SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Spatially and temporally synchronized atomic force and
total internal reflection fluorescence microscopy for
imaging and manipulating cells and biomolecules
Miklós SZ Kellermayer 1*, Árpád Karsai 1, András Kengyel 1, Attila Nagy 1, Pasquale Bianco 1, Tamás Huber 1, Ágnes Kulcsár 2, Csaba Niedetzky 3, Roger Proksch 4 and László Grama 1
1 University of Pécs
2 University of Heidelberg
3 Supertech Ltd
4 Asylum Research
* To whom correspondence should be addressed. E-mail: miklos.kellermayer.jr{at}aok.pte.hu.
Submitted on March 20, 2006
Revised on April 1, 2006
Accepted on 6 July 2006
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Abstract |
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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 co-aligned to achieve spatial synchrony. At each point of scan the sample topography (AFM) 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.
Key Words:
actin, fluorescence microscopy, molecular mechanics, monolayer cells, titin, topography
