Abstract
BackgroundFluorescent 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. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts.ResultsIn 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.ConclusionsOur 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.AvailabilityCGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.
Highlights
Introduction of a confinement areaIt is assumed that the cell cannot exit the area between P1 and PN with a margin. (4) Maximum displacement constraint
Transgenic NIH3T3 cell lines stably expressing a short-lived nuclear yellow fluorescent protein (Venus) from circadian regulatory elements of the Rev-erba locus (Rev-VNP), or luciferase protein driven by circadian Bmal1 promoter (Bmal1-luc), have been established and exploited to unravel different aspects of mammalian circadian clockwork machinery [3,4,8]
Cell cycle analysis An extra feature we have developed for Circadian Gene Express (CGE), granulometry, allows the accurate follow up of protein subnuclear localization, the discrimination between condensed and diffused protein expression pattern
Summary
Introduction of a confinement areaIt is assumed that the cell cannot exit the area between P1 and PN with a margin. (4) Maximum displacement constraint. It is assumed that the cell displacement between consequent frames cannot exceed a certain step size Δ 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. Most of the available software and common methods based on intensity-threshold segmentation and frame-toframe 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. A manual analysis is unreasonably time-consuming and subject to errors in observer judgment
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