3‐D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series

A confocal laser scanning microscope (CLSM) collects information from a thin, focal plane and ignores out‐of‐focus information. The operator configures separate channels (laser, filters, detector settings) for each fluorochrome used in a particular experiment. Then, 3‐D reconstructions are made from Z‐series of confocal images: one series per channel. Channel signal separation is extremely important and measures to avoid bleaching are vital. Post‐acquisition deconvolution of the image series is then performed to increase resolution. In the 3‐D reconstruction program described in this unit, reconstructions can be inspected in real time from any viewing angle. By altering viewing angles and by switching channels off and on, the spatial relationship of 3‐D‐reconstructed structures with respect to structures seen in other channels can be studied. Since each brand of CLSM, computer program, and 3‐D reconstruction package has its own proprietary set of procedures, a general approach is provided wherever possible.