Live imaging of whole mouse embryos during gastrulation : migration analyses of epiblast and mesodermal cells

During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination

Light-sheet microscopy reveals dorsoventral asymmetric membrane dynamics of Amoeba proteus during pressure-driven locomotion

Amoebae are found all around the world and play an essential role in the carbon cycle in the environment. Therefore, the behavior of amoebae is a crucial factor when considering the global environment. Amoebae change their distribution through amoeboid locomotion, which are classified into several modes. In the pressure-driven mode, intracellular hydrostatic pressure generated by the contraction of cellular cortex actomyosin causes the pseudopod to extend. During amoeboid locomotion, the cellular surface exhibits dynamic deformation. Therefore, to understand the mechanism of amoeboid locomotion, it is important to characterize cellular membrane dynamics. Here, to clarify membrane dynamics during pressure-driven amoeboid locomotion, we developed a polkadot membrane staining method and performed light-sheet microscopy in Amoeba proteus, which exhibits typical pressure-driven amoeboid locomotion. It was observed that the whole cell membrane moved in the direction of movement, and the dorsal cell membrane in the posterior part of the cell moved more slowly than the other membrane. In addition, membrane complexity varied depending on the focused characteristic size of the membrane structure, and in general, the dorsal side was more complex than the ventral side. In summary, the membrane dynamics of Amoeba proteus during pressure-driven locomotion are asymmetric between the dorsal and ventral sides

Ultrasensitive Imaging of Ca2+ Dynamics in Pancreatic Acinar Cells of Yellow Cameleon-Nano Transgenic Mice

Yellow Cameleons are genetically encoded Ca2+ indicators in which cyan and yellow fluorescent proteins and calmodulin work together as a fluorescence (Förster) resonance energy transfer Ca2+-sensor probe. To achieve ultrasensitive Ca2+ imaging for low resting Ca2+ or small Ca2+ transients in various organs, we generated a transgenic mouse line expressing the highest-sensitive genetically encoded Ca2+ indicator (Yellow Cameleon-Nano 15) in the whole body. We then focused on the mechanism of exocytotic events mediated by intracellular Ca2+ signaling in acinar cells of the mice with an agonist and observed them by two-photon excitation microscopy. In the results, two-photon excitation imaging of Yellow Cameleon-Nano 15 successfully visualized intracellular Ca2+ concentration under stimulation with the agonist at nanomolar levels. This is the first demonstration for application of genetically encoded Ca2+ indicators to pancreatic acinar cells. We also simultaneously observed exocytotic events and an intracellular Ca2+ concentration under in vivo condition. Yellow Cameleon-Nano 15 mice are healthy and no significant deteriorative effect was observed on physiological response regarding the pancreatic acinar cells. The dynamic range of 165% was calculated from Rmax and Rmin values under in vivo condition. The mice will be useful for ultrasensitive Ca2+ imaging in vivo

Media 1: Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser

Originally published in Biomedical Optics Express on 01 October 2014 (boe-5-10-3311

Media 1: Light sheet-excited spontaneous Raman imaging of a living fish by optical sectioning in a wide field Raman microscope

Originally published in Optics Express on 16 July 2012 (oe-20-15-16195

Media 3: Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser

Originally published in Biomedical Optics Express on 01 October 2014 (boe-5-10-3311

Three-dimensional tracking of mesodermal nuclei.

<p>(A) Computationally reconstructed trajectories (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064506#pone.0064506.s010" target="_blank">Movie S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064506#pone.0064506.s011" target="_blank">S7</a>). White mark at the end of trajectory indicates the latest point. Red, blue, and green arrows at the lower left indicate the anterior–posterior axis, right–left axis, and proximal–distal axis, respectively. (B) Histograms of one-step velocity (top row) and the ratio of actual migration path length to linear displacement over 30 min (bottom row). Nuclear positions at the first time-point were classified into two areas according to two sets of criteria, lateral (cyan) or distal (magenta) and forward (orange) or rear (green). (C) Comparison of triangles connected neighboring nuclei at first time point and after 3 hours (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064506#pone.0064506.s012" target="_blank">Movie S8</a>). Nuclear positions at the first time-point were classified into two areas, lateral (cyan) or distal (magenta). After 3 hours, the areas of the triangles become larger and some triangles overlap each other, indicating that nuclei in the mesoderm do not migrate in straight lines.</p