RESUMO
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.
Assuntos
Citoesqueleto de Actina/metabolismo , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Subfragmentos de Miosina/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Conectina , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia de Força Atômica/instrumentação , Microscopia de Fluorescência/instrumentação , Proteínas Musculares/metabolismo , Proteínas Musculares/ultraestrutura , Subfragmentos de Miosina/ultraestrutura , Miosinas/ultraestrutura , Proteínas Quinases/metabolismo , Proteínas Quinases/ultraestruturaRESUMO
The interactions between two poly(allylamine)/poly(styrene sulfonate) multilayers made of 4.5 and 5 bilayers are investigated by the surface force apparatus (SFA). As the two surfaces approach, one reaches a threshold point where a repulsion sets in, until they become barely compressible. Repetitive load/unload cycles show that, once compressed, the films remain almost in their compressed state. This indicates that the poly(allylamine)/poly(styrene sulfonate) films are in a glassy state, in marked difference with the SFA findings on poly-(L-lysine)/poly-L-glutamic acid) multilayers. These results are discussed in the light of linearly and exponentially growing films.