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Author:
Date:
2015-12-05
[Abstract] The method consists of imaging developing pollen grains as they form within intact, immature Arabidopsis thaliana anthers. Using two-photon excitation in the infrared wavelength range, the intrinsic fluorescence (autofluorescence) of developing pollen grains and surrounding sporophytic tissues of the anther wall, including the tapetum, middle layer, endothecium and epidermis, can be visualized in the three-dimensional space of an intact anther. In contrast to conventional confocal microscopy, the application of red-shifted light by two-photon microscopy improves depth penetration into specimens, while the scattering of light and subsequent phototoxicity is minimized, making this a superior method for imaging the developing pollen grains and tapetal cells enclosed within anthers. ...
[摘要] 该方法包括当它们在完整的未成熟拟南芥花药内形成时使发育的花粉粒成像。使用在红外波长范围内的双光子激发，发育花粉颗粒和花药壁的周围孢子体组织（包括绒毡层，中间层，内皮和表皮）的固有荧光（自发荧光）可以在三维空间的完整的花药。与传统的共聚焦显微镜相反，通过双光子显微镜应用红移光改善深度穿透到标本，同时光的散射和随后的光毒性最小化，使这是成像发展中的花粉粒和绒毡层细胞的优良方法封闭在花药内。所描述的技术被优化用于检测花粉壁的自体荧光组分，包括花粉球蛋白和花粉外壳，并且提供关于野生型和花粉壁突变植物的花药中的自发荧光代谢物的空间和发育数据（Quilichini等al。，2014）。活的，完整的花药的双光子成像的使用具有潜在的未来研究，目的在于了解花粉发育期间配子体和孢子体组织之间的空间关系以及代谢物或荧光标记的蛋白质在发育的花药内的分布。
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