Counting unstained, confluent cells by modified bright-field microscopy. (July 2013)

• Record Type: Journal Article
• Authors: Drey, L. Louis; Graber, Michael C.; Bieschke, Jan
• Is Part Of: Biotechniques. Volume 55:Number 1(2013)
• Page Start: 28
• Page End: 33
• Publication Date: 2013-07
• Subjects: brightfield -- microscopy -- fluorescence -- cell culture -- cell counting -- pinhole -- monochromatic -- PBS -- defocusing; Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000114056
• Languages: English
• ISSNs: 0736-6205

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.