Lattice Selective Plane Illumination Microscopy (L-SPIM) for the analysis of subcellular dynamics in living specimens.

从维多利亚Aequorea水母中发现的绿色荧光蛋白彻底改变了我们研究组织细胞的方式，也改变了我们研究活细胞复杂环境中信号事件、细胞器动力学和细胞器相互作用的方法。与绿色荧光蛋白生物学的发展平行，荧光成像方法和显微系统也取得了进展，这使得跟踪荧光标记的细胞、细胞器和细胞骨架元件、量化它们的丰度和探测它们的流动性和相互作用成为可能。然而，利用传统的激光扫描显微镜对复杂的三维组织进行长期成像仍然是荧光显微镜中最重要的障碍之一，因为这种显微镜会引起光毒性和光漂白。选择性平面照明显微镜（SPIM）已经被开发出来，可以通过扫描样品进行长期成像，而激光引起的损伤（如光毒性和光漂白）要小得多。然而，组织深处的亚细胞结构仍然是一个有待解决的挑战。由于Lattice SPIM的最新发展，现在可以在延长的扫描时间内对组织深处的小结构（如囊泡或微管）进行成像，而不会损坏标本。绿色荧光蛋白生物学和成像方法的结合为研究活细胞中细胞器的动力学特性提供了巨大的机会。埃克塞特大学研究生物学和生物医学研究的许多方面，从真菌相关的植物疾病研究到脊椎动物胚胎发育的信号生物学。在过去，显微方法，如共聚焦显微镜和电子显微镜已被用于研究亚细胞相互作用。这些观察结果现在可以在复杂生物体的活细胞中实时补充和扩展。因此，最新一代的Lattice SPIM是细胞生物学的游戏规则改变者——在细菌、真菌、植物和动物中。现在可以使用荧光显微镜在数小时到数天的时间尺度上生成活细胞中细胞器的动态图，这在以前是不可能的。在这个申请中，我们寻求支持购买一个现代化的莱迪思SPIM，这将补充埃克塞特生物成像中心现有的设施。

The discovery of the green fluorescent protein from the jellyfish Aequorea victoria has revolutionised our way in which we study cells in a tissue but also our approaches to investigate signalling events, organelle dynamics and interactions of organelles within the complex environment of the living cell. Parallel to the developments in GFP biology, there have been advances in fluorescence imaging methods and microscopical systems that make it possible to follow fluorescently labelled cells, organelles and cytoskeleton elements, to quantify their abundance and to probe their mobility and interactions. However, long-term imaging of complex 3D tissues with conventional laser-scanning microscopes is still one of the most significant obstacles in fluorescent microscopy as this microscopy induces phototoxicity and photobleaching. Selective plane illumination microscopes (SPIM) has been developed allowing long-term imaging by scanning the specimen with much less laser-induced damage such as phototoxicity and photobleaching. However, subcellular structures deep in tissue are still a challenge to resolve. Due to the recent development of Lattice SPIM, it is now possible to image small structures such as vesicles or microtubules over an extended scanning time deep in tissue without damaging the specimen. The combined advances in GFP biology and imaging methods are providing a massive opportunity for investigating the kinetic properties of organelles in living cells. The University of Exeter studies many aspects of biological and biomedical research, reaching from fungal-related plant disease research to signalling biology in vertebrate embryonic development. In the past, microscopical approaches such as confocal microscopy and electron microscopy have been used to investigate subcellular interactions. These observations can now be complemented and extended in real-time in living cells in complex organisms. The latest generation of Lattice SPIM is, therefore, a game-changer for cell biology - in bacteria, fungi, plants and animals. It is now possible to generate dynamic maps of organelles in living cells using a fluorescence microscope over a timescale of hours to days, which has not previously been possible. In this application, we seek support to purchase a modern Lattice SPIM, which will complement the existing facilities at the Exeter Bioimaging Centre.