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Multidimensional single-cell analysis based on fluorescence microscopy and automated image analysis

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Abstract

A single-cell analytical technology was developed for evaluating fast-growing cultures of green algae. The main part of the single-cell analysis is an epifluorescence microscopy-based cytometric approach combined with an automated image analysis algorithm and a single-threshold discrimination procedure. The reliability of the technique in terms of object recognition, evaluating particle size, and determining chlorophyll was successfully proven via reference analyses. The microscopy technique was used to determine the size of single cells, the amount of chlorophyll, and the density of chlorophyll in a model algal culture (Acutodesmus o.). The algal cells showed unexpected heterogeneity in all single-cell parameters, and exhibited a high correlation between cell size and amount of chlorophyll but a very low correlation between cell size and chlorophyll density. For a given cell size, the cell-to-cell heterogeneity of the relative chlorophyll density showed a spread of 0.02–0.08. This points to large variations in the architecture and the physiological state of the photosynthetic apparatus in the cells. This complex situation should be considered in future systems biology approaches focusing on the relationships between biomass accumulation, photosynthetic activity, and central carbon metabolism.

Analysis of cell-to-cell heterogeneity obtained from microscopic images

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Acknowledgements

We acknowledge the financial support of the German Federal Ministry of Food and Agriculture (BMEL) (grant 2814ERA03G) and the German Federal Ministry of Economics and Energy (BMWi) (CORNET AiF 129 EN). Additionally, the authors thank Ina Färber and Maja Eigner for maintaining the algal culture collection and for assisting with the experiments.

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Authors and Affiliations

  1. Institute for Food and Environmental Research (ILU e. V.), Arthur-Scheunert-Allee 40-41, 14558, Nuthetal, Germany

    Michael Sandmann, Martin Lippold, Franziska Saalfrank, Chimezie Progress Odika & Sascha Rohn

  2. Hamburg School of Food Science, University Hamburg, Grindelallee 117, 20146, Hamburg, Germany

    Sascha Rohn

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  1. Michael Sandmann
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  2. Martin Lippold
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  3. Franziska Saalfrank
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  4. Chimezie Progress Odika
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  5. Sascha Rohn
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Contributions

M.S. and M.L. programmed the object recognition and the single-cell analysis algorithms. M.L. performed the cell cultivation and the analyses. F.S. carried out the reference pigment analysis. C.O.P. performed the reference analysis based on fluorescent beads and helped with the image analysis. M.S. and S.R. planned and supervised the experiments. M.S. wrote the manuscript. All authors participated in the study design and reviewed the manuscript.

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Correspondence to Michael Sandmann.

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Sandmann, M., Lippold, M., Saalfrank, F. et al. Multidimensional single-cell analysis based on fluorescence microscopy and automated image analysis. Anal Bioanal Chem 409, 4009–4019 (2017). https://doi.org/10.1007/s00216-017-0344-4

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  • DOI: https://doi.org/10.1007/s00216-017-0344-4

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