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Detecting cardiac contractile activity in the early mouse embryo using multiple modalities.

Chen CM, Miranda AM, Bub G, Srinivas S - Front Physiol (2015)

Bottom Line: Here, we describe approaches for visualizing contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients.We describe an algorithm for enhancing this image data and quantifying contractile activity from it.Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology Anatomy and Genetics, University of Oxford Oxford, UK ; Wellcome Trust London, UK.

ABSTRACT
The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodeling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodeling is controlled but also the origin of congenital heart defects (CHDs). Here, we describe approaches for visualizing contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

No MeSH data available.


Related in: MedlinePlus

Stills from time-lapse sequences of Ca2+ transients in three stages of early heart development. The stages shown are pre-linear heart tube, linear heart tube, and early looping heart tube. The pericardial sac was removed to allow entry of the Ca2+ sensitive dye Rhod-2. The scale bar represents 100 microns. Also see Supplementary Movie 3.
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Figure 3: Stills from time-lapse sequences of Ca2+ transients in three stages of early heart development. The stages shown are pre-linear heart tube, linear heart tube, and early looping heart tube. The pericardial sac was removed to allow entry of the Ca2+ sensitive dye Rhod-2. The scale bar represents 100 microns. Also see Supplementary Movie 3.

Mentions: We isolated embryos at several stages of development and exposed the cardiac tissue by removing the overlaying endoderm and pericardial sac. This is essential to allow the calcium sensitive dye to penetrate the developing myocardium. Next, we incubated embryos in 5 μM calcium dye Rhod-2 for 15 min, and then immobilized the embryos for imaging as for brightfield microscopy. Embryos were imaged with a laser scanning confocal microscope using parameters that allowed for scanning at 10 frames per second. We detected clear calcium transients progressing across the forming linear and looping heart tube, indicating that excitation contraction coupling is likely already in place at this stage of development (Figure 3 and Supplementary Movie 3). In order to visualize the calcium transients more clearly, we performed an image subtraction to remove the baseline fluorescence from each frame of the time-lapse movie, so that the change in intensity of the calcium dye (“ΔRhod-2”) was more clearly visible (Figure 3 and Supplementary Movie 3).


Detecting cardiac contractile activity in the early mouse embryo using multiple modalities.

Chen CM, Miranda AM, Bub G, Srinivas S - Front Physiol (2015)

Stills from time-lapse sequences of Ca2+ transients in three stages of early heart development. The stages shown are pre-linear heart tube, linear heart tube, and early looping heart tube. The pericardial sac was removed to allow entry of the Ca2+ sensitive dye Rhod-2. The scale bar represents 100 microns. Also see Supplementary Movie 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4285868&req=5

Figure 3: Stills from time-lapse sequences of Ca2+ transients in three stages of early heart development. The stages shown are pre-linear heart tube, linear heart tube, and early looping heart tube. The pericardial sac was removed to allow entry of the Ca2+ sensitive dye Rhod-2. The scale bar represents 100 microns. Also see Supplementary Movie 3.
Mentions: We isolated embryos at several stages of development and exposed the cardiac tissue by removing the overlaying endoderm and pericardial sac. This is essential to allow the calcium sensitive dye to penetrate the developing myocardium. Next, we incubated embryos in 5 μM calcium dye Rhod-2 for 15 min, and then immobilized the embryos for imaging as for brightfield microscopy. Embryos were imaged with a laser scanning confocal microscope using parameters that allowed for scanning at 10 frames per second. We detected clear calcium transients progressing across the forming linear and looping heart tube, indicating that excitation contraction coupling is likely already in place at this stage of development (Figure 3 and Supplementary Movie 3). In order to visualize the calcium transients more clearly, we performed an image subtraction to remove the baseline fluorescence from each frame of the time-lapse movie, so that the change in intensity of the calcium dye (“ΔRhod-2”) was more clearly visible (Figure 3 and Supplementary Movie 3).

Bottom Line: Here, we describe approaches for visualizing contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients.We describe an algorithm for enhancing this image data and quantifying contractile activity from it.Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology Anatomy and Genetics, University of Oxford Oxford, UK ; Wellcome Trust London, UK.

ABSTRACT
The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodeling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodeling is controlled but also the origin of congenital heart defects (CHDs). Here, we describe approaches for visualizing contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

No MeSH data available.


Related in: MedlinePlus