Regensburg 2016 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 48: Posters - Bioimaging and Spectroscopy

BP 48.10: Poster

Mittwoch, 9. März 2016, 17:00–19:00, Poster C

Confocal Light-Sheet Microscopy: Separation of ballistic and diffusive fluorescence photons — •Tobias Meinert and Alexander Rohrbach — Laboratory for Bio-and Nano-Photonics, University of Freiburg, Germany

In the last ten years light-sheet microscopy has got more and more attention in biological research and is on its way to become the standard technology for long time observation of thick samples. However, the observation of strongly scattering objects suffers from strong imaging artefacts. In particular, the scattering of coherent illumination light generates strong image artifacts. Microscopy with Self-Reconstructing Beams (MISERB), such as Bessel beams, has proven to be a powerful tool to reduce scattering artifacts. By imaging 150 µ m thick Arabidopsis root tips, these effects become well visible.

Reduced contrast due to strong side loops in the Bessel beam profile can be compensated effectively by confocal line detection. Independently of the illumination beam, a general source of reduced contrast is the scattering of fluorescent photons emitted from layers deep inside the object. This effect usually becomes very dominant at an imaging depth of a few 10 µ m. In this presentation a so-called object point spread function (PSFobj) is introduced, which describes the blurring of images due to the object itself. By estimating this function, it is possible to separate the influence of ballistic (unscattered) and diffusive (multiply scattered) photons on the imaging process. Removing the influences of the diffusive photons, a so far unreached image quality in strongly scattering media is obtained.