Journal
JOURNAL OF MICROSCOPY
Volume 248, Issue 3, Pages 292-298Publisher
WILEY
DOI: 10.1111/j.1365-2818.2012.03673.x
Keywords
Confocal microscopy; inner ear; mouse; segmentation; three-dimensional reconstruction; sTSLIM
Categories
Funding
- Roy J. Carver foundation
- NIH
- NIDCD [RO1-DC055095590]
- ICTS [TL1 CTSA UL1RR024979]
- NIH [DC010362]
- University of Iowa Carver College of Medicine
- Medical Scientist Training Program
- Office of the Vice President for Research
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Three-dimensional (3D) reconstructions of the vertebrate inner ear have provided novel insights into the development of this complex organ. 3D reconstructions enable superior analysis of phenotypic differences between wild type and mutant ears but can result in laborious work when reconstructed from physically sectioned material. Although nondestructive optical sectioning light sheet microscopy may ultimately prove the ideal solution, these technologies are not yet commercially available, or in many instances are not monetarily feasible. Here we introduce a simple technique to image a fluorescently labelled ear at different stages throughout development at high resolution enabling 3D reconstruction of any component of the inner ear using confocal microscopy. We provide a step-by-step manual from tissue preparation to imaging to 3D reconstruction and analysis including a rationale and troubleshooting guide at each step for researchers with different equipment, protocols, and access to resources to successfully incorporate the principles of this method and customize them to their laboratory settings.
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