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

水母Aequorea Victoria绿色荧光蛋白的发现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的信号事件、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相平行的是，荧光成像方法和显微系统也取得了进展，使得能够跟踪荧光标记的细胞、细胞器和细胞骨架元素，量化它们的丰度，并探索它们的流动性和相互作用。然而，使用传统的激光扫描显微镜对复杂的3D组织进行长期成像仍然是荧光显微镜最重要的障碍之一，因为这种显微镜会导致光毒性和光漂白。选择性平面照明显微镜(SPIM)已经开发出来，可以通过扫描样品进行长期成像，而光毒性和光漂白等激光损伤要小得多。然而，组织深处的亚细胞结构仍然是一个需要解决的挑战。由于格子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(如小泡或微管)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共同进步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，共聚焦显微镜和电子显微镜等显微方法被用来研究亚细胞相互作用。这些观察现在可以在复杂生物体的活细胞中实时补充和扩展。因此，最新一代的晶格SPIM改变了细胞生物学--细菌、真菌、植物和动物--的游戏规则。现在，使用荧光显微镜可以在几小时到几天的时间尺度上生成活细胞中细胞器的动态图谱，这在以前是不可能的。在此应用程序中，我们寻求支持购买一个现代化的Lattice SPIM，它将补充埃克塞特生物成像中心的现有设施。

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.