Spinning disk confocal laserscanning microscope

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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.