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A Software Solution for Recording Circadian Oscillator Features in Time-Lapse Live Cell Microscopy

D. Sage, M. Unser, P. Salmon, C. Dibner

Cell Division, vol. 5, no. 17, pp. 1-9, July 6, 2010.


Background: Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/ dedicated tracking software. The latter however only applicable in cases where the cell does not change its position during the whole experiment, and thus are limiting.

Results: In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets.

Conclusions: Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series.

The associated software is available here.

@ARTICLE(http://bigwww.epfl.ch/publications/sage1001.html,
AUTHOR="Sage, D. and Unser, M. and Salmon, P. and Dibner, C.",
TITLE="A Software Solution for Recording Circadian Oscillator Features
	in Time-Lapse Live Cell Microscopy",
JOURNAL="Cell Division",
YEAR="2010",
volume="5",
number="17",
pages="1--9",
month="July 6,",
note="")

© 2010 BioMed Central Ltd.. Personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works must be obtained from BioMed Central Ltd.. This material is presented to ensure timely dissemination of scholarly and technical work. Copyright and all rights therein are retained by authors or by other copyright holders. All persons copying this information are expected to adhere to the terms and constraints invoked by each author's copyright. In most cases, these works may not be reposted without the explicit permission of the copyright holder.
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