Nano-Mechanics and Membrane Receptor Mapping of CD 8 + T Cells

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1 Current Nanoscience, 2009, 5, Nano-Mechanics and Membrane Receptor Mapping of CD 8 + T Cells Yangzhe Wu 1, 3 *, Yi Hu 1, Hongsong Lu 2, Jiye Cai 1 *, Xianhui He 2 and Hongxia Zhao 1 1 Chemistry Department, 2 Institution for Tissue Transplantation and Immunology, Jinan University, Guangzhou PR China 3 Biological and Irrigation Engineering Department, Utah State University, Logan, Utah USA Abstract: The investigation of nano-mechanical properties and nano-architectures of CD 8 + T cells activated in vitro will help further interpret the immune response/recognition of these cells at nanoscale resolution. In this work, the local biophysical properties of human peripheral blood CD 8 + T cells were obtained, revealing the adhesion force of membrane was increased after CD 8 + T cells were stimulated with phorbol dibutyrate plus ionomycin, though the measured local stiffness for resting and activated cells was similar. The 3-D distribution pattern and grayscale map together revealed that membrane receptors (CD 8 and activation marker CD 69) distributed non-uniformly on the membrane and were shown as micro-, nano-scale clusters or domains. This work describes a relatively simple approach for exploiting the distribution pattern of receptor molecules on a single T cell surface. Keywords: Force spectroscopy, molecule recognition, receptor mapping, nano-mechanics, CD 8 + T cell. 1. INTRODUCTION CD + 8 T cells (also known as killer T cell) belong to a sub-group of T lymphocytes, and they protect the host by killing pathogens or viruses infected cells and dysfunctional or damaged cells via the release of perforin and granzyme [1]. CD + 8 T cells are well known to play an indispensable role in immune response, such as cytotoxic role. They express specific receptor molecules, CD 8, which recognizes specific antigenic epitopes that links to MHC-I molecules. T cell activation initiates T cell receptor (TCR) recognizing antigen and may lead to many potential changes of biophysical properties on the whole cell [1], such as the vibrations of cell surface charge [1], cell morphology, and membrane viscoelasticity [2], and even spatial distribution of TCR itself. Up to now, though the immune function of antigen-antibody complexes of CD 8 had been well documented biologically, the knowledge about the spatial distribution of CD 8 on membrane at nanoscale resolution remains largely obscure, and the potential changes in nano-mechanical properties of CD + 8 T cells that are activated in vitro by PDB plus ION and how to measure such cell surface recognition events at molecular level are unclear too. If assuming the nano-mechanical properties of CD + 8 T cells alter upon in vitro activation, then these variations would promote the understanding of the immune functions such as immune regulation and immune response of these cells. However, because of the inability of measurement of living cell with high spatial resolution, conventional microscopy such as electron microscopes and optical microscopy cannot achieve the nanoscale resolution which is desired in observation of cell surface ultrastructure changes. Fortunately, however, atomic force microscopy (AFM), a powerful and highly sensitive force quantification technique, has been demonstrated to measure non-specific interaction between tip and cell membrane [3, 4], specific interaction of ligand-receptor [3-7] and cell-cell interaction [3, 8-12] at single molecule or cell level. The introduction of AFM into biological application has opened a new door for nano-biology [13-15] research. The unique advantages of AFM mainly include the nanoscale resolution of surface structure imaging, the ability of nano-mechanics measurement of sample, the non-invasive, and the very simple process of sample preparation, etc [3, 4]; however, the biggest disadvantage of AFM is that it cannot recognize what it s observing, which has obstructed its application in biology for a long time, fortunately, however, specific molecule (e.g. ligand) modified AFM tip [3, 16] has resolved this problem. *Address correspondence to these authors at Chemistry Department, Jinan University, Guangzhou Guangdong, PR China; Tel (Fax): ; s: tjycai@jnu.edu.cn and y.wu@usu.edu To date, AFM-based molecule force spectroscopy has been widely applied in cell nano-mechanical property measurements (such as stiffness, elasticity, adhesion force, and cell spring constant and so on), ligand-receptor interaction, unfolding force of peptide