Abstract
SummaryCell counting is commonly used to determine proliferation rates in cell cultures and for adherent cells it is often a ‘destructive’ process requiring disruption of the cell monolayer resulting in the inability to follow cell growth longitudinally. This process is time consuming and utilises significant resource. In this study a relatively inexpensive, rapid and widely applicable phase contrast microscopy‐based technique has been developed that emulates the contrast changes taking place when bright field microscope images of epithelial cell cultures are defocused. Processing of the resulting images produces an image that can be segmented using a global threshold; the number of cells is then deduced from the number of segmented regions and these cell counts can be used to generate growth curves. The parameters of this method were tuned using the discrete mereotopological relations between ground truth and processed images. Cell count accuracy was improved using linear discriminant analysis to identify spurious noise regions for removal.The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum. The approach developed has broad applicability and utility for researchers with standard laboratory imaging equipment.
Highlights
Epithelial cells typically provide a barrier or lining function and can form stratified structures, for example in skin and masticatory mucosa, where a robust response to mechanical stress and chemical irritants is essential to maintainingIndirect spectrophotometric and fluorometric methods to determine cell numbers have been developed
The proposed cell counting technique was validated by comparing the results with a manual count of cells in images, and subsequently applied to generate growth curves for oral keratinocyte cultures supplemented with a range of concentrations of foetal calf serum
Because the effect of defocusing causes the image to lose contrast detail, we investigated whether convolution filtering could be used on Phase contrast (PC) images to emulate the contrast changes in the defocused bright field microscopy and enable segmentation of regions corresponding with cells to estimate numbers
Summary
Epithelial cells typically provide a barrier or lining function and can form stratified structures, for example in skin and masticatory mucosa, where a robust response to mechanical stress and chemical irritants is essential to maintainingIndirect spectrophotometric and fluorometric methods to determine cell numbers have been developed. Epithelial cells typically provide a barrier or lining function and can form stratified structures, for example in skin and masticatory mucosa, where a robust response to mechanical stress and chemical irritants is essential to maintaining. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyletetrazolium bromide (MTT) assay is a relatively simple and inexpensive assay that can be applied to large sample numbers (several other tetrazolium compounds are commercially available which operate ) (Riss et al, 2016). The assay involves the addition of the MTT substrate to cell cultures, where the enzymatic activity in viable cells reduces MTT to formazan (a purple water-insoluble dye) and its concentration is determined spectrophotometrically (Mosmann, 1983). Cell numbers are estimated indirectly using a calibration curve. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society
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