Spinning disk confocal laserscanning microscope

将获得的显微镜应该能够对单个细胞进行活细胞成像，也可以对三维组织中的细胞进行成像。它旨在供在布伦斯韦格工业大学新成立的制药工程中心(PVZ，制药工程中心)共同工作的多个团体使用。因此，它的测量原理应该允许灵活地适应这些小组所采用的各种细胞模型的不同要求。在Z维，分辨率应该足以分辨每个单元的多个平面，而在X/Y维，需要高分辨率，但不一定是超分辨率。总体而言，这些特征应该在空间分辨率和时间分辨率之间提供平衡。应该可以达到10赫兹以上的帧速率(例如，记录胞浆钙的变化以及线粒体或分泌颗粒的移动性)，而不需要引起光漂白或光毒性的荧光激发强度。避免毒性也与对持续灌流的细胞或组织进行长期测量有关。这些特征应该伴随着足以处理3D模型器官(器官)的穿透深度。旋转圆盘共聚焦显微镜满足了这些要求。与一次顺序激发一个图像点的共焦激光扫描显微镜的“常规”变体不同，旋转盘(尼普科夫盘)借助包含螺旋图案的针孔的旋转盘一次激发多个荧光图像点。图像生成从一开始就是二维的，允许使用相机芯片作为检测元件。最初，旋转盘原理受到低光透射率的影响。有两项发展使旋转圆盘成为高灵敏度荧光检测仪器。首先，激发光通过平行第二盘中对应的微透镜图案聚焦在针孔图案上。其次，最近发展起来的sCMOS相机大大提高了检测效率，这种相机结合了非常高的灵敏度和较小的像素尺寸。由于物面中的每个点在图像生成期间被多次扫描(取决于盘的旋转速度)，因此每个图像点和时间的激发能量比传统的激光扫描显微镜低得多。这使得能够在没有光毒性和/或高图像采集率的情况下进行长期观察。后一种特征可用于细胞内的快速z堆叠，从而能够在没有混叠的情况下描述3D样本中的功能变化。综上所述，在目前的发展状态下，旋转磁盘原理已经导致了一种非常通用的方法来描述高空间和时间分辨率的功能变化。

The microscope to be acquired should enable the live cell imaging of single cells but also of cells within a three-dimensional tissue. It is intended to be used by a number of groups which work together in the newly founded Zentrum für Pharmaverfahrenstechnik (PVZ , Center of Pharmaceutical Engineering) of the TU Braunschweig. Thus, its measuring principle should allow for the flexible adaption to the diverse requirements of the various cellular models employed by these groups. In the Z-dimension the resolution should be sufficient to resolve multiple planes per cell, while in the X/Y-dimension a high resolution but not necessarily super-resolution is desirable. Overall, the characteristics should provide a balance between spatial resolution and temporal resolution. Frame rates of more than 10 Hz should be achievable (e.g. to document changes in the cytosolic calcium together with the mobility of mitochondria or secretory granules) without requiring a strength of fluorescence excitation which causes photobleaching or phototoxicity. The avoidance of toxicity is also relevant for long-term measurements of continuously perifused cells or tissues. These features should come along with a depth of penetration that is sufficient to handle 3D model organs (organoids). These requirements are met by the spinning disk confocal microscopy. In contrast to the "conventional" variant of the confocal laser scanning microscope which sequentially excites the fluorescence of one image point at a time, the spinning disk (Nipkow disk) variant excites a multitude of fluorescent image points at a time by virtue of a rotating disk containing a spiral pattern of pinholes. The image generation is two-dimensional from the beginning on, permitting the use of a camera chip as the detecting element. Originally, the spinning disk principle suffered from the low light transmission. Two developments have contributed to make the spinning disk an instrument for high sensitivity fluorescence detection. First, the excitation light is focused on the pinhole pattern by a corresponding pattern of microlenses in a parallel second disc. Second, the detection efficiency was greatly improved by the recent evolution of sCMOS cameras, which combine a very high sensitivity with a small pixel size. Since each point in the object plane is scanned multiple times during image generation (depending on the rotation velocity of the disk), the excitation energy per image point and time is much lower than in the conventional laser scanning microscopes. This enables long-term observations without phototoxicity and/or high rates of image acquisition. The latter feature can be used for fast z-stacking within a cell and thus enables the description of functional changes in 3D specimen without aliasing. Taken together, the spinning disk principle in its current state of development has resulted in a very versatile method to describe functional changes with high spatial and temporal resolution.