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Quantitative phase microscopy: a new tool for measurement of cell culture growth and confluency in situ

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Abstract

Quantitative phase microscopy (QPM) is a recently developed computational approach that provides quantitative phase measurements of specimen images obtained under bright-field conditions without phase- or interference-contrast optics. To perform QPM, an in-focus bright-field image is acquired, together with one positive and one negative de-focus image. An algorithm is then applied to produce a specimen phase map. In this investigation we demonstrate that manipulation of the phase map intensity histogram using novel, non-subjective thresholding and segmentation methods provides enhanced delineation of cells in culture. QPM was utilised to measure the growth behaviour of cultured airway smooth muscle cells over a 92-h period. There was a high degree of correlation between parallel QPM-derived confluency measurements and haemocytometry-derived counts of airway smooth muscle cells over this time period. Using QPM, translucent cells can be visualised with improved cell boundary definition allowing precise and reproducible measurements of cell culture confluency. Quantitative phase imaging provides a rapid, optically simple and non-destructive approach for measurement of cellular morphology. Further development of the QPM-based analysis methodology has the potential to provide even more refined measures of cellular growth.

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Acknowledgements

The authors thank Professor Keith Nugent and Associate Professor Ann Roberts for valuable advice in relation to image generation and analysis throughout the investigations undertaken, and are grateful to Mr. David Stewart of Zeiss Australia for assistance with optical and software components. Funding support from the Australian Research Council (Strategic Partnerships in Industry—Research and Training Scheme), Iatia Ltd. and GlaxoSmithKline (UK) is acknowledged. The QPM system utilized for these investigations is marketed by Iatia Ltd (“QPm”), http://www.iatia.com.au.

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Authors and Affiliations

  1. Department of Physiology, University of Melbourne, 3010, Parkville, Victoria , Australia

    Claire L. Curl, Peter J. Harris & Lea M. D. Delbridge

  2. Department of Pharmacology, University of Melbourne, 3010, Parkville, Victoria, Australia

    Trudi Harris & Alastair G. Stewart

  3. School of Physics, University of Melbourne, 3010, Parkville, Victoria, Australia

    Catherine J. Bellair

  4. Imaging Division, Iatia Ltd., 2/935 Station St., 3129, Box Hill Nth, Victoria, Australia

    Brendan E. Allman

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  1. Claire L. Curl
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  2. Trudi Harris
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  3. Peter J. Harris
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  4. Brendan E. Allman
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  5. Catherine J. Bellair
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  6. Alastair G. Stewart
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  7. Lea M. D. Delbridge
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Correspondence to Lea M. D. Delbridge.

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Curl, C.L., Harris, T., Harris, P.J. et al. Quantitative phase microscopy: a new tool for measurement of cell culture growth and confluency in situ. Pflugers Arch - Eur J Physiol 448, 462–468 (2004). https://doi.org/10.1007/s00424-004-1248-7

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  • DOI: https://doi.org/10.1007/s00424-004-1248-7

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