Fluorescence microscopy is one of the most important methods in the field of modern biomedical research due to its potentially high specificity and sensitivity. Modern dyeing methods allow the specific marking of a variety of molecules with different dyes in the same sample. This information can be used to draw valuable conclusions about the localization, interaction and migration of molecules in biological samples. For this purpose, different molecules must be detectable in the sample and the detection must be possible in a spatially clearly defined volume without disturbing signals from above or below levels. The latter enable so-called confocal laser scanning microscopes, in which the emission light is selectively detected from the focus plane (by means of an optical pinhole). Thanks to this capability, confocal microscopes are probably the most widely used microscopes in the field of biomedical research, which also enable single-molecule imaging techniques such as fluorescence correlation spectroscopy (FCS). However, the "classic" confocal microscopes suffer from various limitations: On the one hand, the selective scanning of the confocal image leads to rather slow imaging rates, on the other hand, the number of fluorophores that can be used in parallel is limited, as is the achievable spatial resolution. The spatial resolution is limited by the physics of light diffraction. In recent decades, various so-called superresolution techniques have been developed to overcome this limitation. The different solutions differ in the achievable spatial resolution, but also in the temporal resolution, the size of the imaging field and the amount of light with which the sample must be exposed. Experiments with living samples require gentle lighting conditions and often high temporal resolution, which limits the choice of microscopy techniques. For the planned experiments, the microscope should provide a high spatial resolution imaging method (to resolve molecular complexes such as membrane pores) with minimal photo-bleaching (to keep phototoxicity low) and fast recording speeds for FRAP, FLIP and FCS studies. In addition, many planned experiments require high temporal resolution to visualize dynamic changes in cellular properties such as membrane potential and macromolecule assembly without drastically reducing the field of view or losing signal quality by increasing the speed of the confocal microscope's point scanner to very high speeds. These requirements are met by the concept of "image scanning microscopy", in which the classic point detector of a confocal microscope is replaced by a detector array similar to a camera. This allows for better resolution as well as greater efficiency of signal detection through a computational step known as "pixel reassignment." The presence of such a detector can also be used to increase the temporal resolution. For this purpose, the illumination must be extended from a single point to a short line covering the area of several "pixel rows" that can be imaged simultaneously, resulting in higher frame rates (this is sometimes referred to as "multiplexing"). The simultaneous marking of samples with several dyes is often complicated by the width of the emission spectra, which can overlap and lead to a mixing of signals in different detection channels. This problem can be solved by a method called "spectrally resolved detection", in which the microscope can sort the detected photons according to a known reference spectrum and thereby separate different color channels within a captured image ("spectral segregation"). For this purpose, the microscope must be capable of spectrally resolved detection, where the number of detection channels is equal to or greater than the number of markings, and have a high detection efficiency (QE > 40% for green light). This is important because the detected signal is divided into several detection channels, resulting in a low signal in each individual channel. The Zeiss LSM 980 Airyscan2 is the only confocal microscope that meets all of the above requirements.
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