To address certain questions of neuronal development, in particular the process of neuronal circuit formation, spatial resolution at the EM level is essential. Almost 30 years ago this was demonstrated in "The Mind of the Worm" (White et al. 1986)1, an electron microscopical (EM) reconstruction of the nervous system of C. elegans based on ultrathin serial sections. This work provided the reference for C. elegans neuroanatomy down to the level of individual synaptic connections. Since then very few 3D reconstructions of worms have been published, obviously due to the immense effort in time and labor it takes to accomplish such a task. A recent development at our institute offers a novel solution to the problem of reliable large scale serial sectioning for electron microscopy. A scanning electron microscope was equipped with an ultramicrotome inside the vacuum chamber, which allows to image the block-face of a sample (Denk and Horstmann 2004)2. Several hundred sections of a sample can be produced and imaged reliably avoiding tedious collection of serial sections by hand and simplifying alignment of sections. We are testing whether this machine would make EM analysis of some of our axon guidance mutants feasible, in particular addressing questions of synapse formation in mutants with axonal outgrowth defects. Block-face imaging precludes poststaining of sections and we have to find a way to introduce sufficient contrast during fixation. Currently we are optimizing fixation protocols on mixed populations of worms using high pressure freezing and freeze substitution. We will present first data stacks and discuss the potential use of the method.1 White et al. (1986) Phil. Trans. R. Soc. Lond. B314, 1-3402 Denk W, Horstmann, H (2004) PLoS Biol 2 (11):
e329