Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes

Abstract

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.

Wavefront Coding Fluorescence Microscopy Using High Aperture Lenses

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Detection efficiency in total internal reflection fluorescence microscopy

“…This allows accurately modeling and predicting the measured fluorescence signal as recently shown in detail by Enderlein et al [6]. The physical concepts of focusing electromagnetic waves and detecting dipole waves are well-known and described for instance by Török, Higdon and Enderlein [7,8,9,10]. In analogy to the classical Debye formulation [11] and the seminal work by Wolf and Richards [12,13], we recently reformulated the calculation of the electromagnetic field in the focus of high numerical aperture objectives based on a Fourier or chirp z transform [14] and achieved unprecedented calculation speed and flexibility.…”

Full wave model of image formation in optical coherence tomography applicable to general samples

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