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SummaryA theory for multiphoton fluorescence imaging in high aperture scanning optical microscopes employing finite sized detectors is presented. The effect of polarisation of the fluorescent emission on the imaging properties of such microscopes is investigated. The lateral and axial resolutions are calculated for one-, two-and three-photon excitation of p-quaterphenyl for high and low aperture optical systems. Significant improvement in lateral resolution is found to be achieved by employing a confocal pinhole. This improvement increases with the order of the multiphoton process. Simultaneously, it is found that, when the size of the pinhole is reduced to achieve the best possible resolution, the signal-to-noise ratio is not degraded by more than 30%. The degree of optical sectioning achieved is found to improve dramatically with the use of confocal detection. For two-and three-photon excitation axial full width half-maximum improvement of 30% is predicted.

SummaryWe consider the effect of detector size on the polarization extinction coefficient in three geometries of confocal microscopes. We find that a single mode optical fibre-based reciprocal system employing circularly polarized light offers great ease of alignment together with an extremely high extinction coefficient.