Protocol | Published:

Quantifying cellular interaction dynamics in 3D fluorescence microscopy data

Nature Protocols volume 4, pages 13051311 (2009) | Download Citation



The wealth of information available from advanced fluorescence imaging techniques used to analyze biological processes with high spatial and temporal resolution calls for high-throughput image analysis methods. Here, we describe a fully automated approach to analyzing cellular interaction behavior in 3D fluorescence microscopy images. As example application, we present the analysis of drug-induced and S1P1-knockout-related changes in bone–osteoclast interactions. Moreover, we apply our approach to images showing the spatial association of dendritic cells with the fibroblastic reticular cell network within lymph nodes and to microscopy data regarding T–B lymphocyte synapse formation. Such analyses that yield important information about the molecular mechanisms determining cellular interaction behavior would be very difficult to perform with approaches that rely on manual/semi-automated analyses. This protocol integrates adaptive threshold segmentation, object detection, adaptive color channel merging, and neighborhood analysis and permits rapid, standardized, quantitative analysis and comparison of the relevant features in large data sets.

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This research was supported by the Intramural Research Program of NIAID, NIH. M.I. was supported by a fellowship grant from the International Human Frontier Science Program.

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Author notes

    • Masaru Ishii
    •  & Marc Bajénoff

    Present addresses: Laboratory of Biological Imaging, Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita 565-0871, Japan (M.I.), INSERM U924, Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique, Université de Nice-Sophia-Antipolis, Valbonne, France (M.B.).


  1. Program in Systems Immunology and Infectious Disease Modeling, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    • Frederick Klauschen
    • , Ronald N Germain
    •  & Martin Meier-Schellersheim
  2. Institute of Pathology, Universitätsmedizin Berlin, Berlin, Germany.

    • Frederick Klauschen
  3. Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    • Masaru Ishii
    • , Hai Qi
    • , Marc Bajénoff
    • , Jackson G Egen
    •  & Ronald N Germain


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F.K. designed, implemented and tested the method. F.K., M.M.-S. and R.N.G. prepared the paper. M.M.-S. and R.N.G. supervised the project. M.I., H.Q., M.B., J.G.E. and F.K. generated and provided experimental data.

Corresponding authors

Correspondence to Frederick Klauschen or Martin Meier-Schellersheim.

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