Dynamics of protrusions during amoeboid movements.

<p>By using the images obtained by ezDSLM, velocities of extending or retracting protrusions were measured. (A) Mean velocities of protrusions during extension or retraction are shown in box plots. Blue and black dots indicate mean values and outliers, respectively. The mean velocities of extension and retraction were significantly different by t-test (p<0.01). Data from 25 protrusions for each category are shown. (B) Time-evolved velocities of extending protrusions. Data from 5 protrusions are shown as mean±SD.</p

High-Speed Imaging of Amoeboid Movements Using Light-Sheet Microscopy

<div><p>Light-sheet microscopy has been developed as a powerful tool for live imaging in biological studies. The efficient illumination of specimens using light-sheet microscopy makes it highly amenable to high-speed imaging. We therefore applied this technology to the observation of amoeboid movements, which are too rapid to capture with conventional microscopy. To simplify the setup of the optical system, we utilized the illumination optics from a conventional confocal laser scanning microscope. Using this set-up we achieved high-speed imaging of amoeboid movements. Three-dimensional images were captured at the recording rate of 40 frames/s and clearly outlined the fine structures of fluorescent-labeled amoeboid cellular membranes. The quality of images obtained by our system was sufficient for subsequent quantitative analysis for dynamics of amoeboid movements. This study demonstrates the application of light-sheet microscopy for high-speed imaging of biological specimens.</p> </div

Setup of ezDSLM for observation of amoeboid movements.

<p>(A) Side view of the instrument (not to scale). Optical axes of objectives for excitation (Ex) and emission (Em) are shown in dashed lines. The illumination optics were derived from the confocal laser scanning microscope (CLSM). Specimens were placed on the sample holder (SH) in the chamber unit (CU) filled with medium. The CU containing the objective for emission detection is mounted on a movable motor stage. BF, barrier filter; TL, tube lens; CCD, CCD camera. (B) Photograph of the chamber unit. (C) Photograph of the sample holder. Amoebae were placed on the cover glass for imaging (arrow head).</p

Schematic of ezDSLM (A, B).

<p>Line scan (A) to make an ‘apparent’ light-sheet (B). This step is similar to that in the conventional DSLM. (C) Scan of the light-sheet to obtain three-dimensional image of specimen. The objective for emission detection is synchronized with the movement of the light-sheet scanning. Ex, objective for excitation; Em, objective for emission detection; Sp, specimen.</p

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

Live imaging of primary ocular vasculature formation in zebrafish

<div><p>Ocular vasculature consists of the central retinal and ciliary vascular systems, which are essential to maintaining visual function. Many researchers have attempted to determine their origins and development; however, the detailed, stepwise process of ocular vasculature formation has not been established. In zebrafish, two angioblast clusters, the rostral and midbrain organizing centers, form almost all of the cranial vasculature, including the ocular vasculature, and these are from where the cerebral arterial and venous angioblast clusters, respectively, differentiate. In this study, we first determined the anatomical architecture of the primary ocular vasculature and then followed its path from the two cerebral angioblast clusters using a time-lapse analysis of living <i>Tg(flk1</i>:<i>EGFP)</i>^<i>k7</i> zebrafish embryos, in which the endothelial cells specifically expressed enhanced green fluorescent protein. We succeeded in capturing images of the primary ocular vasculature formation and were able to determine the origin of each ocular vessel. In zebrafish, the hyaloid and ciliary arterial systems first organized independently, and then anastomosed via the inner optic circle on the surface of the lens by the lateral transfer of the optic vein. Finally, the choroidal vascular plexus formed around the eyeball to complete the primary ocular vasculature formation. To our knowledge, this study is the first to report successful capture of circular integration of the optic artery and vein, lateral transfer of the optic vein to integrate the hyaloidal and superficial ocular vasculatures, and formation of the choroidal vascular plexus. Furthermore, this new morphological information enables us to assess the entire process of the primary ocular vasculature formation, which will be useful for its precise understanding.</p></div