STED Imaging in Drosophila Brain Slices
Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.
Key wordsSTED Drosophila melanogaster Immunofluorescence Cryostat sectioning Brain slice
We are indebted to H. Leonhardt and the BioImaging Network Munich for generous support. We thank Marianne Braun and Ursula Weber for excellent help with technical procedures, and Aljoscha Nern, Gerald M. Rubin, and Barry Dickson for providing transgenic flies.
- 12.Rodríguez AdV, Didiano D, Desplan, C (2011) Power tools for gene expression and clonal analysis in Drosophila. NatureMethods 9(1):47–55Google Scholar
- 13.Venken KJT, Simpson JH, Bellen HJ (2011) Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72: 202–230Google Scholar
- 14.Nern A, Pfeiffer BD, Svoboda K, Rubin GM (2011) Multiple new site-specific recombinases for use in manipulating animal genomes. Proceedings of the National Academy of Sciences of the United States of America 108(34):14198–14203Google Scholar
- 15.Nern A, Pfeiffer BD, Rubin GM (2015) Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system. Proceedings of the National Academy of Sciences of the United States of America 112(22):E2967–E2976Google Scholar
- 16.Bellen HJ, Tong C, Tsuda H (2010) 100 years of Drosophila research and its impact on vertebrate neuroscience: a history lesson for the future. Nat. Rev. Neurosci. 11:514–522Google Scholar
- 17.Kazama H (2015) Systems neuroscience in Drosophila: conceptual and technical advantages. Neuroscience 296:3–14Google Scholar
- 18.Maisak MS, Haag J, Ammer G, Serbe E, Meier M, Leonhardt M, Schilling T, Bahl A, Rubin GM, Nern A, Dickson BJ, Reiff DF, Hopp E, Borst A (2013) A directional tuning map of Drosophila elementary motion detectors. Nature 500:212–216Google Scholar
- 19.Wagh DA, Rasse TM, Asan E, Hofbauer A, Schwenkert I, Duerrbeck H, Bucher S, Dabauvalle MC, Schmidt M, Qin G, Wichmann C, Kittel R, Sigrist SJ, Bucher E (2006) Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila. Neuron 49(6):833–844Google Scholar