Sheehan Moira J, Pawlowski Wojciech P
Nature Source Genetics, Ithaca, New York, USA.
Methods Enzymol. 2012;505:125-43. doi: 10.1016/B978-0-12-388448-0.00015-2.
Progression of meiosis has been traditionally reconstructed from microscopic images collected from fixed cells. This approach has clear shortcomings in accurately portraying the dynamics of meiotic processes. Studies conducted in recent years mostly in unicellular fungi have shown that chromosomes in meiotic prophase exhibit dynamic motility that cannot be accurately examined using fixed cell imaging. However, in contrast to yeast, research on meiotic chromosome dynamics in multicellular eukaryotes has been lagging. This was in part because meiocytes in multicellular eukaryotes reside deep within reproductive organs and are often refractory to culturing. Here, we describe a method in which intact, live-plant reproductive organs (anthers) are cultured to enable monitoring chromosome dynamics of meiocytes using multiphoton excitation (MPE) microscopy. The method was developed for use in maize but can be applied to other plant species and adapted for use in other taxa in which meiocytes are embedded in multicellular reproductive structures. MPE microscopy allows visualization of meiocytes embedded within native tissue in planta and thus meiocytes remain intact for the entire imaging procedure. We detail the kinds of time-lapse movies that can be captured and analyzed using this technique and also highlight software packages that can be utilized for analysis of movies chromosome dynamic in live meiocytes.
减数分裂的进程传统上是根据从固定细胞收集的显微图像来重建的。这种方法在准确描绘减数分裂过程的动态方面存在明显缺陷。近年来主要在单细胞真菌中进行的研究表明，减数分裂前期的染色体表现出动态运动，使用固定细胞成像无法准确检测到这种运动。然而，与酵母不同的是，多细胞真核生物中减数分裂染色体动态的研究一直滞后。部分原因是多细胞真核生物中的减数分裂细胞位于生殖器官深处，通常难以培养。在这里，我们描述了一种方法，即培养完整的活植物生殖器官（花药），以便使用多光子激发（MPE）显微镜监测减数分裂细胞的染色体动态。该方法是为玉米开发的，但可应用于其他植物物种，并适用于减数分裂细胞嵌入多细胞生殖结构的其他分类群。MPE显微镜可以观察植物体内天然组织中嵌入的减数分裂细胞，因此减数分裂细胞在整个成像过程中保持完整。我们详细介绍了使用该技术可以捕获和分析的延时电影类型，并强调了可用于分析活减数分裂细胞中染色体动态电影的软件包。
