situ in living cells. The latter approach has been enabled by the use of a modified commercial AFM, linked to a confocal microscope, which has allowed adhesion forces between ligands and receptors in cells to be measured and mapped, and downstream cellular responses analysed. We review the application of AFM to cell biology and, in particular, to the study of ligand–receptor interactions and draw examples from our own work and that of others to show the utility of AFM, including for the exploration of cell surface functionalities. We also identify shortcomings of AFM in comparison to “standard” methods, such as receptor auto-radiography or immuno-detection, that are widely applied in cell biology and pharmacological analysis.     

Towards nanomicrobiology using atomic force microscopy

At the cross-roads of nanoscience and microbiology, the nanoscale analysis of microbial cells using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Over the past decade, there has been tremendous progress in our use of AFM to observe membrane proteins and live cells at high resolution. Remarkable advances have also been made in applying force spectroscopy to manipulate single membrane proteins, to map surface properties and receptor sites on cells and to measure cellular interactions at the single-cell and single-molecule levels. In addition, recent developments in cantilever nanosensors have opened up new avenues for the label-free detection of microorganisms and bioanalytes.     

Methods for reducing nonspecific interaction in antibody-antigen assay via atomic force microscopy

We developed a method to measure the rupture forces between antibody and antigen by atomic force microscopy (AFM). Previous studies have reported that in the measurement of antibody–antigen interaction using AFM, the specific intermolecular forces are often obscured by nonspecific adhesive binding forces between antibody immobilized cantilever and substrate surfaces on which antigen or nonantigen are fixed. Here, we examined whether detergent and nonreactive protein, which have been widely used to reduce nonspecific background signals in ordinary immunoassay and immunoblotting, could reduce the nonspecific forces in the AFM measurement. The results showed that, in the presence of both nonreactive protein and detergent, the rupture forces between anti-ferritin antibodies immobilized on a tip of cantilever and ferritin (antigen) on the substrate could be successfully measured, distinguishing from nonspecific adhesive forces. In addition, we found that approach/retraction velocity of the AFM cantilever was also important in the reduction of nonspecific adhesion. These insights will contribute to the detection of specific molecules at nanometer scale region and the investigation of intermolecular interaction by the use of AFM.     

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.     

Nonspecific interactions in AFM force spectroscopy measurements

Sample-probe contact duration (dwell time) and loading force are two important parameters for the atomic force microscopy (AFM) force spectroscopy measurements of ligand-receptor interaction. A prolonged contact time may be required to initiate ligand-receptor binding as a result of slow on-rate kinetics or low reactant density. In general, increasing contact duration promotes nonspecific interactions between the substrate and the functionalized cantilever and, thus, masking the detection of the specific interactions. To reduce the nonspecific interactions in AFM force measurements requiring extended substrate-probe contact, we investigated the interaction of bovine serum albumin (BSA)-functionalized cantilever with BSA-coated glass, polyethylene glycol (PEG)-functionalized glass, Pluronic-treated Petri dishes and agarose beads. The frequency of nonspecific interaction between the BSA-functionalized cantilever and the different samples increased with loading force and dwell time. This increase in nonspecific adhesion can be attributed to the interaction mediated by forced unfolding of BSA. By reducing the loading force, the contact duration of the AFM probe with an agarose bead can be extended to a few minutes without nonspecific adhesion.