Two-for-one: The authors discuss a powerful new tool that, in a single dynamic force microscopy scan, maps the topography of a surface and simultaneously recognizes specific target sites (see figure). Topography and recognition images correlated nicely at a lateral resolution of a few nanometers. This clever technique has great potential for a broad range of applications involving the detection of particular binding sites in native biological systems.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59±2 pN, which correlated nicely to the 51±1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.

Non-specific adsorption of proteins at solid/liquid interfaces is a major problem in the use of synthetic biomaterials and in ultrasensitive detection methods. Grafting surfaces with a dense layer of poly(ethylene glycol) (PEG) or other polymers is a most widely used strategy to solve this task. While such modified surfaces have been characterized by their ability to resist protein adsorption, the polymer layers themselves have rarely been studied in fine detail. Atomic force microscopy (AFM) using the pulsed force mode (PFM), is an ideal technique to investigate structural features and physiochemical properties of surfaces because topology and adhesion are simultaneously detected with high lateral resolution. In the present study, PFM-AFM was applied to thoroughly characterize different stages of glass derivatization, up to the formation of a dense PEG layer. Lateral inhomogeneities in topology and/or adhesion were observed at all stages before PEG attachment. The covalently bound PEG, however, was seen to form a densely packed monolayer with maximal thickness, smooth surface, and weak adhesion. Thus, PFM-AFM appears to be a valuable tool for the characterization of protein-repelling surfaces in solution. (C) 2005 Wiley-Liss, Inc.

We studied the frequency response of a magnetically driven atomic force microscope (AFM) cantilever close to a sample surface in liquids. Amplitude-frequency (tuning) curves showed pronounced differences in dependence on the tip-sample separation (from 1 to 50 microm), with significant shifts of the resonance peak. A model was developed in which the cantilever was described in a full shape manner and the hydrodynamic forces acting on the cantilever were approximately calculated. The slight inclination of the cantilever to the surface (alpha approximately 15 degrees) leads to a force profile along the cantilever. Therefore, the mathematical problem can be strictly solved only numerically. For an approximate analytical solution, the hydrodynamic force profile was approximated by a constant force along the cantilever for large separations and by a point force acting on the tip of the cantilever for small separations. The theoretical results calculated within this model agreed well with the experimental data and allowed to determine the cantilever mass in liquid M*, the joint mass at the tip end m*t, and the coefficient of viscous interaction of the cantilever with free liquid, gamma(infinity).

Tether them! A matrix of proteins that are covalently bound to mica with only one short tether per protein was created in order to fulfil all requirements for molecular recognition force microscopy of single proteins (see picture). This immobilization technique produces a high density of individual proteins on a flat surface in liquid with extremely low surface roughness.