Enhancing the Axial Resolution in Far-field Light Microscopy: Two-photon 4Pi Confocal Fluorescence Microscopy

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Abstract
We demonstrate theoretically and experimentally a fourfold increase in axial point resolution in far-field light microscopy. The resolution enhancement is achieved by coherently illuminating the specimen with two opposing objective lenses (4Pi confocal microscopy) and applying two-photon excitation. The point spread function and the axial resolution of this set-up are calculated in optical units. The axial resolution is measured and compared with predictions, both for the confocal and for the 4Pi confocal set-up, in single as well as in two-photon excitation mode.