Berlin 2015 – scientific programme

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BP: Fachverband Biologische Physik

BP 1: Imaging

BP 1.2: Talk

Monday, March 16, 2015, 10:00–10:15, H 1028

Reflected light-sheet microscopy reveals single molecules in Drosophila melanogaster embryo — •Ferdinand Greiss¹, Myrto Deligiannaki², Christophe Jung², Ulrike Gaul², and Dieter Braun¹ — ¹System Biophysics, Department of Physics, Ludwig Maximilians University, Amalienstr. 54, 80799 Munich, Germany — ²Gene Center, Department of Biochemistry, Ludwig Maximilans University, Feodor-Lynen-Str. 25, 81377 Munich, Germany

Searching the growing field of light microscopy for dynamic high resolution imaging in vivo suggests total internal reflection microscopy (TIRF) as one of the most popular candidates. However, TIRF has the major limitation that it is constrained to the imaging depth of only several hundred nanometers close to the coverslip surface. The need for alternative imaging techniques is therefore evident.

Here we report a method to reveal single molecules at various imaging depths and thicker tissues. We adapted and optimized reflected light-sheet microscopy (RLSM) as described by Gebhardt et al. (2013) and used it to study the epidermal barrier of Drosophila embryos.

Epidermal cells seal their intermembrane space by septate junctions, which impede free diffusion through the paracellular route. We were able to detect single 10 kDa Alexa647-conjugated Dextran diffusing in and around epidermal tissue. As discovered with ensemble measurements, mutants with disrupted septate junctions exhibit leaky epithelial barrier. We were able to confirm these findings with our optical setup on the single molecule level.