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Spatially and Temporally Synchronized Atomic Force and Total Internal Reflection Fluorescence Microscopy for Imaging and Manipulating Cells and Biomolecules

Miklós S. Z. Kellermayer ^*, Árpád Karsai ^*, András Kengyel ^*, Attila Nagy ^*, Pasquale Bianco ^*, Tamás Huber ^*, Ágnes Kulcsár , Csaba Niedetzky , Roger Proksch  and László Grama ^*

^* Department of Biophysics, University of Pécs, Faculty of Medicine, Pécs H-7624, Hungary; Institute of Physical Chemistry, University of Heidelberg, 69120 Heidelberg, Germany; Supertech Ltd., Pécs H-7624, Hungary; and Asylum Research, Santa Barbara, California 93117

Correspondence: Address reprint requests to Miklós S. Z. Kellermayer, Tel.: 36-72-536-271; Fax: 36-72-536-261; E-mail: miklos.kellermayer.jr{at}aok.pte.hu.

The atomic force microscope is a high-resolution scanning-probe instrument which has become an important tool for cellular and molecular biophysics in recent years but lacks the time resolution and functional specificities offered by fluorescence microscopic techniques. To exploit the advantages of both methods, here we developed a spatially and temporally synchronized total internal reflection fluorescence and atomic force microscope system. The instrument, which we hereby call STIRF-AFM, is a stage-scanning device in which the mechanical and optical axes are coaligned to achieve spatial synchrony. At each point of the scan the sample topography (atomic force microscope) and fluorescence (photon count or intensity) information are simultaneously recorded. The tool was tested and validated on various cellular (monolayer cells in which actin filaments and intermediate filaments were fluorescently labeled) and biomolecular (actin filaments and titin molecules) systems. We demonstrate that with the technique, correlated sample topography and fluorescence images can be recorded, soft biomolecular systems can be mechanically manipulated in a targeted fashion, and the fluorescence of mechanically stretched titin can be followed with high temporal resolution.