Figure 1. Illustration of the confocal microscope principle. A collimated beam of excitation light (blue) is tightly focused in the sample by a microscope objective. The left image shows that the emission light resulting from the focal spot will be focused through the pinhole and most light will reach the detector. The right hand side shows the case where the emitted light arises from a point that is located outside the focal volume. This light will not be focused through the pinhole and only a very small amount of light is able to reach the detector. As a result, the out of focus fluorescent light is mainly blocked by the pinhole and therefore the background signal is significantly reduced.[1]
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