Spinning disk confocal laserscanning microscope

所获得的显微镜应能对单个细胞和三维组织内的细胞进行活细胞成像。它打算被一些在布伦瑞克工业大学新成立的制药工程中心（PVZ，制药工程中心）共同工作的小组使用。因此，它的测量原则应该允许灵活地适应这些群体所采用的各种细胞模型的不同要求。在z维度中，分辨率应该足以解决每个单元格的多个平面，而在X/ y维度中，高分辨率但不一定是超分辨率是理想的。总的来说，这些特征应该在空间分辨率和时间分辨率之间提供一个平衡。大于10hz的帧率应该是可以实现的（例如，记录胞质钙的变化以及线粒体或分泌颗粒的流动性），而不需要引起光漂白或光毒性的荧光激发强度。避免毒性也适用于连续浸润的细胞或组织的长期测量。这些特征应该伴随着足以处理3D模型器官（类器官）的穿透深度。旋转圆盘共聚焦显微镜满足了这些要求。与“传统”的共聚焦激光扫描显微镜不同的是，旋转圆盘（尼普科夫圆盘）通过包含针孔螺旋图案的旋转圆盘，一次依次激发一个图像点的荧光。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，旋转圆盘原理受到光透射率低的困扰。两个发展有助于使旋转盘成为高灵敏度荧光检测仪器。首先，激发光通过平行第二圆盘上相应的微透镜聚焦在针孔图案上。其次，最近的sCMOS相机的发展大大提高了检测效率，它结合了非常高的灵敏度和小像素尺寸。由于在成像过程中对物体平面上的每个点进行多次扫描（取决于磁盘的旋转速度），因此每个图像点和时间的激发能量远低于传统的激光扫描显微镜。这使得长期观察没有光毒性和/或高图像采集率。后一种特性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样品的功能变化。综上所述，旋转盘原理在其目前的发展状态导致了一种非常通用的方法，以高空间和时间分辨率描述功能变化。

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.