Automated analysis of time-lapse fluorescence microscopy images: from live cell images to intracellular foci

MOTIVATION Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. RESULTS Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening. AVAILABILITY AND IMPLEMENTATION The described system was implemented in MATLAB (The MathWorks, Inc., USA) and compiled to run within the MATLAB environment. The routines together with four sample datasets are available at http://celmia.bigr.nl/. The software is planned for public release, free of charge for non-commercial use, after publication of this article.

[1]  Thomas Martin Deserno,et al.  Bildverarbeitung für die Medizin: Grundlagen, Modelle, Methoden, Anwendungen , 1997, Bildverarbeitung für die Medizin.

[2]  Kannappan Palaniappan,et al.  Segmentation and Classification of Cell Cycle Phases in Fluorescence Imaging , 2009, MICCAI.

[3]  B. Roysam,et al.  Automated Cell Lineage Construction: A Rapid Method to Analyze Clonal Development Established with Murine Neural Progenitor Cells , 2006, Cell cycle.

[4]  Vladimir Kolmogorov,et al.  Computing geodesics and minimal surfaces via graph cuts , 2003, Proceedings Ninth IEEE International Conference on Computer Vision.

[5]  Abhijit G. Shanbhag,et al.  Utilization of Information Measure as a Means of Image Thresholding , 1994, CVGIP Graph. Model. Image Process..

[6]  Akiko Inagaki,et al.  Dynamic localization of human RAD18 during the cell cycle and a functional connection with DNA double-strand break repair. , 2009, DNA repair.

[7]  Karl Rohr,et al.  Non-rigid Temporal Alignment of 2D and 3D Multi-channel Microscopy Image Sequences of Human Cells , 2007, Bildverarbeitung für die Medizin.

[8]  Kate Hardy,et al.  Are we ignoring potential dangers of in vitro fertilization and related treatments , 2002 .

[9]  Aaron Ponti,et al.  Replication foci dynamics: replication patterns are modulated by S‐phase checkpoint kinases in fission yeast , 2007, The EMBO journal.

[10]  Patrick Bouthemy,et al.  Space-Time Adaptation for Patch-Based Image Sequence Restoration , 2007, IEEE Transactions on Pattern Analysis and Machine Intelligence.

[11]  Anne E Carpenter,et al.  Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins , 2006, Nature Methods.

[12]  Nikos Paragios,et al.  Non-rigid registration using distance functions , 2003, Comput. Vis. Image Underst..

[13]  W. V. van Cappellen,et al.  Nuclear Dynamics of PCNA in DNA Replication and Repair , 2005, Molecular and Cellular Biology.

[14]  H. Leonhardt,et al.  Dynamics of DNA Replication Factories in Living Cells , 2000, The Journal of cell biology.

[15]  Takeo Kanade,et al.  Cell population tracking and lineage construction with spatiotemporal context , 2008, Medical Image Anal..

[16]  W. V. van Cappellen,et al.  Dynamics of relative chromosome position during the cell cycle. , 2004, Molecular biology of the cell.

[17]  Thomas Boudier,et al.  Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing , 2005, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[18]  Karl Rohr,et al.  Nonrigid Registration of 3-D Multichannel Microscopy Images of Cell Nuclei , 2008, IEEE Transactions on Image Processing.

[19]  S. West,et al.  Interplay between human DNA repair proteins at a unique double‐strand break in vivo , 2006, The EMBO journal.

[20]  Stefano Soatto,et al.  Deformotion: Deforming Motion, Shape Average and the Joint Registration and Approximation of Structures in Images , 2003, International Journal of Computer Vision.

[21]  N. Thomas.  Lighting The Circle of Life: Fluorescent Sensors for Covert Surveillance of the Cell Cycle , 2003, Cell cycle.

[22]  George Iliakis,et al.  Computational Methods for Analysis of Foci: Validation for Radiation-Induced γ-H2AX Foci in Human Cells , 2006, Radiation research.

[23]  Jens Rittscher,et al.  Spatio-temporal cell cycle phase analysis using level sets and fast marching methods , 2009, Medical Image Anal..

[24]  Y. Adachi,et al.  Phosphorylation and Rapid Relocalization of 53BP1 to Nuclear Foci upon DNA Damage , 2001, Molecular and Cellular Biology.

[25]  Michal Kozubek,et al.  Fast point-based 3-D alignment of live cells , 2006, IEEE Transactions on Image Processing.

[26]  M Kozubek,et al.  High-resolution cytometry of FISH dots in interphase cell nuclei. , 1999, Cytometry.

[27]  Nathalie Harder,et al.  Automated Analysis of the Mitotic Phases of Human Cells in 3D Fluorescence Microscopy Image Sequences , 2006, MICCAI.

[28]  Wiro J. Niessen,et al.  Advanced Level-Set-Based Cell Tracking in Time-Lapse Fluorescence Microscopy , 2010, IEEE Transactions on Medical Imaging.

[29]  Ioannis Pitas,et al.  Automated evaluation of her-2/neu status in breast tissue from fluorescent in situ hybridization images , 2005, IEEE Transactions on Image Processing.

[30]  Jan Flusser,et al.  Image registration methods: a survey , 2003, Image Vis. Comput..

[31]  Roland Eils,et al.  Analyzing motion and deformation of the cell nucleus for studying co-localizations of nuclear structures , 2006, 3rd IEEE International Symposium on Biomedical Imaging: Nano to Macro, 2006..

[32]  F. E. Grubbs.  Procedures for Detecting Outlying Observations in Samples , 1969 .

[33]  J. Ellenberg,et al.  4D imaging to assay complex dynamics in live specimens. , 2003, Nature cell biology.

[34]  Christophe Zimmer,et al.  Segmenting and tracking fluorescent cells in dynamic 3-D microscopy with coupled active surfaces , 2005, IEEE Transactions on Image Processing.

[35]  H. Netten,et al.  Fluorescent dot counting in interphase cell nuclei , 1996 .

[36]  Meng Wang,et al.  Novel cell segmentation and online SVM for cell cycle phase identification in automated microscopy , 2008, Bioinform..

[37]  R. Yu,et al.  Single-cell quantification of molecules and rates using open-source microscope-based cytometry , 2007, Nature Methods.