Live-cell Imaging and Quantitative Analysis of Meiotic Divisions in Caenorhabditis elegans Males
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Author:
Date:
2020-10-20
[Abstract] Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode Caenorhabditis elegans, removal of spermatocytes from gonads has damaging effects, as most of the extracted spermatocytes show a high variability in the timing of meiotic divisions or simply arrest during the experiment. Therefore, we developed a live-cell imaging approach for in situ filming of spermatocyte meiosis in whole immobilized C. elegans males, thus allowing an observation of male germ cells within an unperturbed environment. For this, we make use of strains with fluorescently labeled chromosomes and centrosomes. Here we describe how to immobilize male worms for live-imaging. Further, we describe ...
[摘要] [摘要] 用相差显微镜对一些物种的精母细胞进行了减数分裂的实时成像。对于秀丽隐杆线虫来说,去除生殖腺中的精母细胞具有破坏性作用,因为大多数精母细胞在减数分裂的时间上表现出高度的变异性,或者只是在实验中停止。因此,我们开发了一种活体细胞成像方法,用于原位拍摄固定化线虫雄性精母细胞减数分裂过程,从而可以在不受干扰的环境中观察雄性生殖细胞。为此,我们利用带有荧光标记染色体和中心体的菌株。在这里我们描述如何固定男性蠕虫进行实时成像。此外,我们描述了获取和处理数据的工作流程,以获得有关精母细胞减数分裂I和II中染色体分离动态的定量信息。此外,我们最新开发的方法允许我们在电子显微镜中重新定位胶片上的主轴,而不管蜗杆的初始3D方位如何,并以统计稳健的方式分析活蠕虫的纺锤动力学。我们的实时成像方法也适用于秀丽隐杆线虫雌雄同体,并且可以扩展到其他荧光标记的线虫或其他完全透明的小型模型生物。 [背景] ...
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Fluorescent Labeling of Rat-tail Collagen for 3D Fluorescence Imaging
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Author:
Date:
2018-07-05
[Abstract] Rat tail collagen solutions have been used as polymerizable in vitro three-dimensional (3D) extracellular matrix (ECM) gels for single and collective cell migration assays as well as spheroid formation. These 3D hydrogels are a relatively inexpensive, simple to use model system that can mimic the in vivo physical characteristics of numerous tissues within the body, namely the skin. While confocal imaging techniques such as fluorescence reflection and two-photon microscopy are able to visualize collagen fibrils during 3D imaging without fluorescence, other imaging modalities require direct conjugation of fluorescent dyes to collagen. Here we detail how to generate 3D collagen gels labeled with a fluorescent dye. Furthermore, we go through the steps required to ...
[摘要] 大鼠尾胶原溶液已被用作可聚合的体外三维(3D)细胞外基质(ECM)凝胶,用于单一和集体细胞迁移测定以及球状体形成。 这些3D水凝胶是相对便宜,易于使用的模型系统,其可以模拟体内许多组织(即皮肤)的体内物理特征。 虽然诸如荧光反射和双光子显微镜的共焦成像技术能够在没有荧光的3D成像期间可视化胶原原纤维,但是其他成像模式需要荧光染料直接缀合到胶原。 在这里,我们详细介绍了如何生成用荧光染料标记的3D胶原凝胶。 此外,我们还经历了可重复生成适用于活细胞3D成像技术的明亮胶原水凝胶所需的步骤。
【背景】自20世纪50年代以来,Paul Weiss和Beatrice Garber最初观察到增加血浆浓度(纤维蛋白)对间充质细胞形态的影响(Weiss和Garber,1952),开始研究细胞迁移和细胞与周围微环境的相互作用。在随后的几十年中,生物化学家开始深入研究从鼠尾胶原中纯化提取物,并开始将其用作高度可聚合的3D基质(Fitch et al。,1955; Gross et al。,1955; Chandrakasan et al。,1976)。直到20世纪90年代,3D矩阵才真正对细胞生物学界有用,尤其是研究细胞迁移(Friedl et ...
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