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Laser Scanning Microscopy and Quantitative Image Analysis of Neuronal Tissue

Volume 87 of the series Neuromethods pp 163-188

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Zebrafish Brain Development Monitored by Long-Term In Vivo Microscopy: A Comparison Between Laser Scanning Confocal and 2-Photon Microscopy

  • Nicolas DrossAffiliated withNikon Imaging Center, University of HeidelbergCenter for Organismal Studies, Heidelberg University
  • , Carlo Antonio BerettaAffiliated withDepartment of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University
  • , Peter BankheadAffiliated withNikon Imaging Center, University of HeidelbergCenter for Organismal Studies, Heidelberg University
  • , Matthias CarlAffiliated withDepartment of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg University
  • , Ulrike EngelAffiliated withNikon Imaging Center, University of HeidelbergCenter for Organismal Studies, Heidelberg University

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Abstract

Zebrafish is an attractive model organism to study vertebrate brain development. Its transparency makes it possible to follow development using live imaging. In a transgenic line where a subset of neurons is labeled by GFP expression, their migration, proliferation and the extension of axons can be observed by laser scanning confocal microscopy (LSCM) or 2-photon microscopy (2PM). However, when the whole brain is imaged, LSCM might result in phototoxicity. In contrast, 2PM allows for image acquisition over several days at intervals shorter than an hour. In this article, we describe a method to image a large region of the brain (500 × 500 μm) spanning 300 μm in depth by 2PM over 2 days or more. The results are compared with those obtained by the more widespread LSCM. Visualization and analysis of the resulting data is challenging, as they exceed the size that can be loaded into standard rendering software. We propose a routine to reduce the size by maximum projection while keeping and displaying three-dimensional information by a color code within ImageJ.

Key words

Zebrafish Axon extension Laser scanning confocal microscopy 2-Photon microscopy Phototoxicity Photobleaching In vivo Long-term imaging