Regensburg 2007 – scientific programme

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BP: Fachverband Biologische Physik

BP 16: Poster Session I

BP 16.21: Poster

Tuesday, March 27, 2007, 17:00–19:30, Poster D

Single molecule microscopy using focal plane illumination — •Jörg Ritter, Werner Wendler, and Ulrich Kubitscheck — Institute of Physical and Theoretical Chemistry, Bonn, Germany

Single molecule fluorescence microscopy performed in spatially extended samples severely suffers from a high fluorescence background. To overcome this problem we used a focal plane instead of the conventional epi-illumination. By means of a custom made cylindrical lens system (NA 0.33) we created a light sheet with a Rayleigh length of 37 μm, a FWHM width of 8.3 μm, and a FWHM thickness of 2 μm within the object plane of a detection objective lens. In this manner a simple optical sectioning microscope was created (Voie et al., 1993). The light sheet was produced inside a water chamber, where the sample was fixed within an agarose gel cylinder on a micrometer stage (Huisken et al., 2004). Fluorescence light was detected perpendicular to the illumination plane by a water-dipping microscope objective lens (60X, NA 1.0) and imaged onto an EMCCD. Only the plane of interest was illuminated and affected by photobleaching. Movement of the stage allowed the acquisition of 3D image stacks. Excitation in the focal plane only resulted in a striking reduction of fluorescence background. The axial resolution was determined by the light sheet thickness and the resolving power of the detection objective lens, and was determined as 1.35 μm FWHM at 680 nm (theoretical expectation, 1.17 μm). The penetration depth of the optical sectioning was limited by the working distance of the water-dipping microscope objective (2.5 mm).