Three‐dimensional counting: An accurate and direct method to estimate numbers of cells in sectioned material

Abstract

We introduce a way to count and measure cells in an optically defined volume of tissue called a counting box. This method—direct three‐dimensional counting (3DC)—eliminates the need for correction factors, such as that introduced by Abercrombie (Anat. Rec. 94:239–247, '46), to determine the number of cells per unit volume (N_v). Problems caused by irregular cell shape and cell size, nonrandom orientation, and splitting of cells by the knife during sectioning are overcome. Furthermore, 3DC is insensitive to large variations in section thickness. The innovative feature of 3DC is the definition of a counting box with top and bottom sides located inside the section a precise distance away from the cut surfaces of the tissue. The positions of the top and bottom sides of the counting box are delimited by using a digital length gauge and a Z‐axis control unit. Sections of tissue between 8 and 100 μ thick are examined with a high numerical aperture objective in combination with video‐enhanced differential interference contrast optics (DIC). Cells are marked on a television screen while the microscopist scans systematically from the top to the bottom of the counting box. Cells that are located completely inside the box and cells that only cross through its top, right, or back sides are counted. All cells that cross the planes that define the bottom, left, and front sides of the counting box are not counted. Direct 3DC provides an accurate, simple, and reliable way to count cells, nuclei, nucleoli, or other objects in sectioned material. To build the counting system requires a standard transmitted light microscope equipped with DIC optics, a video camera, a position sensor, and a Z‐axis control unit.