or protein molecules, and others [3, 4]. However, there are only a few reports on force spectroscopy used in mapping cell surface properties. Wagh et al. reported on an application of force spectroscopy to map elasticity in epithelial cells [17], and Gunning et al mapped the adhesion force of Caco-2 cell monolayers with Alexa 488 WGA-functionalized AFM tip [5]. The force spectroscopy can help further interpret the molecular recognition event at single cell level, and also provide insightful views for immune activation at nanoscale resolution. By analyzing the force-distance curves and measuring ligand-receptor interaction, one can not only detect the changes of membrane nano-mechanical properties in the context of T cell recognition of antigens, but also achieve molecular level detection of cell membrane molecules [3]. Therefore, the obtained results may provide new insights into the immune response of T cells and ultimately into immunity. In previous work, the membrane nanostructures and antigen-antibody interaction of CD + 4 T cells were investigated using AFM [18]. In the present work, the nanostructures and adhesion force of both resting and activated CD + 8 T cells are acquired by AFM; furthermore, the spatial distribution mode of both CD 8 molecules and activation marker CD 69 molecules on cell membrane are obtained via the specific force spectroscopy-based 3-D reconstruction. This work will help further understand the biophysical properties (morphology, adhesion force, and stiffness), receptor-ligand interaction, and the activation-related biological behaviors of CD + 8 T cells. 2. MATERIALS AND METHODS 2.1. CD + 8 T Cells Isolation and Sample Preparation Specific CD + 8 T cells were isolated from peripheral venous blood of healthy, drug free adult donors according to the manual of RosetteSep human CD + 8 T lymphocyte enrichment cocktail ( In brief, 1) 100 L of RosetteSep human CD + 8 T lymphocyte enrichment cocktail was fully mixed with 2mL of whole blood, and incubated for 20 min at room temperature; 2) diluted with 2 ml of PBS containing 2% bovine serum albumin (BSA) gently; 3) layered the diluted solution on the top of 3 ml density medium (Ficoll), then centrifuged at 1200 g for 20 min; 4) removed the enriched cells from the density medium : blood plasma interface, and then washed these enriched cells with 2% PBS (centrifuged at 425 g for 10 min) (repeated once). The isolated T lymphocytes ( ) were separated into two groups: control group and activation group. The latter /09 $ Bentham Science Publishers Ltd.

2 466 Current Nanoscience, 2009, Vol. 5, No. 4 Wu et al Functionalization of the AFM Tip To measure the specific interaction between CD 8 molecules and anti-cd 8 molecules and to further determine the distribution of CD 8 molecules on the cell membrane, the contact AFM probes (Si 3 N 4 cantilevers) were functionalized referring to the methods described by Wojcikiewicz et al. [20]. In brief, the AFM tips were soaked in acetone for 5 min and then irradiated with UV for 20 min; tips were incubated in 100 l drop of biotin-bsa (0.5 mg/ml in 0.1M Na- HCO 3, ph 8.8) solution overnight at 37 C and then washed three times with PBS (ph 7.2) to remove unbound protein; incubated tips in a 100 l of streptavidin (0.5 mg/ml in PBS) at room temperature for 20 min; washed tips with PBS to remove unbound streptavidin; finally, the streptavidin-functionalized tips were incubated in 10 g/ml biotinylated monoclonal anti-human CD 8 antibody solution at 4 C overnight. Similarly, anti CD 69 molecules were also linked onto AFM tip. The sketch procedures of AFM tip functionalization are shown in Fig. (1). The functionalized tips were stored in PBS at 4 C before the specific force measurements. Fig. (1). The schematic illustration of the functionalization procedures of AFM tip (not to scale). were stimulated with phorbol dibutyrate (PDB, mol/ml; Calbiochem Co.) plus ionomycin (ION, 0.5 g/ml; Sigma) for 24h. The cells of both groups were cultured in RPMI 1640 for the following experiments AFM Measurements of CD + 8 T Cells Atomic force microscope (AFM) (Autoprobe CP Research, USA) was used to acquire morphology, membrane nanostructures and adhesion force of CD + 8 T cells in air (Fig. S1 in supplementary material), and to measure specific interaction of antigen-antibody in PBS solution (ph 7.2) (Fig. S3 in supplementary material). Because the resolution of cell images acquired in air is significant higher than that acquired in liquid (living cells in PBS will maintain motility for a period of time), therefore, cellular morphology