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.

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules / Kellermayer, Miklós S. Z; Karsai, Árpád; Kengyel, András; Nagy, Attila; Bianco, Pasquale; Huber, Tamás; Kulcsár, Ágnes; Niedetzky, Csaba; Proksch, Roger; Grama, László. - In: BIOPHYSICAL JOURNAL. - ISSN 0006-3495. - STAMPA. - 91:(2006), pp. 2665-2677. [10.1529/biophysj.106.085456]

Spatially and temporally synchronized atomic force and total internal reflection fluorescence microscopy for imaging and manipulating cells and biomolecules

BIANCO, PASQUALE;
2006

Abstract

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.
2006
91
2665
2677
Kellermayer, Miklós S. Z; Karsai, Árpád; Kengyel, András; Nagy, Attila; Bianco, Pasquale; Huber, Tamás; Kulcsár, Ágnes; Niedetzky, Csaba; Proksch, Roger; Grama, László
File in questo prodotto:
File Dimensione Formato  
Kellermayer 2006.pdf

accesso aperto

Descrizione: Articolo principale
Tipologia: Pdf editoriale (Version of record)
Licenza: Open Access
Dimensione 1.55 MB
Formato Adobe PDF
1.55 MB Adobe PDF

I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1051821
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 49
  • ???jsp.display-item.citation.isi??? 44
social impact