Automated imaging of extended tissue volumes using confocal microscopy

• Published in: Microscopy research and technique 2005-08, Vol.67 (5), p.227-239
• Main Authors: Sands, Gregory B., Gerneke, Dane A., Hooks, Darren A., Green, Colin R., Smaill, Bruce H., Legrice, Ian J.
• Format: Article
• Language: English
• Subjects: Animals; Automation; Brain - ultrastructure; confocal microscopy; Heart Ventricles - ultrastructure; Imaging, Three-Dimensional - instrumentation; Imaging, Three-Dimensional - methods; Microscopy, Confocal - instrumentation; Microscopy, Confocal - methods; microstructure; Rats; tissue processing; visualisation; volume imaging
• ISSN: 1059-910X; 1097-0029
• DOI: 10.1002/jemt.20200

Confocal microscopy enables constitutive elements of cells and tissues to be viewed at high resolution and reconstructed in three dimensions, but is constrained by the limited extent of the volumes that can be imaged. We have developed an automated technique that enables serial confocal images to be acquired over large tissue areas and volumes. The computer‐controlled system, which integrates a confocal microscope and an ultramill using a high‐precision translation stage, inherently preserves specimen registration, and the user control interface enables flexible specification of imaging protocols over a wide range of scales and resolutions. With this system it is possible to reconstruct specified morphological features in three dimensions and locate them accurately throughout a tissue sample. We have successfully imaged various samples at 1‐μm voxel resolution on volumes up to 4 mm3 and on areas up to 75 mm2. Used in conjunction with appropriate embedding media and immuno‐histochemical probes, the techniques described in this paper make it possible to routinely map the distributions of key intracellular structures over much larger tissue domains than has been easily achievable in the past. Microsc. Res. Tech. 67:227–239, 2005. © 2005 Wiley‐Liss, Inc.