A lattice lightsheet microscope for imaging highly dynamic processes in living cells and organisms.

光学显微镜是生物学研究的核心。自从光学显微镜在17世纪发明以来，它已经揭示了细胞如何功能，生长，分裂和死亡，以及单个细胞如何共同产生组织和生物体的深刻见解。用于研究活细胞中蛋白质功能的荧光探针的发展，加上显微镜设计的非凡进步，标志着通过光学显微镜对细胞功能进行前所未有的深入了解的时代。最近的两次诺贝尔奖就是一个标志。其中一项是在2008年因从水母中发现绿色荧光蛋白而获奖，这种荧光蛋白可以通过基因工程与感兴趣的蛋白质结合。嵌合蛋白可以在活细胞中表达和观察。从那时起，已经鉴定出发出不同颜色荧光的蛋白质，使研究人员能够同时跟踪同一细胞中的多种蛋白质，从而提供有关细胞行为的重要信息。2014年，第二届诺贝尔奖颁发给了超分辨率显微镜的发展。这些方法提供了一种观察结构细节的方法，比使用衍射限制技术更有可能，衍射限制技术的分辨率是由恩斯特·阿贝（Ernst Abbe） 19世纪的方程设定的。2014年的获奖者之一埃里克·贝齐格（Eric Betzig）设计了一种名为晶格光片显微镜（LLSM）的显微镜。这项技术突破有许多优点。它可以非常快速地捕捉3D图像，这意味着现在可以用3D跟踪细胞内快速移动的细胞结构。对于移动较慢的结构，它提供了在活细胞内进行超分辨率成像的选择。最后，或许也是最重要的一点，晶格光片的照明对细胞、组织和生物体非常温和，因为它造成的光损伤很小。这意味着细胞过程可以被跟踪的时间比以前更长，速度更快，分辨率更高。这项突破性的技术现已由智能成像创新公司（3i）商业化，在这里，我们建议使用LLSM在几分钟到几天的时间尺度上对活细胞内各种不同的细胞结构进行成像。我们将能够分析快速移动的成分，如核内体和mRNA颗粒的行为，以及神经细胞中快速轴突运输的货物。我们还将对细胞骨架的结构成分——肌动蛋白丝和微管——进行成像，因为它们在细胞分裂、迁移和细胞间通讯等各种过程中起作用。LLSM对不同厚度样品成像的能力将使我们能够在从单细胞到组织样品或3D培养的细胞的样品中遵循这些过程。此外，我们还可以用它来观察果蝇和斑马鱼胚胎发育过程中的细胞行为。LLSM将使至少24组拥有BBSRC大量资金的高产科学家受益。这也将加强下一代研究人员在复杂的光显微技术和解释结果所需的数据分析方面的培训。此外，所产生的定量数据将用于加强生物学家、数学家和计算机科学家之间的合作，从而促进跨学科研究。

Light microscopy lies at the heart of biological research. Ever since the light microscope was invented in the seventeenth century, it has been revealing profound insights into how cells function, grow, divide and die, and how individual cells work together to generate tissues and organisms. The development of fluorescent probes for studying protein function in living cells coupled with extraordinary progress in microscope design have signalled an era of unprecedented insight into cell function via light microscopy. This has been marked by two recent Nobel Prizes. One was awarded in 2008 for the discovery of green fluorescent protein from jelly fish, which can be joined to a protein of interest by genetic engineering. The chimeric protein can then be expressed and observed in living cells. Since then, proteins that fluoresce in different colours have been identified, allowing researchers to follow multiple proteins in the same cell at the same time, providing vital information about cell behaviour. The second Nobel Prize was awarded in 2014 for the development of super-resolution microscopy. These methods gave a way of seeing structures with great level of detail than possible using diffraction-limited techniques, where the resolution is set by Ernst Abbe's nineteenth century equation.One of the 2014 winners, Eric Betzig, has gone on to design a microscope called the lattice lightsheet microscope (LLSM). This technological breakthrough has many advantages. It captures images in 3D very rapidly, meaning that cellular structures that move very fast inside the cell can now be followed in 3D. For slower-moving structures, it offers the option of super-resolution imaging within living cells. Lastly, and perhaps most importantly, the lattice light-sheet illumination is very gentle on cells, tissues and organisms, because it causes minimal photo-damage. This means that cellular processes can be followed for longer times than previously possible, and at faster speeds and higher resolution.This ground-breaking technology is now commercially available from Intelligent Imaging Innovations (3i), and here we propose to use the LLSM to image a wide range of different cellular structures within living cells over timescales ranging from minutes to days. We will be able to analyse the behaviour of fast-moving components such as endosomes and mRNA particles, and the cargoes transported by fast axonal transport in nerve cells. We will also image structural components of the cell's cytoskeleton - actin filaments and microtubules - as they work in processes as varied as cell division, migration and cell-cell communication. The LLSM's ability to image samples of different thickness will allow us to follow these processes in samples ranging from single cells through to cells in tissue samples or 3D cultures. In addition, we can use it to watch cell behaviour in developing embryos of fruit fly and zebrafish. The LLSM will benefit at least 24 groups of highly productive scientists holding significant BBSRC funding. This will also enhance the training of the next generation of researchers in a sophisticated light microscopic technique and the data analysis needed to interpret the results. In addition, the quantitative data generated will be used enhance collaborations between biologists, mathematicians and computer scientists, so promoting interdisciplinary research.