Highly sensitive bioluminescence and fluorescence imaging system for large fields of view

主要设备将作为成像系统，在分子植物研究中读取整个器官和生物水平的转基因荧光或生物发光报告线。申请的装置将在新的尺度上进行成像，这将允许在生物体的背景下监测对例如生物或非生物压力对活样本的系统反应。所研究的过程对于理解植物对非生物和生物胁迫条件的生理反应是相关的，因为它们越来越多地发生在逐步的气候变化中。转基因报告系使用例如基因编码的生物传感器(特定地响应于生理参数而改变其性质的荧光蛋白)来表达靶向不同的亚细胞室，或者以荧光素酶为报告基因的启动子或蛋白质融合。这种实验方法允许监测亚细胞过程，如局部氧化还原状态的变化或系统背景下基因表达的调节。利用本方案中应用的主要设备，可以在大视场内记录植物等整个生物体的生物发光或荧光(约。20厘米)。为此，将使用冷却到-90°C的高性能CCD摄像机(16位)，该摄像机具有必要的灵敏度和非常低的背景噪声(暗电流)。摄像头设置在一个防光盒中，盒子里也有荧光所需的照明。为了覆盖尽可能多的荧光蛋白和基因编码的生物传感器，必须包括合适的激发和发射过滤器。仪器和采集参数的调整通过外部PC上的制造商专用软件进行控制。拟议中的设备将首次在德国用于植物科学。

The major equipment will be used as an imaging system to read out transgenic fluorescent or bioluminescent reporter lines at the level of whole organs and organisms in molecular plant research. The applied for setup will enable imaging on a new scale, which will allow for the monitoring of systemic reactions to e.g. biotic or abiotic stress on the living sample in an organismic context. The processes studied are relevant for understanding the physiological responses of plants to abiotic and biotic stress conditions, as they increasingly occur with progressive climate change. The transgenic reporter lines used express e.g. genetically encoded biosensors (fluorescent proteins that change their properties specifically in response to a physiological parameter) with targeting to different subcellular compartments, or promoter or protein fusions with luciferase as reporter. This experimental approach allows monitoring of subcellular processes such as local changes in redox status or regulation of gene expression in a systemic context. With the major equipment applied for in this proposal, the bioluminescence or fluorescence of whole organisms such as plants can be recorded in large fields of view (approx. 20 cm). For this purpose, a high-performance CCD camera (16 bit) cooled to -90°C will be used, which has the necessary sensitivity and very low background noise (dark current). The camera setup is located in a light-proof box, which also contains the necessary illumination for fluorescence. To cover as many fluorescent proteins and genetically encoded biosensors as possible, suitable excitation and emission filters must be included. The adjustment of the instrument and acquisition parameters are controlled via a manufacturer-specific software from an external PC. The proposed device would be used for the first time in Germany in plant sciences.