title: Automated method for the rapid and precise estimation of adherent cell culture characteristics from phase contrast microscopy images.
abstract: The quantitative determination of key adherent cell culture characteristics such as confluency, morphology, and cell density is necessary for the evaluation of experimental outcomes and to provide a suitable basis for the establishment of robust cell culture protocols. Automated processing of images acquired using phase contrast microscopy (PCM), an imaging modality widely used for the visual inspection of adherent cell cultures, could enable the non-invasive determination of these characteristics. We present an image-processing approach that accurately detects cellular objects in PCM images through a combination of local contrast thresholding and post hoc correction of halo artifacts. The method was thoroughly validated using a variety of cell lines, microscope models and imaging conditions, demonstrating consistently high segmentation performance in all cases and very short processing times (<1 s per 1,208 × 960 pixels image). Based on the high segmentation performance, it was possible to precisely determine culture confluency, cell density, and the morphology of cellular objects, demonstrating the wide applicability of our algorithm for typical microscopy image processing pipelines. Furthermore, PCM image segmentation was used to facilitate the interpretation and analysis of fluorescence microscopy data, enabling the determination of temporal and spatial expression patterns of a fluorescent reporter. We created a software toolbox (PHANTAST) that bundles all the algorithms and provides an easy to use graphical user interface. Source-code for MATLAB and ImageJ is freely available under a permissive open-source license.
authors.affiliation: Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, United Kingdom; Centre for Mathematics and Physics in the Life Sciences and Experimental Biology, University College London, London, United Kingdom.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Jaccard
authors.forename: Lewis D
authors.initials: LD
authors.lastname: Griffin
authors.forename: Ana
authors.initials: A
authors.lastname: Keser
authors.forename: Rhys J
authors.initials: RJ
authors.lastname: Macown
authors.forename: Alexandre
authors.initials: A
authors.lastname: Super
authors.forename: Farlan S
authors.initials: FS
authors.lastname: Veraitch
authors.forename: Nicolas
authors.initials: N
authors.lastname: Szita
mesh_terms: D000818:Animals; D057205:Automation, Laboratory / Q000379:methods*; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002468:Cell Physiological Phenomena; D006224:Cricetinae; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D051379:Mice; D008858:Microscopy, Phase-Contrast / Q000379:methods*
keywords: adherent cells; cell density; confluency; image-processing; morphology; on-line monitoring; phase contrast microscopy
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