Screening microscope with confocal functionality

我们正在申请一种具有共焦功能的筛查显微镜。在生理细胞/组织培养条件下，可以以非常好的时间分辨率同时分析多达96(384)个平行条件。在完整的96(384)井中可以分辨单个细胞以及具有非常好的共聚焦分辨率的亚细胞结构。所有物镜(包括具有高数值孔径的浸水物镜)都可以在测量过程中完全自动改变，因此筛选实验可以与(自动)选定区域的高分辨率共焦测量相结合。并行测量不仅具有巨大的时间优势，而且还使我们有可能最大限度地利用来自癌症患者的材料。此外，3R原则(替换、减少、改进)在动物实验研究中得到了很好的实施，通过与动物细胞/组织的平行测量。特别是人类或小鼠免疫与癌细胞的相互作用的分析将在我们系进行。对于这些实验，需要在96井(或384井)中进行测量，同时获取共焦数据。需要平行的96孔(或384孔)实验是因为这样一个事实，即对献血者或癌症患者的免疫细胞进行的长达8小时(甚至24-48小时)的实验中，许多不能连续进行，因为1)50种不同的条件对8小时的实验大约需要20天，2)这些细胞不会持续那么长时间，而且3.)这些测量结果将不再具有可比性。这项资助的所有申请者必须对原代人类和/或小鼠单细胞或有机体进行并行测量，最好从几小时到几天进行测量。同时，必须在相同的测量中分析亚细胞结构，以了解分子过程。使用所要求的具有共焦功能的屏幕显微镜，可以进行亚细胞荧光测量并从XY分辨率低于200 nm的堆叠图像中进行3D重建。因此，具有共焦功能的屏幕显微镜非常适合执行本文所述的项目。它相对容易使用，因此可以预期，该仪器将被来自霍姆堡和萨尔布吕肯的许多团体使用。通常，对非常大量的数据进行分析是一个主要问题。作为2017-2022年资助的BMBF项目的一部分，我们可以为这里的所有申请者提供半自动分析。为了在未来尽可能地自动化这些半自动分析，我们刚刚聘请了一名全职生物信息学家。

We are applying for a screening microscope with confocal functionality. Up to 96 (384) parallel conditions can be analyzed simultaneously with very good time resolution under physiological cell/tissue culture conditions. Single cells in the complete 96 (384) well can be resolved as well as subcellular structures with very good confocal resolution. All objectives (including the water immersion objectives with high numerical aperture) can be changed fully automatically during the measurement, so that screening experiments can be combined with high-resolution confocal measurements of (automatically) selected areas. Parallelized measurements not only have enormous time advantages, but also make it possible to make optimal use of the material from - in our case - cancer patients. Furthermore, the 3R principle (Replace, Reduce, Refine) is very well implemented in animal experimental research by parallelized measurements with cells/tissues of one animal.In particular, analyses of the interactions of human or murine immune and cancer cells will be performed in our department. For these experiments, it is necessary to measure in 96-well (or 384-well) while simultaneously acquiring confocal data. The need for parallel 96-well (or 384-well) experiments arises from the fact that many of the 8 (or even 24-48) hour-long experiments with immune cells from blood donors or cancer patients cannot be performed consecutively because 1.) 50 different conditions would take approximately 20 days for 8-hour experiments, 2.) the cells would not last that long, and 3.) the measurements would no longer be comparable. All applicants of this grant must perform parallelized measurements of primary human and/or murine single cells or organoids, preferably measured over hours to days. At the same time, subcellular structures must be analyzed in the same measurement to understand molecular processes. With the requested screening microscope with confocal functionality, subcellular fluorescence measurements and 3D reconstructions from stacked images with an xy resolution of less than 200 nm are possible.The screening microscope with confocal function is thus ideally suited to perform the projects described here. It is relatively easy to use, so it is to be expected that the instrument will be used by very many groups from Homburg and Saarbrücken. Often the analysis of the very large amount of data is a major problem. As part of a BMBF project funded from 2017 - 2022, we can support all applicants here with semi-automatic analyses. In order to automate these semi-automatic analyses as best as possible in the future, we have just a hired full-time bioinformatician.