Counting unstained, confluent cells by modified bright-field microscopy

We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. Refraction by each cell body generates a sharp, bright spot when the image is defocused. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions using free, readily available image-analysis tools. Contrast may be enhanced by swelling cell bodies with a brief incubation in PBS. The procedure was benchmarked against manual and automated counting of fluorescently labeled cell nuclei. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown. On average, bright-field images produced the same counts as fluorescence images, with less than 5% error. This method will allow routine cell counting using a plain bright-field microscope without cell-line modification or cell staining.