Physical Sciences of Imaging in the Biomedical Sciences Doctoral Training Centre, University of Birmingham, Edgbaston, Birmingham, B5 7EG, U.K.
School of Dentistry, University of Birmingham, Edgbaston, Birmingham, B5 7EG, U.K.
J Microsc. 2018 Sep;271(3):345-354. doi: 10.1111/jmi.12726. Epub 2018 Jul 12.
Cell 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.
细胞计数通常用于确定细胞培养物的增殖率，对于贴壁细胞，它通常是一种“破坏性”的过程，需要破坏细胞单层，导致无法纵向跟踪细胞生长。这个过程既耗时又耗费大量资源。在这项研究中，开发了一种相对廉价、快速且广泛适用的相差显微镜为基础的技术，它模拟了当上皮细胞培养物的明场显微镜图像散焦时发生的对比度变化。处理得到的图像会产生一个可以使用全局阈值进行分割的图像；然后，从分割的区域数量中推断出细胞数量，这些细胞计数可以用于生成生长曲线。该方法的参数使用真实值和处理图像之间的离散单纯拓扑关系进行调整。使用线性判别分析来识别虚假噪声区域并将其去除，从而提高细胞计数的准确性。通过将结果与手动计数图像中的细胞进行比较，验证了所提出的细胞计数技术，并随后将其应用于生成添加了一系列胎牛血清浓度的口腔角质形成细胞培养物的生长曲线。该方法具有广泛的适用性和实用性，适用于拥有标准实验室成像设备的研究人员。
