Universität zu Köln

Rieckher, Matthias ORCID: 0000-0002-1945-9474, Psycharakis, Stylianos E., Ancora, Daniele ORCID: 0000-0002-1232-4112, Liapis, Evangelos, Zacharopoulos, Athanasios, Ripoll, Jorge ORCID: 0000-0001-8856-7738, Tavernarakis, Nektarios ORCID: 0000-0002-5253-1466 and Zacharakis, Giannis (2018). Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics. Biotechnol. J., 13 (1). WEINHEIM: WILEY-V C H VERLAG GMBH. ISSN 1860-7314

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Abstract

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Rieckher, Matthias UNSPECIFIED orcid.org/0000-0002-1945-9474 UNSPECIFIED Psycharakis, Stylianos E. UNSPECIFIED UNSPECIFIED UNSPECIFIED Ancora, Daniele UNSPECIFIED orcid.org/0000-0002-1232-4112 UNSPECIFIED Liapis, Evangelos UNSPECIFIED UNSPECIFIED UNSPECIFIED Zacharopoulos, Athanasios UNSPECIFIED UNSPECIFIED UNSPECIFIED Ripoll, Jorge UNSPECIFIED orcid.org/0000-0001-8856-7738 UNSPECIFIED Tavernarakis, Nektarios UNSPECIFIED orcid.org/0000-0002-5253-1466 UNSPECIFIED Zacharakis, Giannis UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-204484
DOI:	10.1002/biot.201700419
Journal or Publication Title:	Biotechnol. J.
Volume:	13
Number:	1
Date:	2018
Publisher:	WILEY-V C H VERLAG GMBH
Place of Publication:	WEINHEIM
ISSN:	1860-7314
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language CAENORHABDITIS-ELEGANS; OPTICAL TOMOGRAPHY; SCATTERING LAYERS; DAF-16; DEEP; SPHEROIDS; SPECIMENS; BIOLOGY; MEDIA; TIME Multiple languages Biochemical Research Methods; Biotechnology & Applied Microbiology Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/20448