Regensburg 2002 – wissenschaftliches Programm

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SYBM: Physik biologischer Materie

SYBM 401: Physics of biological matter: Poster

SYBM 401.9: Poster

Dienstag, 12. März 2002, 18:00–19:30, H37

Simultaneous Force Spectroscopy and optical TIR Microscopy studies on the binding of fluorescent TOPRO-3 to single DNA molecules — •Andrew Hards, Andreas Zumbusch, and Christoph Braeuchle — LMU Muenchen Institut fuer Physikalische Chemie

Abstract Using a combined optical imaging and atomic force spectroscopy setup, the simultaneous fluorescence imaging and mechanical stretching of individual DNA molecules was possible.

Phage DNA molecules, were immobilised on the glass surface and intercalated with the fluorescence dye TOPRO-3. Individual DNA strands could be elongated on the glass substrate by shear flow and consequently imaged with an inverted optical microscope using an attached fluorescence detecting CCD camera. With an AFM placed on top of the inverted microscope, individual DNA strands were then addressed and stretched with the AFM tip. To reduce background signal and eliminate autofluorescence from the tip, a recently developed imaging method called TIR (Total Internal Reflection) microscopy was employed.

Simultaneous force-extention curves and imaging sequences revealed insights into the nature of the bonds between the intercalated dye molecules and the DNA base pairs. After applying a definite mechanical stress to the strand, the spectroscopic changes in the emitted fluorescence and the binding characteristics of the dyes were examined.