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Working principle of confocal and two-photon microscopy. (a) In a confocal microscope, all emitted fluorescent light from the focal plane focused at the confocal pinhole and passed to the detector, whilst out-of-focus signal will hit the edge of the pinhole and be physically blocked from reaching the detector. (b) In two-photon microscopy, the two-photon effect only occurs at the focal point, therefore out-of-focus signal is limited and no pinhole is needed.
