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Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

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ABSTRACT

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos.

No MeSH data available.


Left-right symmetry breaking of the C. elegans embryo at 4-6 cell stage.(a–d) 3D volume renderings of a C. elegans embryo (OD95) viewed from the front (a,b) and back (c,d) at two time points of 1.5 min apart at the 4 to 6 cell stage (Cells are segmented and only the cell membrane is rendered). All cells are segmented and rendered in different colours for clearer visualization. (e–h) 3D volume renderings of the segmented EMS, ABa, ABp and P2 cells at the earlier, and (i–l) the later time point, showing the intracellular activities and interactions of different cells. The white, red and yellow arrows mark the activities related to the EMS cell retreating, the ABa cell contractile ring and the ABp cell contractile ring. Scale bars, 5 μm (a–d), 3 μm (e–l).
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f3: Left-right symmetry breaking of the C. elegans embryo at 4-6 cell stage.(a–d) 3D volume renderings of a C. elegans embryo (OD95) viewed from the front (a,b) and back (c,d) at two time points of 1.5 min apart at the 4 to 6 cell stage (Cells are segmented and only the cell membrane is rendered). All cells are segmented and rendered in different colours for clearer visualization. (e–h) 3D volume renderings of the segmented EMS, ABa, ABp and P2 cells at the earlier, and (i–l) the later time point, showing the intracellular activities and interactions of different cells. The white, red and yellow arrows mark the activities related to the EMS cell retreating, the ABa cell contractile ring and the ABp cell contractile ring. Scale bars, 5 μm (a–d), 3 μm (e–l).

Mentions: To evaluate the live imaging performance of TLS-SPIM, we imaged endogenously labelled myosin II particle activities in a live C. elegans embryo (LP162) every 8.5 s for 150 time points (Fig. 2, Supplementary Movie 8). A Bessel light sheet, generated by scanning an incoherent seven Bessel beam array, was tiled at three positions to image the embryo (Supplementary Movie 1). Both the spatial and temporal resolution of our results are sufficient to track and analyze the myosin II particle movements in 3D, by which the behaviour of the actomyosin network and its impact on embryonic cells can be studied. In another example, we imaged a C. elegans embryo (OD95) with the same light sheet every 30 s in two channels for 167 time points (Supplementary Movie 9). Both high spatial resolution and good SNR were maintained through the process, and the embryo hatched successfully in the end (Supplementary Fig. 17, Supplementary Movie 10). The result shows comparable spatial resolution, SNR, imaging speed and much longer imaging time compared with the results obtained from similar specimens imaged by Bessel SPIM and Lattice light-sheet microscopy in previous publications67. The result allows us to extract the dynamic information of every individual cell and analyze their relationship over developmental time, facilitating both visualization and understanding of cell behaviours as embryonic development progresses (Figs 3 and 4, Supplementary Movie 11,12). Clearly, TLS-SPIM performs well for 3D live imaging in terms of the spatial resolution, SNR, imaging speed, photobleaching and photodamage when the tiling number is kept low.


Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy
Left-right symmetry breaking of the C. elegans embryo at 4-6 cell stage.(a–d) 3D volume renderings of a C. elegans embryo (OD95) viewed from the front (a,b) and back (c,d) at two time points of 1.5 min apart at the 4 to 6 cell stage (Cells are segmented and only the cell membrane is rendered). All cells are segmented and rendered in different colours for clearer visualization. (e–h) 3D volume renderings of the segmented EMS, ABa, ABp and P2 cells at the earlier, and (i–l) the later time point, showing the intracellular activities and interactions of different cells. The white, red and yellow arrows mark the activities related to the EMS cell retreating, the ABa cell contractile ring and the ABp cell contractile ring. Scale bars, 5 μm (a–d), 3 μm (e–l).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4814582&req=5

f3: Left-right symmetry breaking of the C. elegans embryo at 4-6 cell stage.(a–d) 3D volume renderings of a C. elegans embryo (OD95) viewed from the front (a,b) and back (c,d) at two time points of 1.5 min apart at the 4 to 6 cell stage (Cells are segmented and only the cell membrane is rendered). All cells are segmented and rendered in different colours for clearer visualization. (e–h) 3D volume renderings of the segmented EMS, ABa, ABp and P2 cells at the earlier, and (i–l) the later time point, showing the intracellular activities and interactions of different cells. The white, red and yellow arrows mark the activities related to the EMS cell retreating, the ABa cell contractile ring and the ABp cell contractile ring. Scale bars, 5 μm (a–d), 3 μm (e–l).
Mentions: To evaluate the live imaging performance of TLS-SPIM, we imaged endogenously labelled myosin II particle activities in a live C. elegans embryo (LP162) every 8.5 s for 150 time points (Fig. 2, Supplementary Movie 8). A Bessel light sheet, generated by scanning an incoherent seven Bessel beam array, was tiled at three positions to image the embryo (Supplementary Movie 1). Both the spatial and temporal resolution of our results are sufficient to track and analyze the myosin II particle movements in 3D, by which the behaviour of the actomyosin network and its impact on embryonic cells can be studied. In another example, we imaged a C. elegans embryo (OD95) with the same light sheet every 30 s in two channels for 167 time points (Supplementary Movie 9). Both high spatial resolution and good SNR were maintained through the process, and the embryo hatched successfully in the end (Supplementary Fig. 17, Supplementary Movie 10). The result shows comparable spatial resolution, SNR, imaging speed and much longer imaging time compared with the results obtained from similar specimens imaged by Bessel SPIM and Lattice light-sheet microscopy in previous publications67. The result allows us to extract the dynamic information of every individual cell and analyze their relationship over developmental time, facilitating both visualization and understanding of cell behaviours as embryonic development progresses (Figs 3 and 4, Supplementary Movie 11,12). Clearly, TLS-SPIM performs well for 3D live imaging in terms of the spatial resolution, SNR, imaging speed, photobleaching and photodamage when the tiling number is kept low.

View Article: PubMed Central - PubMed

ABSTRACT

Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos.

No MeSH data available.