and membrane nanostructures were visualized in air using the tapping mode probe. As a contrast, the non-specific and specific interaction force was measured in PBS solution using the contact mode probe. Just as expected, the resolution of cell images acquired in liquid is dramatically lower than those acquired in air (Figs. S1 and S2 in Supplementary Material). As for cells prepared for measurement in air, they were fixed by 2.5% glutaraldehyde (Sigma) in buffer solution for 10 min, then about 10 l of cell suspension was dropped onto cover glass and air dried, and then transferred to AFM scanner stage for measurements. The oscillation frequency and the force constant of the tapping mode probe are khz and 3 N/m, respectively. As for contact mode probe, the oscillation frequency is khz, and the force constant is 0.9 N/m. (these parameters were offered by manufacturer) 2.3. Substrate Treatment and Cell Adsorption To perform the measurement in liquid, the cover glass substrate must be pretreated to attach T cells steadily. The cover glasses were pretreated referring to the method described by Büechi et al. [19]. In brief, freshly cleaned cover glasses (18 18 mm) were immersed in 20% H 2 SO 4 for 2 h, then washed in distilled water, and finally stored in acetone for at least 2 h. The air-dried cover glasses were then incubated with 2% solution in acetone of 3-aminopropyltriethoxysilane (APS, Sigma) for 24 h at 47 C, and then washed in 100% acetone and incubated with 1% aqueous solution of glutaraldehyde for 1h at 4 C. After three further washings with PBS, the cover glasses were used immediately for attachment of CD + 8 T cells. 3. RESULTS AND DISCUSSION 3.1. Surface Adhesion Force of Resting and Activated CD + 8 T Cells Measured in Air To analyze the nano-mechanical properties of resting and activated CD + 8 T cells, force-distance curves of cell membrane were acquired by the contact mode AFM in air (Fig. 2). When the AFM tip scans along cell membrane, the non-specific interaction between bare tip and cell membrane (Fig. 2b), which is one of the factors resulting in the low resolution image (Fig. 2a), can be used to track the potential changes of surface adhesion force of cells by recording force-distance curves (Fig. 2c-2f). The approaching branches of force curves show no obvious snap-in forces (highlighted by blue dashed circles in Fig. 2c, 2e), which implies that, when the measurements are conducted in air, the van der Walls attractive force between bare tip and cell membrane [21] could be negligible. The nonlinear interaction, which mainly results from electrostatic force, van der Waals force, steric force of polymer on membrane, and nonlinear deformation of cells [22-24], in many cases governs the shape of curve occurring at the point that the tip just contacts the sample surface [21, 23]. Because CD + 8 T cells have to circulate around the body with blood, the unapparent nonlinear interaction can let cells avoid adhering onto vascular wall or coagulating. This is supported by the further measurements when the force curves were obtained in PBS liquid, as shown in Figs. (3d and 3j), both the nonlinear interaction and the non-specific interaction (adhesion force) between bare tip and cell membrane are invisible. The retraction branch of force-distance curves (Fig. 2c-2f) represents the required force (also called as pull-off force or rupture force) [4] pulling tip away from cell membrane. According to the force-distance curves, one can determine detachment (or binding) events occurring locations and the rupture force strength required for breaking these detachment events (Fig. 2d-2h). Fig. (2d) shows a typical force curve with three unfolding peaks (black arrows point), and indicates that pull-off distance of detachment events (Fig. 2g) are 16 nm and 23 nm, and their corresponding pull-off force are 367 pn and 395 pn, respectively. Tip snaps out away from cell membrane when the pulling force is over the threshold of the last weak attractive force between tip and membrane molecules (repture force is 223 pn, Fig. 2d). As for the interaction between tip and activated T cells, the force curves indicate that there are five detachment events/peaks with different pull-off distance and pulloff force (Fig. 2f, 2h). The force spectroscopy measurements demonstrate that PDB plus ION stimulation resulted in the increases of the adhesion force of CD + 8 T cells (Fig. 2i-2j). The statistical results (reported as mean±sd) indicate that the adhesion force of resting cell is ± pn, and that of activated cell is ± pn. The difference in adhesion force between rest-

