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Proceedings Paper

Investigation of second harmonic generation and multispectral imaging as new contrast mechanisms in scanning laser optical tomography
Author(s): Lena Nolte; Georgios C. Antonopoulos; Alexander Heisterkamp; Tammo Ripken; Heiko Meyer
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Paper Abstract

Scanning laser optical tomography (SLOT) is a 3D imaging technique, based on the principle of computed tomography to visualize samples up to magnitude of several centimeters. Intrinsic contrast mechanisms as absorption, scattering and autofluorescence provide information about the 3D architecture and composition of the sample. Another valuable intrinsic contrast mechanism is second harmonic generation (SHG), which is generated in noncentrosymmetric materials and commonly used to image collagen in biological samples. The angular dependence of the SHG signal, however, produces artifacts in reconstructed optical tomography datasets (OPT, SLOT). Thus, successful use of this intrinsic contrast mechanism is impaired. We investigate these artifacts by simulation and experiment and propose an elimination procedure that enables successful reconstruction of SHG-SLOT data. Nevertheless, in many cases specific labeling of certain structures is necessary to make them visible. Using multiple dyes in one sample can lead to crosstalk between the different channels and reduce contrast of the images. Also autofluorescence of the sample itself can account for that. By using multispectral imaging in combination with spectral unmixing techniques, this loss can be compensated. Therefore either a spectrally resolved detection path, or spectrally resolved excitation is required. Therefore we integrated a white supercontinuum light source in our SLOT-setup that enables a spectral selection of the excitation beam and extended the detection path to a four channel setup. This enables the detection of three fluorescence channels and one absorption channel in parallel, and increases the contrast in the reconstructed 3D images significantly.

Paper Details

Date Published: 23 February 2018
PDF: 8 pages
Proc. SPIE 10499, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV, 1049913 (23 February 2018);
Show Author Affiliations
Lena Nolte, Laser Zentrum Hannover e.V. (Germany)
Georgios C. Antonopoulos, Laser Zentrum Hannover e.V. (Germany)
Alexander Heisterkamp, Leibniz Univ. Hannover (Germany)
Tammo Ripken, Laser Zentrum Hannover e.V. (Germany)
Heiko Meyer, Laser Zentrum Hannover e.V. (Germany)

Published in SPIE Proceedings Vol. 10499:
Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XXV
Thomas G. Brown; Carol J. Cogswell; Tony Wilson, Editor(s)

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