Perhaps the most versatile and powerful technique for localizing molecules within a cell by light microscopy is fluorescent staining of cells and observation by fluorescence microscopy. A chemical is said to be fluorescent if it absorbs light at one wavelength (the excitation wavelength) and emits light (fluoresces) at a specific longer wavelength. Modern microscopes used for observing fluorescent samples are configured to pass the excitation light through the objective lens into the sample and then selectively observe the emitted fluorescent light coming back through the objective lens from the sample. This is achieved by reflecting the excitation light with a special type of filter, called a dichroic mirror, into the sample and allowing the light emitted at the longer wavelength to pass through to the observer (see Figure 4-9d). Here we discuss several ways in which fluorescence microscopy can be used to examine specific molecules in cells.