3 Nano-Mechanics and Membrane Receptor Mapping Current Nanoscience, 2009, Vol. 5, No Fig. (2). Non-specific interaction measured by contact mode AFM in air. (a) Membrane ultrastructure of CD 8 + T cell; (b) sketch drawing of AFM tip scanning across membrane surface (not to scale), illustrating the interaction between tip and membrane protein (enlarged view); (c, d) representative force-distance curves of resting CD 8 + T cells acquired by bare tip; (e, f) representative force-distance curves of activated CD 8 + T cells acquired by bare tip; (g, h) sketch drawings of non-specific interaction between tip and cell membrane (not to scale), in which the aqua curves between tip and cell membrane intuitively denote the potential bonds formation (or binding events) that correspond to the black arrows shown in Fig. d and f; (i, j) statistical distribution of non-specific force of resting CD 8 + T cells (i) and activated CD 8 + T cells (j). ing and activated cells may be due to the conformational variation of adhesion receptors on T cells, such as selectins and integrins [2, 25], and/or the topographical and nanostructural difference between resting and activated T cells (Fig. S1 and Table 1 in supplementary material). From the results of non-specific interaction between bare tip and cell membrane, we can draw the following conclusions: 1) the adhesion force of CD + 8 T cells was increased due to the PDB plus ION stimulation; 2) PDB plus ION stimulation did not induce the obvious alteration in cell stiffness according to the primary analysis of the slope of approaching portion of force curves (data not shown here); 3) force spectroscopy can be used as a helpful nanotechnology to evaluate the potential changes in nano-mechanics of cells that are in different conditions. In addition, it is worth noting that the measurements of the specific interaction (membrane receptors-ligands) may be affected by non-specific interaction (bare tipmembrane). Therefore, we extended our force spectroscopy measurements of antibody-antigen in liquid to avoid such potential effects of non-specific interaction Nano-Mechanics and Receptor Detection of Resting and Activated CD + 8 T Cells In Situ Furthermore, the force-distance curve analysis was performed in PBS (ph 7.2) solution to map the spatial distribution of functional sites at which membrane CD 8 antigens are expressed on living CD + 8 T cells that can specifically capture CD 8 antibodies (Fig. 3a-3f). According to the specific interaction force between anti-cd 8 antibodies and surface CD 8 antigen, the 3-D map (Fig. 3a) of CD 8 molecules distributing on cell membrane (Fig. 3b) was reconstructed. In Fig. (3a), the force value decreases from Red color to Aqua color, implying the changes in the number of CD 8 molecules on cell membrane. The 3-D reconstruction (Fig. 3a) and the grayscale map (Fig. 3e) of CD 8 molecule together demonstrate that CD 8 molecules distribute non-uniformly on cell membrane and apparently concentrate into nanoclusters or nanodomains. Moreover, only partial CD 8 molecules on CD + 8 T cell expose their high affinity sites in a certain orientation at which they can capture or interact with specific antibodies; therefore, even though force spectroscopy cannot necessarily provide the exact amount of membrane receptors, the distribution pattern of specific receptors could be qualitatively determined. It should be noted that this observed distribution pattern of CD 8 molecules on T cell membrane is similar to that of CD 4 molecules (another TCR) [18] and TCR [26]. On the other hand, the force spectroscopy based 3-D reconstruction provides a direct visualization approach for mapping the distribution pattern of membrane surface receptors on single living cell surface. According to the retraction branch of force-distance curve (Fig. 3f), the number of specific binding events and the rupture force of

