Quantification of dendritic spine populations using image analysis and a tilting disector.

A series of image analysis routines, stochastic geometry methodology, and a design-based stereological procedure have been developed to quantify objectively the length, layout, and the true density of neuronal dendritic spines observed at the light (or confocal) microscope level. First, the image of a dendritic fragment of interest (in the plane of view) is scaled to a standard brightness scale, and the dendritic profile is separated from the background using a computerized thresholding algorithm that analyzes the histogram of grey levels. Secondly, the resulting binary image of the dendrite is transformed to a midline skeleton that underlies the dendritic geometry. Thirdly, skeletal branch lengths are directly computed (in pixels), thus giving objective measures of visible spine lengths and inter-spine distances along the dendritic stem. These raw data are the basis for (1) an estimation of the distribution of 3D spine lengths, and (2) a nearest neighbour analysis of the spine layout along the dendrite. A design-based stereological routine, the tilting disector, is suggested for unbiased estimation of the true (3D) density of spines along dendrites. The routine involves tilting the dendritic fragment of interest around its longitudinal axis for a known angular sector and scoring the number of spines seen in one angular position and unseen in the other position. Data from a study of neuronal dendrites in the chick forebrain are presented.