Adding a narrow-spectrum color filter to the light path improves coherence and contrast in our images. This could be seen as preventing chromatic dispersion from the cell-body lenses. Although a green filter was used initially to enhance contrast with the pink cell culture medium, experiments with a red monochromatic filter (Kodak Wratten filter #61) yielded equally good results (not shown).
Swelling cell bodies by treating with PBS enhanced cell curvature and thus enhanced the lensing effect, substantially improving cell counting accuracy. Under our experimental conditions, short-term incubation in PBS had no measurable effect on growth parameters, permitting two or more independent counts over the course of an experiment. While counting accuracy was improved at high cell densities, the technique reached its limit when imaging cells had grown into multiple layers.
Previously published strategies to enhance image contrast for bright-field microscopy have used contrast differentials from multiple z-stacked images (9, 10), or have determined cell density through measuring the size of confluent areas and multiplying by a calculated density factor (17). These strategies are best suited for automated image acquisition. The method presented here permits cell counting by use of a simple bright-field microscope with very minor hardware modifications. It allows repeated, quick, and simple counting in routine cell culture environments and should be a versatile quality-control tool for a variety of mammalian cell culture applications.
The SH-EP cell line was a gift from R. König, F Westermann and M. Schwab (DKFZ, Heidelberg, Germany). We gratefully acknowledge the use of the fluorescence microscope of S. Sakiyama- Elbert, BME Washington University in St. Louis. LLD wishes to thank G.F. Schreiner, Washington University Medical School, for giving access to his laboratory and equipment while the imaging method was refined. This research was in part financially supported by the DRC at Washington University (NIH Grant No. 5 P30 DK020579), the German Science Foundation (DFG, BI 1409/1-1), and by the German Ministry for Science and Education (BMBF, NGFN-Plus 01GS08132, GERAMY 01GM1107C). This paper is subject to the NIH Public Access Policy.
The authors declare no competing interests.
Correspondence Address correspondence to Jan Bieschke, Washington University in St. Louis, Department of Biomedical Engineering, St. Louis, MO. E-mail: [email protected]">[email protected]References
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