4 468 Current Nanoscience, 2009, Vol. 5, No. 4 Wu et al. Fig. (3). Specificity force spectroscopy-based 3-D reconstruction. AFM images and force-distance curves of resting (a-f) and activated (g-l) CD 8 + T cells o- btained in PBS solution. (a) Force spectroscopy based 3-D reconstruction map of CD 8 molecules distributing on cell membrane (b) of CD8+ T cells; (c) sketch drawing (not to scale) of specific interaction between anti-cd 8 molecules (green crosses) on tip and CD 8 molecules on cell membrane (aqua U-shape), which intuitively illustrates the separation of tip from cell surface and the potential bond numbers that are shown by brown broken arrows in (f); (d) two representative force-distance curves acquired using bare tip and anti-cd 8 antibodies functionalized tip in PBS solution, respectively; (e) grayscale map of CD 8 molecule distribution on membrane. (g) Force spectroscopy based 3-D reconstruction map of CD 69 molecule distributing on cell membrane (h) of activated CD8+ T cells; (i) sketch drawing (not to scale) of specific interaction between anti-cd 69 molecules (purple crosses) on tip and CD 69 molecules on cell membrane (aqua U-shape), which evidently displays the separation of tip from cell surface and the potential bond numbers that are shown by brown broken arrows in (l); (j) two representative force-distance curves acquired by bare tip and anti-cd 69 antibodies functionalized tip in PBS solution, respectively; (k) grayscale map of CD 69 molecule distribution on membrane. receptor CD 8 antibody bonds (Fig. 3c) can also be quantitatively determined. Fig. (3f) depicts a typical force-distance curve presenting six rupturing events (brown arrows) when anti-cd 8 functionalized tip is pulling away from cell membrane, and the force required for rupturing the bindings and the pulling length of every binding peptide molecule are respectively shown in Fig. (3f) (arrows and numbers). The absence or presence of specific TCRs on CD + 8 T cells must therefore reflect the overall specific surface properties of cells as well. To estimate the potential variation of membrane property of CD + 8 T cells due to PDB plus ION stimulation, we then conducted the force-distance curve analysis to determine the spatial distribution of binding sites at which responsive marker CD 69 molecules expressed on activated CD + 8 T cells (Fig. 3g-3l). The 3-D reconstruction (Fig. 3g) and the grayscale map (Fig. 3k) together suggest that CD 69 molecules also distribute non-uniformly on cell membrane and are shown as nanoclusters or nanodomains. And the four specific binding events (brown arrows) are observed between anti- CD 69 functionalized tip and cell membrane, as shown in Fig. (3l). Similarly, the rupture force of CD 69 -anti CD 69 binding and the pulling length of every binding event were also measured, as shown in Fig. (3l). Interestingly, comparison of force curves suggests that the cell stiffness of activated CD 8 + T cells is slightly smaller than that of resting CD 8 + T cells, which probably implies the cytoskeleton rearrangement upon the expression of specific receptors; their similar stiffness, however, may be responsible for their similar capability of circulation or infiltration. 4. CONCLUSIONS The present work is the first report on the application of single molecule force spectroscopy of antigen-antibody interaction to map receptor sites on living CD + 8 T cell membrane surfaces. In this re-

5 Nano-Mechanics and Membrane Receptor Mapping Current Nanoscience, 2009, Vol. 5, No port, the membrane nanostructures and the local biophysical properties of human peripheral blood CD 8 + T cells were obtained, quantitatively revealing the variation of CD 8 + T cells before and after stimulation of phorbol dibutyrate (PDB) plus ionomycin (ION), for example, cell volume, cell diameter, cell height, mean height of membrane surface nano-domains, average roughness of membrane nanostructures (see Supplementary Materials), and adhesion force increased after PDB plus ION stimulation. Furthermore, the 3-D distribution pattern and grayscale map together reveal that membrane receptors (CD 8 and activation marker CD 69 ) distribute nonuniformly on the membrane and are shown as micron-, nano- clusters or domains. In conclusion, AFM-based nanotechnology not only leads to the visualization of cell membrane nanostructures, but can also help us determine the adhesion force of cell membrane and the spatial distribution of receptor on living T cells, which therefore could lead us to further understand the biological function of CD 8 + T cells. ACKNOWLEDGEMENT This work is supported by the general project of National Natural Science Foundation of China (NSFC) (NO , NO & NO ) (J. C.), the general project of NSFC (NO ) and the major project of NSFC (NO ) (X. H.). We also thank Dr. Anhong Zhou (Department of Biological and Irrigation Engineering, Utah State University) for proof reading this manuscript. SUPPLEMENTARY MATERIAL Supplementary material is available on the publishers Web site along with the published article. REFERENCES [1] Mekala, D.J.; Geiger, T.L. Functional segregation of the TCR and antigen- MHC complexes on the surface of CTL. J. 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