Dataset Information

Measurement and 3D-visualization of cell-cycle length using double labelling with two thymidine analogues applied in early heart development.

ABSTRACT: Organ development is a complex spatial process in which local differences in cell proliferation rate play a key role. Understanding this role requires the measurement of the length of the cell cycle at every position of the three-dimensional (3D) structure. This measurement can be accomplished by exposing the developing embryo to two different thymidine analogues for two different durations immediately followed by tissue fixation. This paper presents a method and a dedicated computer program to measure the resulting labelling indices and subsequently calculate and visualize local cell cycle lengths within the 3D morphological context of a developing organ. By applying this method to the developing heart, we show a large difference in cell cycle lengths between the early heart tube and the adjacent mesenchyme of the pericardial wall. Later in development, a local increase in cell size was found to be associated with a decrease in cell cycle length in the region where the chamber myocardium starts to develop. The combined application of halogenated-thymidine double exposure and image processing enables the automated study of local cell cycle parameters in single specimens in a full 3D context. It can be applied in a wide range of research fields ranging from embryonic development to tissue regeneration and cancer research.

SUBMITTER: de Boer BA

PROVIDER: S-EPMC3473012 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Measurement and 3D-visualization of cell-cycle length using double labelling with two thymidine analogues applied in early heart development.

de Boer Bouke A BA van den Berg Gert G Soufan Alexandre T AT de Boer Piet A J PA Hagoort Jaco J van den Hoff Maurice J B MJ Moorman Antoon F M AF Ruijter Jan M JM

PloS one 20121016 10

Organ development is a complex spatial process in which local differences in cell proliferation rate play a key role. Understanding this role requires the measurement of the length of the cell cycle at every position of the three-dimensional (3D) structure. This measurement can be accomplished by exposing the developing embryo to two different thymidine analogues for two different durations immediately followed by tissue fixation. This paper presents a method and a dedicated computer program to ...[more]

PMID: 23091641

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Project description:UnlabelledBackgroundPrimary cilia length is an important measure of cell and tissue function. While accurate length measurements can be calculated from cells in 2D culture, measurements in tissue or 3D culture are inherently difficult due to optical distortions. This study uses a novel combination of image processing techniques to rectify optical distortions and accurately measure cilia length from 3D images.MethodsPoint spread functions and experimental resolutions were calculated from subresolution microspheres embedded in 3D agarose gels for both wide-field fluorescence and confocal laser scanning microscopes. The degree of axial smearing and spherical aberration was calculated from xy:xz diameter ratios of 3D image data sets of 4 ?m microspheres that had undergone deconvolution and/or Gaussian blurring. Custom-made 18 and 50 ?m fluorescent microfibers were also used as calibration objects to test the suitability of processed image sets for 3D skeletonization. Microfiber length in 2D was first measured to establish an original population mean. Fibers were then embedded in 3D agarose gels to act as ciliary models. 3D image sets of microfibers underwent deconvolution and Gaussian blurring. Length measurements within 1 standard deviation of the original 2D population mean were deemed accurate. Finally, the combined method of deconvolution, Gaussian blurring and skeletonization was compared to previously published methods using images of immunofluorescently labeled renal and chondrocyte primary cilia.ResultsDeconvolution significantly improved contrast and resolution but did not restore the xy:xz diameter ratio (0.80). Only the additional step of Gaussian blurring equalized xy and xz resolutions and yielded a diameter ratio of 1.02. Following image processing, skeletonization successfully estimated microfiber boundaries and allowed reliable and repeatable measurement of fiber lengths in 3D. We also found that the previously published method of calculating length from 2D maximum projection images significantly underestimated ciliary length.ConclusionsThis study used commercial and public domain image processing software to rectify a long-standing problem of 3D microscopy. We have shown that a combination of deconvolution and Gaussian blurring rectifies optical distortions inherent in 3D images and allows accurate skeletonization and length measurement of microfibers and primary cilia that are bent or curved in 3D space.

Project description:Photoacoustic (PA) imaging (or optoacoustic imaging) is a novel biomedical imaging method in biological and medical research. This modality performs morphological, functional, and molecular imaging with and without labels in both microscopic and deep tissue imaging domains. A variety of innovations have enhanced 3D PA imaging performance and thus has opened new opportunities in preclinical and clinical imaging. However, the 3D visualization tools for PA images remains a challenge. There are several commercially available software packages to visualize the generated 3D PA images. They are generally expensive, and their features are not optimized for 3D visualization of PA images. Here, we demonstrate a specialized 3D visualization software package, namely 3D Photoacoustic Visualization Studio (3D PHOVIS), specifically targeting photoacoustic data, image, and visualization processes. To support the research environment for visualization and fast processing, we incorporated 3D PHOVIS onto the MATLAB with graphical user interface and developed multi-core graphics processing unit modules for fast processing. The 3D PHOVIS includes following modules: (1) a mosaic volume generator, (2) a scan converter for optical scanning photoacoustic microscopy, (3) a skin profile estimator and depth encoder, (4) a multiplanar viewer with a navigation map, and (5) a volume renderer with a movie maker. This paper discusses the algorithms present in the software package and demonstrates their functions. In addition, the applicability of this software to ultrasound imaging and optical coherence tomography is also investigated. User manuals and application files for 3D PHOVIS are available for free on the website (www.boa-lab.com). Core functions of 3D PHOVIS are developed as a result of a summer class at POSTECH, "High-Performance Algorithm in CPU/GPU/DSP, and Computer Architecture." We believe our 3D PHOVIS provides a unique tool to PA imaging researchers, expedites its growth, and attracts broad interests in a wide range of studies.

Dataset Information

Measurement and 3D-visualization of cell-cycle length using double labelling with two thymidine analogues applied in early heart development.

Publications

Measurement and 3D-visualization of cell-cycle length using double labelling with two thymidine analogues applied in early heart development.

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