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Simultaneous Topography and Recognition Imaging Using Force Microscopy

Cordula M. Stroh ^*, Andreas Ebner ^*, Manfred Geretschläger , Günter Freudenthaler ^*, Ferry Kienberger ^*, A. S. M. Kamruzzahan ^*, Sandra J. Smith-Gill , Hermann J. Gruber ^* and Peter Hinterdorfer ^*

^* Institute for Biophysics and Institute of Experimental Physics, Atomic Physics and Surface Science, Johannes Kepler University of Linz, A-4040 Linz, Austria; and Basic Research Laboratory, Division of Basic Sciences, Frederick Cancer Research and Development Center, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201 USA

Correspondence: Address reprint requests to Peter Hinterdorfer, Institute for Biophysics, University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria. Tel.: 43-732-2468-9265; Fax: 43-732-2468-9280; E-mail: peter.hinterdorfer{at}jku.at.

We present a method for simultaneously recording topography images and localizing specific binding sites with nm positional accuracy by combining dynamic force microscopy with single molecule recognition force spectroscopy. For this we used lysozyme adsorbed to mica, the functionality of which was characterized by enzyme immunoassays. The topography and recognition images were acquired using tips that were magnetically oscillated during scanning and contained antibodies directed against lysozyme. For cantilevers with low Q-factor (1 in liquid) driven at frequencies below resonance, the surface contact only affected the downward deflections (minima) of the oscillations, whereas binding of the antibody on the tip to lysozyme on the surface only affected the upwards deflections (maxima) of the oscillations. The recognition signals were therefore well separated from the topographic signals, both in space (z 5 nm) and time (0.1 ms). Topography and recognition images were simultaneously recorded using a specially designed electronic circuit with which the maxima (U_up) and the minima (U_down) of each sinusoidal cantilever deflection period were depicted. U_down was used for driving the feedback loop to record the height (topography) image, and U_up provided the data for the recognition image.

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