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Proceedings Paper

Epi wide-field fluorescence microscopy through scattering media (Conference Presentation)
Author(s): Matthias Hofer; Christian Soeller; Jacopo Bertolotti; Sophie Brasselet

Paper Abstract

The reconstruction of objects hidden behind scattering media has been partly triggered by the striving for applying those techniques in biological imaging where fluorescence serves as an image contrast. Object reconstructions rely on speckle correlations that are rooted in the spatial and spectral memory effect of the medium. In the majority of previous seminal experiments in the field, laser light transmission was used as object contrast and millimetric sized objects were separated by cm distances from the scattering media. An open question therefore was to what extent microscopic-sized fluorescent structures can be reconstructed at the proximity and behind a scattering media, in a conventional epi wide field microscope as found in imaging laboratories. Here, we make use of the magnification properties of the scattering medium itself to resolve a scattered fluorescent image on a sCMOS camera. Due to the epi configuration, light is scattered in the excitation as well as the detection. Hence, we show that the use of the full fluorescence bandwidth greatly facilitates the detection of scattered signals in a single-shot at short integration times. Along those lines we present a phase retrieval algorithm of Fienup-type that is less sensitive to low signal conditions and takes four times less iterations than comparable phase retrieval algorithms. Backed by a complementary Fourier smoothing image processing technique that no longer requires an autocorrelation of the scattered light image to separate out the speckle noise, we are able to provide high quality micrometric fluorescent object reconstructions in a simple conventional inverted microscope.

Paper Details

Date Published: 4 March 2019
Proc. SPIE 10886, Adaptive Optics and Wavefront Control for Biological Systems V, 1088611 (4 March 2019); doi: 10.1117/12.2509710
Show Author Affiliations
Matthias Hofer, Institut Fresnel (France)
Christian Soeller, Univ. of Exeter (United Kingdom)
Jacopo Bertolotti, Univ. of Exeter (United Kingdom)
Sophie Brasselet, Institut Fresnel (France)

Published in SPIE Proceedings Vol. 10886:
Adaptive Optics and Wavefront Control for Biological Systems V
Thomas G. Bifano; Sylvain Gigan; Na Ji, Editor(s)

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