Combining multichannel confocal laser scanning microscopy with serial section reconstruction to analyze large tissue volumes at cellular resolution

Abstract

Current advances in imaging techniques have extended the possibility of visualizing small structures within large volumes of both fixed and live specimens without sectioning. However, technical limits still hamper the use of these approaches to investigate deep anatomical regions. The alternative to intact structures imaging is serial section reconstruction, but this technique requires a high number of sections to acquire and register in order to reconstruct large volume of tissue at cellular resolution. Here, we present a method to combine multichannel confocal laser scanning microscopy (CLSM) and serial section reconstruction in order to reconstruct large volumes of tissue at cellular resolution using only few sections. In this method a series of thick sections is imaged with CLSM and the resulting stacks of images are registered and 3D reconstructed. This approach is compatible with all major fluorescent labeling techniques, does not requires specific optics, and is based on existing freeware software. © 2014 Springer Science+Business Media New York.