title: Counting unstained, confluent cells by modified bright-field microscopy.
abstract: 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.
authors.affiliation: Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
authors.forename: L Louis
authors.initials: LL
authors.lastname: Drey
authors.forename: Michael C
authors.initials: MC
authors.lastname: Graber
authors.forename: Jan
authors.initials: J
authors.lastname: Bieschke
mesh_terms: D002452:Cell Count / Q000379:methods*; D045744:Cell Line, Tumor; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D016014:Linear Models; D008853:Microscopy / Q000379:methods*; D012984:Software