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Quantifying cellular interaction dynamics in 3D fluorescence microscopy data

Abstract

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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Figure 1: Automated threshold segmentation.
Figure 2: Adaptive channel merging.
Figure 3: Analysis of the impact of variations in the derivative constant on the normalized interface area (ratio of the interface area/total bone surface area) and on the difference between 'wild-type' and 'knockout' groups.
Figure 4: Segmentation example results.

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Acknowledgements

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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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.

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Correspondence to Frederick Klauschen or Martin Meier-Schellersheim.

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Klauschen, F., Ishii, M., Qi, H. et al. Quantifying cellular interaction dynamics in 3D fluorescence microscopy data. Nat Protoc 4, 1305–1311 (2009). https://doi.org/10.1038/nprot.2009.129

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