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Three-dimensional fluorescence imaging with an automated light-field microscope
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Paper Abstract

In addition to the two-dimensional intensity distribution in the image plane, light field microscopes capture information about the angle of the incident radiation. This information can be used to extract depth information about the object, calculate all-in-focus images and perform three-dimensional reconstructions from a single exposure. In combination with automated microscopy setups, this makes the technique a promising tool for high-throughput, three-dimensional cell assay evaluation which could substantially improve drug development and screening. To this end, we have developed a novel generalized calibration and three-dimensional reconstruction scheme for a lightfield fluorescence microscope setup. The scheme can handle Keplerian and Galilean light field camera configurations added to infinity corrected microscopes configured to be telecentric as well as non-telecentric or hypercentric. The latter provides a significant advantage over the state of the art as it allows for an application specific optimization of lateral and axial resolution, field-of-view, and depth-of-focus. The reconstruction itself is performed iteratively using an expectation maximization algorithm. Super-resolved reconstructions can be achieved by including experimentally measured pointspread- functions. To reduce the required computational power, sparsity and periodicity of the system matrix relating object space to light field space is exploited. This is particularly challenging for the non-telecentric cases, where the voxel size of the reconstructed object space depends on the axial coordinate. We provide details on the experimental setup and the reconstruction algorithm, and present results on the experimental verification of theoretical performance parameters as well as successful reconstructions of fluorescent beads and three-dimensional cell spheroids.

Paper Details

Date Published: 21 February 2019
PDF: 10 pages
Proc. SPIE 10883, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI, 108831B (21 February 2019); doi: 10.1117/12.2508401
Show Author Affiliations
J. Steinbrener, CTR Carinthian Tech Research AG (Austria)
J. Pribošek, CTR Carinthian Tech Research AG (Austria)
M. Baumgart, CTR Carinthian Tech Research AG (Austria)
T. Bereczki, CTR Carinthian Tech Research AG (Austria)
K. Harms, CTR Carinthian Tech Research AG (Austria)
D. Ripper, IMC Fachhochschule Krems GmbH (Austria)
A. Tortschanoff, CTR Carinthian Tech Research AG (Austria)
A. Kenda, Molecular Devices (Austria) GmbH (Austria)

Published in SPIE Proceedings Vol. 10883:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXVI
Thomas G. Brown; Tony Wilson, Editor(s)

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