Open Access Highly Accessed Research article

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy

Tomoko Watanabe1, Anisha Thayil2, Alexander Jesacher2, Kate Grieve2, Delphine Debarre2, Tony Wilson2, Martin Booth2 and Shankar Srinivas1*

Author Affiliations

1 Department of Physiology Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK

2 Department of Engineering Science, University of Oxford, Oxford, OX1 3PJ, UK

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BMC Cell Biology 2010, 11:38  doi:10.1186/1471-2121-11-38

Published: 3 June 2010

Additional files

Additional file 1:

Aberration correction improves HGM image quality. (A) Schematic of the adaptive harmonic generation microscope. Lx, lens; Mx, mirror; BSx, beam splitter; DM, deformable mirror; O, objective; S, specimen; C, condenser; D, dichroic; PMTx, photomultiplier tubes for THG (blue) and SHG/TPF (green) signal detection. He-Ne laser (dashed outline) is used for DM characterization and this path is disabled during imaging. (B) Representative THG image of 5.5 dpc mouse embryo, a region of size 30 μm × 30 μm × 15 μm, approximately 90 μm deep in the sample before aberration correction (C) after correcting system induced aberrations. Scale bar is 10 μm. (D) The correction phase function applied to the DM. This consists mainly of spherical aberration, probably due to incomplete coverglass thickness compensation in the objective lens. Compensating system aberrations results in an over all signal improvement of nearly 40%.

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Additional file 2:

SHG and THG image of mouse zygote. Opacity rendering of SHG (magenta) and THG (grey) image volumes of the surface of a mouse zygote. The meiotic spindle of the second polar body is visible in SHG. THG reveals LD and sperm.

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Additional file 3:

THG image of nucleolus. Opacity rendering of a THG image volume of LD and a nucleolus. The spherical nucleolus is within the nucleus, that is marked by an absence of THG signal.

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Additional file 4:

THG image of 2-cell mouse embryos. Opacity rendering of a THG image volume of densely packed LD in a two cell embryo.

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Additional file 5:

Time-lapse of embryo development: extended focus projection. Extended focus projection of a mouse embryo imaged with THG (grey) and SHG (magenta). Imaging was started at the compacted morula stage and development followed till the blastocyst stage. Images were captured at 20 minute intervals. The majority of THG signal is generated by lipid droplets, which become fewer and larger over the course of development. Use keyboard arrow keys to navigate frame by frame through the animation. Scale = 20 μm.

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Additional file 6:

Time-lapse of embryo development: optical section. Optical sections of the mouse embryo in additional file 5 (M4-emb-proj.mov), imaged with THG (grey) and SHG (magenta). Imaging was started at the compacted morula stage and development followed till the blastocyst stage. Images were captured at 20 minute intervals. The majority of THG signal is generated by lipid droplets, which become fewer and larger over the course of development. Use keyboard arrow keys to navigate frame by frame through the animation. Scale = 20 μm.

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Additional file 7:

Time-lapse of LD aggregate moving and changing shape. Images were captured at 10 minute intervals. LD aggregates move as single units while continuously changing shape. The top panel is an opacity volume rendering while the bottom panel shows optical sections in the image plane (X-Y plane) and X-Z and Y-Z projections. At the centre of the opacity rendering is the LD that is followed in the optical sections. One can see the LD aggregate continuously changing shape while moving, and the component LD appearing to 'jostle' each other. LD aggregates persist and behave as single units for over 7 hours. Use keyboard arrow keys to navigate frame by frame through the animation. Scale = 5 μm.

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Additional file 8:

LD aggregates stained with LipidTox. Two examples of LD aggregates from a live blastocyst stained with LipidTox and imaged by confocal microscopy. (A' and B') are different 3D views of the LD in (A and B) respectively. Corresponding LD are numbered in the two views, and the orientation is shown at bottom left. (A'' and B'') Optical section of the LD in (A and B) respectively, showing that LD are aggregates of smaller LD. LD numbers correspond to those in the volume renderings. Scale bar in (A'') = 2 μm and in (B'') = 5 μm.

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Additional file 9:

Time-lapse of LD in embryo at 1 second interval - change movie. Optical section of a mouse compacted morula imaged with THG at 1 second interval. The high-contrast lipid droplets do not move appreciably, but appear to jiggle in place. Use keyboard arrow keys to navigate frame by frame through the animation. Scale = 20 μm.

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Additional file 10:

Nuclear stain of control and Nocodazole treated embryos. Opacity renderings of morulae cultured in DMSO (carrier control) and Nocodazole overnight. Embryos were fixed and stained with DAPI to visualise DNA. The control morula (at left) developed normally, and underwent several rounds of division. One can see interphase nuclei as regular spheroids and one cell in metaphase towards the top right of the embryo. In contrast, the nocodazole treated embryo suffered metaphase arrest, as evident from their bumpy appearance due to condensed chromosomes and the presence of only 8 staining bodies.

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Additional file 11:

Time-lapse of embryo development in cytochalasin D change movie. Extended focus projection of THG signal from a mouse morula cultured in the presence of cytochalasin. Images were captured at 10 minute intervals. The embryo undergoes decompaction as a result of cytochalasin treatment. LD continue to move actively and aggregate into larger LD. Use keyboard arrow keys to navigate frame by frame through the animation. Scale = 20 μm.

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Additional file 12:

Time-lapse of embryo development in nocodazole change movie. Optical section of THG signal from a mouse morula cultured in the presence of nocodazole. Images were captured at 10 minute intervals. The embryo fails to form a blastocoel cavity as a result of nocodazole treatment. LD continue to move actively and aggregate into larger LD. Use keyboard arrow keys to navigate frame by frame through the animation. Scale = 20 μm.

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