Computer-aided 3-dimensional reconstruction of stained neurones viewed in wholemount

Programming details of a simple microcomputer-based system for aiding three-dimensional reconstruction of neuronal anatomy are described. The X and Y axes are obtained by tracing the neural outline onto a digitizing tablet using a camera lucida, whereas the Z axis is obtained by digitizing the microscope focus position. The computer accepts data into a storage array when the pen is down, and the digitizer pen or microscope focus is being moved. A 'pen-up' flag is inserted when the pen is raised, as occurs when moving from tracing one dendrite to another. Thus, the neural structure is coded as a 'stick diagram' within the computer in the form of a list of coordinate triplets, each representing a point in space occupied by part of the neurone. Sequential triplets in the list represent contiguous points in space (e.g., a dendrite), whereas a pen-up flag character inserted in the list indicates that no structural connection exists between the preceding and following coordinate points. Routines are presented for developing a transformation matrix which allows the database containing the stick diagram to be scaled, translated, and rotated in three dimensions. The stick diagram can then be drawn as a two-dimensional orthogonal projection, plotting the transformed X-Y coordinates with the points joined by a line unless separated by a pen-up flag. In this manner, the neural structure can be presented as if viewed from any angle.