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Interferometric Resolution and Efficiency Enhancement for Scanning Fluorescence Microscopes

Dr Rainer Heintzmann

King's College, London, UK

Tuesday 16 September 2008, 3:30 pm, EN103 (Ground Floor, EN Building), Hawthorn.

Compared to wide field fluorescence microscopy, confocal microscopy can improve the lateral as well as the axial resolution. This is usually achieved by introducing a confocal pinhole and closing it to a size below the diffraction limited spot in the image plane, inevitably decreasing the detection efficiency. Sandeau et al. (2006) suggested a method to improve the lateral resolution for 4Pi microscopes. We present a more general approach for improving the lateral resolution and detection efficiency for scanning fluorescence microscopes by introducing an interferometer with partial image inversion to the detection pathway. We show that the resulting detection transfer function is essentially the absolute square of the system's amplitude transfer function, magnified to twice its spatial frequency range. Simulations for a confocal system yield a lateral full width at half maximum (FWHM) resolution of 168 nm (135 nm after subtraction of the two images acquired in the different interferometer outputs), as compared to 218 nm without the interferometer. As the detection point spread function is independent of z, this method is also well suited for extended focus imaging. Here simulations for Bessel beam excitation yield a resolution of 146 nm (116 nm after image subtraction), as compared to 199 nm for integrating detection without an interferometer. First experimental results using a laser sent though a mono-mode fiber as a source will be presented.

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