Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17708-13. doi: 10.1073/pnas.1108494108. Epub 2011 Oct 17.
The Caenorhabditis elegans embryo is a powerful model for studying neural development, but conventional imaging methods are either too slow or phototoxic to take full advantage of this system. To solve these problems, we developed an inverted selective plane illumination microscopy (iSPIM) module for noninvasive high-speed volumetric imaging of living samples. iSPIM is designed as a straightforward add-on to an inverted microscope, permitting conventional mounting of specimens and facilitating SPIM use by development and neurobiology laboratories. iSPIM offers a volumetric imaging rate 30× faster than currently used technologies, such as spinning-disk confocal microscopy, at comparable signal-to-noise ratio. This increased imaging speed allows us to continuously monitor the development of C, elegans embryos, scanning volumes every 2 s for the 14-h period of embryogenesis with no detectable phototoxicity. Collecting ∼25,000 volumes over the entirety of embryogenesis enabled in toto visualization of positions and identities of cell nuclei. By merging two-color iSPIM with automated lineaging techniques we realized two goals: (i) identification of neurons expressing the transcription factor CEH-10/Chx10 and (ii) visualization of their neurodevelopmental dynamics. We found that canal-associated neurons use somal translocation and amoeboid movement as they migrate to their final position in the embryo. We also visualized axon guidance and growth cone dynamics as neurons circumnavigate the nerve ring and reach their targets in the embryo. The high-speed volumetric imaging rate of iSPIM effectively eliminates motion blur from embryo movement inside the egg case, allowing characterization of dynamic neurodevelopmental events that were previously inaccessible.
秀丽隐杆线虫胚胎是研究神经发育的强大模型,但传统的成像方法要么速度太慢,要么光毒性太大,无法充分利用该系统。为了解决这些问题,我们开发了一种倒置选择性平面照明显微镜(iSPIM)模块,用于对活样本进行非侵入式高速体积成像。iSPIM 设计为倒置显微镜的简单附加组件,允许常规安装标本,并促进发育和神经生物学实验室使用 SPIM。iSPIM 提供的体积成像速度比目前使用的技术(如旋转盘共聚焦显微镜)快 30 倍,同时保持可比的信噪比。这种更高的成像速度使我们能够连续监测秀丽隐杆线虫胚胎的发育,在胚胎发生的 14 小时期间,每隔 2 秒扫描一个体积,而不会检测到光毒性。在整个胚胎发生过程中收集约 25,000 个体积,实现了对细胞核位置和身份的整体可视化。通过将双色 iSPIM 与自动化谱系分析技术相结合,我们实现了两个目标:(i)鉴定表达转录因子 CEH-10/Chx10 的神经元,(ii)可视化它们的神经发育动力学。我们发现,管相关神经元在迁移到胚胎中的最终位置时使用体位移位和阿米巴样运动。我们还观察到神经元在绕过神经环并到达胚胎中的目标时的轴突导向和生长锥动力学。iSPIM 的高速体积成像速度有效地消除了卵壳内胚胎运动的运动模糊,从而能够对以前无法获取的动态神经发育事件进行特征描述。