Although various light microscopic techniques (e.g. 1- and 2-Photon laser scanning microcopy) add valuable insights the required information to establish a detailed and complete functional circuit diagram at synaptic resolution can only be obtained with electron microscopy (EM). To this end several 3D EM techniques had been developed over the past few years. To study the anatomy of the fly visual system we cooperate with Winfried Denk and use the Serial-Block-Face-Scanning-Electron–Microscopy technique (SBEM) introduced in 2004 by him (Denk & Horstmann, 2004). This technique combines block-face imaging of backscattered electrons (BSE detection) under low voltage and low vacuum conditions with serial tissue sectioning inside the vacuum chamber of a scanning electron microscope (Fig. 1a). This combination of serial ultra-thin sectioning with high resolution EM-imaging opens the possibility to obtain large 3D EM volume data sets (Fig. 1b) containing for example the complete neuronal circuitry of the Drosophila melanogaster optic lobe (app. 160 x 150 x 200 µm).
Prior to sectioning and imaging with the SBEM the fly brains are labeled with a variety of heavy metal stains and embedded in epoxy resins. To this end we develop optimized staining and embedding techniques to achieve on the one hand a sufficiently high signal to noise ratio in BSE imaging and on the other hand are able to reach the necessary voxel resolution (< 10nm in X/Y and < 25nm in Z) to detect and subsequently reconstruct all neuronal processes as well as chemical synapses.
The vast amount of data created within one SBEM experiment (several TB) creates the necessity to develop new ways for data handling, visualization and reconstruction of neurons and synapses. In this part of the project we use the software tool Knossos (www.knossostool.org) and cooperate with the Research Group Structure of Neocortical Circuits at the Institute.
To dissect the neuronal circuits and further understand the computations leading to the Lobula Plate Tangential cells (LPTCs) direction selective motion vision response we currently reconstruct the complete neuronal network of the Drosophila melanogaster Lobula Plate. The Lobula Plate connectome will provide us with new insights on the intrinsic structure – function relationship as well as guide our identification of presynaptic input elements and postsynaptic targets of LPTCs.
Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2, e329 (2004).