Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

J MICROSC-OXFORD 193: 127-141 Part 2 FEB 1999 Higdon PD, Torok P, Wilson T

A theory for multiphoton fluorescence imaging in highaperture scanning optical microscopes employing finitesized detectors is presented. The effect of polarisation of thefluorescent emission on the imaging properties of suchmicroscopes is investigated. The lateral and axial resolutionsare calculated for one-, two- and three-photonexcitation of p-quaterphenyl for high and low apertureoptical systems. Significant improvement in lateral resolutionis found to be achieved by employing a confocalpinhole. This improvement increases with the order of themultiphoton process. Simultaneously, it is found that, whenthe size of the pinhole is reduced to achieve the best possibleresolution, the signal-to-noise ratio is not degraded by morethan 30%. The degree of optical sectioning achieved isfound to improve dramatically with the use of confocaldetection. For two- and three-photon excitation axial fullwidth half-maximum improvement of 30% is predicted.

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