Three-dimensional reconstruction of neurons in the lamprey spinal cord in whole-mount, using a confocal laser...

When investigating the detailed morphology of nerve cells, three-dimensional structural information is often of great value. We present here a technique by which 'optical sectioning' and three-dimensional reconstruction of fluorescence-labelled neurons in the lamprey spinal cord has been performed by means of a confocal microscope scanner with a laser beam as the light source. In confocal microscopy only a small spot of the specimen is being illuminated at any one time, and only light from the illuminated spot is detected. This gives several advantages compared with traditional microscopy: (1) Lateral resolution is improved, and any 'halo' effects occurring around structures with intense fluorescence are drastically reduced. In addition (2), a unique depth resolution is obtained due to the strong attenuation of structures that are out of focus. This allows the system presented here to (3) perform a detailed three-dimensional computer reconstruction of the neuron, without any need for physical sectioning of the tissue. The volume of data points sampled can subsequently be treated in various ways, including selection of different viewing angles, enhancement of contours, and background suppression.