Image processing in 3D standing-wave fluorescence microscopy

Vijay Krishnamurthi; Brent Bailey; Frederick Lanni

doi:10.1117/12.237480

10 April 1996 Image processing in 3D standing-wave fluorescence microscopy

Vijay Krishnamurthi, Brent Bailey, Frederick Lanni

Proceedings Volume 2655, Three-Dimensional Microscopy: Image Acquisition and Processing III; (1996) https://doi.org/10.1117/12.237480
Event: Electronic Imaging: Science and Technology, 1996, San Jose, CA, United States

Abstract

Standing-wave fluorescence microscopy, a method which utilizes interference to create a periodic excitation pattern along the optical axis, has been shown to provide improved axial resolution in thin, fluorescently labeled specimens. In each plane of focus, a complete standing wave data set is obtained by acquiring an image at each of three distinct positions of the interference fringes. Thicker specimens require through-focus data consisting of three images per plane. In this report we describe the recovery of information from this data using 3D image processing. The effective optical transfer function (OTF) of the standing wave microscope consists of the conventional OTF and two sidebands which are copies of the conventional OTF shifted axially by the spatial frequency of the interference fringes. The large gaps between the central band and the sidebands lead to significant ringing in the 3D reconstruction if linear deconvolution methods are employed. The use of non-linear, constrained image processing techniques has been shown to allow accurate extrapolation outside the OTF band limit. We demonstrate the extent to which the sidebands enhance recovery of information in the gaps, and provide a comparison between deconvolution using inverse-filtering and maximum-likelihood estimation.

Citation Download Citation

Vijay Krishnamurthi, Brent Bailey, and Frederick Lanni "Image processing in 3D standing-wave fluorescence microscopy", Proc. SPIE 2655, Three-Dimensional Microscopy: Image Acquisition and Processing III, (10 April 1996); https://doi.org/10.1117/12.237480

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