Simultaneous topography and recognition imaging on endothelial cells.

Determining the landscape of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometres) is an important task in many fields of biological science. During the past five years, dynamic recognition imaging (e.g. simultaneous topography and recognition (TREC) imaging) has proven to be a powerful technique in biophysical research. This technique becomes an indispensable tool for high-resolution receptor mapping as it has been successfully demonstrated on different biomolecular model systems. In these studies, the topographical imaging of receptor molecules is combined with molecular recognition by their cognate ligands bound to the atomic force microscope (AFM) tip via a flexible and distensible tether. In this review, we describe the principles of TREC imaging and provide a flavour of its recent application on endothelial cells.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Covalent attachment of proteins to calcite surface for the single molecule experiments.

Two protocols of covalent attachment of proteins onto the calcite surface, viz. one using the metallochelat and second using the aminohexil, are elaborated. Single molecule force spectroscopy method has been used to test their efficiency and practical applicability. Experiments were performed measuring the specific interaction force between bovine serum albumin (BSA) fixed onto the freshly cleaved calcite single crystal surface (procedure under the study here) and its polyclonal antibody (Ab-BSA) immobilized onto an AFM tip using standard and well studied procedure. We found the conditions, when up to 3-3.5% of tip-sample approaches lead to the formation of a single specific bond.

Force-clamp spectroscopy with a small dithering of AFM tip, and its application to explore the energy landscape of single avidin-biotin complex.

We have recently developed a new method for directly measuring the spring constant of single molecules and molecular complexes on a real-time basis [L.A. Chtcheglova, G.T. Shubeita, S.K. Sekatskii, G. Dietler, Biophys. J. 86 (2004) 1177]. The technique combines standard force spectroscopy with a small dithering of tip. Changes in the amplitude of the oscillations are measured as a function of the pulling-off force to yield the spring constant of the complex. In this report, we present the first results of combination of this approach with the force-clamp spectroscopy. The standard atomic-force microscope has been supplemented with an electronic unit, which is capable of realizing an arbitrary force function, and permits the force-loading regime to be interrupted at any time. Using this method, the time needed to rupture a single bond can be measured as a function of the force that is required to maintain the complex in a stretched condition. The energy landscape of the avidin-biotin complex is explored and discussed.