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Optical System and Detector Requirements for Live-Cell Imaging
In designing an optical microscopy system for live-cell investigations, the primary considerations are detector sensitivity (signal-to-noise), the required speed of image acquisition, and specimen viability. The relatively high light intensities and long exposure times that are typically employed in recording images of fixed cells and tissues (where photobleaching is the major consideration) must be strictly avoided when working with living cells. In virtually all cases, live-cell microscopy represents a compromise between achieving the best possible image quality and preserving the health of the cells. Rather than unnecessarily oversampling time points and exposing the cells to excessive levels of illumination, the spatial and temporal resolutions set by the experiment should be limited to match the goals of the investigation.
In principle, the ideal live-cell image acquisition system would be sensitive enough to acquire superior images from weakly fluorescent specimens, while simultaneously being fast enough to record all dynamic processes. In addition, the system would have sufficient resolution to capture the finest specimen detail and a wide dynamic range capable of accurately measuring very minute differences in intensity. Unfortunately, optimizing any one of these criteria is only accomplished at the expense of the others. It is therefore currently impossible to design an all-purpose live-cell imaging system that will be ideal for the entire range of possible investigations. Instead, the researcher must compromise by determining the most important parameters for optimization while still attempting to limit the sacrifice made by those variables that are deemed of lesser interest. In the end, microscope configuration is ultimately determined by the requirements of the imaging mode(s), the necessities of maintaining specimen viability during the experiment, the difficulty level of labeling protocols, and the availability of equipment.