Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

• Published in: Journal of microscopy (Oxford) Vol. 193; no. 2; pp. 127 - 141
• Main Authors: HIGDON, P.D, TÖRÖK, P, WILSON, T
• Format: Journal Article
• Language: English
• Published: Oxford, U.K. and Cambridge, USA Oxford, U.K. and Cambridge, USA : Blackwell Publishing Ltd 01-02-1999; Blackwell Publishing Ltd
• Subjects: Confocal microscopy; fluorescence microscopy; image analysis; imaging
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1046/j.1365-2818.1999.00448.x

A 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.