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The widespread use of fluorescence microscopy along with the vast library of available fluorescent stains and staining
methods has been extremely beneficial to researchers in many fields, ranging from material sciences to plant biology. In
clinical diagnostics, the ability to combine different markers in a given sample allows the simultaneous detection of the
expression of several different molecules, which in turn provides a powerful diagnostic tool for pathologists, allowing a
better classification of the sample at hand. The correct detection and separation of multiple stains in a sample is achieved
not only by the biochemical and optical properties of the markers, but also by the use of appropriate hardware and software
tools. In this chapter, we will review and compare these tools along with their advantages and limitations.