LP-38 Correlative Microscopical Studies of the Secretory and Endocytic Pathway using DAB-Photoconversion

We are particularly interested in the dynamic compartments at the interface at the biosynthetic and endocytic systems. To characterize membranous organelles in these regions, we established a combined light-and electron microscopical technique that allows a detailed analysis and characterization of these crossroads within the cells. Hereby, we took advantage of the in vivo visualisation and time resolution by means of life cell analysis combined with the fine structural resolution at the EM level from the same probes. For this, the fluorescence signal of the lipophilic fluorophore BODIPY (4,4-difluoro-3a,4a-diaza-s-indacene; Molecular Probes) was converted into an electron dense precipitate via diaminobenzidine (DAB)-photoconversion. The technique, first described by Maranto et al, 1982, was used to visualize membranes and organelles of the endocytic pathway. During photoconversion, the DAB in Tris buffer is oxidized by oxygen radicals emitted from the fluorophore during bleaching and precipitated in th