High Throughput Fluorescence Imaging for Plant Sciences

研究细胞和发育生物学依赖于活细胞内的成像结构和分子。通过这种方式，生物过程可以在几十毫秒到几十小时的时间尺度上进行3D跟踪。收集这类图像最广泛应用的方法是共聚焦荧光显微镜，它提供活体组织内的清晰图像，而不需要杀死和物理切片标本。该方法的有效性首先取决于用荧光标签标记特定分子的能力，其次取决于显微镜在排除失焦信息的同时形成高分辨率和对比度图像的能力。结构照明技术的最新进展，如蔡司细胞体系统，已经允许在较低倍率下进行光学切片，这极大地促进了共聚焦成像之前的初始荧光筛选，或者在更大的空间尺度（几平方厘米）上对整个完整植物组织和器官进行成像所需的生理和发育过程的测量。因此，能够跟踪发育和生理反应的理想解决方案需要将用于初始筛选的低倍率荧光与用于详细的细胞和亚细胞分辨率的高分辨率随访相结合。增加一个针对植物标本需求的机器人系统，最大限度地提高了数据收集的效率，以实现筛选项目的高通量，或允许长期无人值守的发育研究操作。这种自动化的高通量荧光筛选在动物研究中是常规的，细胞可以很容易地在多孔培养板中生长，但是专门为处理适合植物系统的空间尺度和发育时间段范围量身定制的管道在英国将是独一无二的。

Research cell and developmental biology relies on imaging structures and molecules within living cells. In this way biological process can be followed in 3D over time scales ranging from tens of milliseconds to tens of hours. The most widely applied method for collecting such images is confocal fluorescence microscopy which provides clear images from within living tissues without the need to kill and physically section the specimen. The effectiveness of the method depends, first, on the ability to tag specific molecules with a fluorescent label and, second, on the ability of the microscope to form an image with high resolution and contrast whilst excluding out-of-focus information. Recent advances in structured illumination techniques, such as the Zeiss apotome system, have allowed optical sectioning at lower-magnifications which greatly facilitates initial fluorescence screening prior to confocal imaging, or measurements of physiological and developmental processes operating at much larger spatial scales (several cm squared) needed to image entire intact plant tissues and organs. Thus the ideal solution to be able to track developmental and physiological responses needs to combine low-magnification fluorescence for initial screening with high-resolution follow-up for detailed cellular and sub-cellular resolution. The addition of a robotic system tailored to the demands of plant specimens maximises the efficiency of data collection needed to achieve high-throughput for screening projects, or to allow long-term unattended operation for developmental studies. Such automated high-throughput fluorescence screening is routine in animal studies, where cells can be easily grown in multi-well culture plates, but a pipeline specifically tailored to handle the range of spatial scales and developmental time periods appropriate for plant systems would be unique in the UK.