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Demonstrating Improved Multiple Transport‐Mean‐Free‐Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics
Author(s) -
Rieckher Matthias,
Psycharakis Stylianos E.,
Ancora Daniele,
Liapis Evangelos,
Zacharopoulos Athanasios,
Ripoll Jorge,
Tavernarakis Nektarios,
Zacharakis Giannis
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700419
Subject(s) - light sheet fluorescence microscopy , microscopy , fluorescence microscope , fluorescence , confocal microscopy , optical sectioning , resolution (logic) , context (archaeology) , green fluorescent protein , biophysics , optics , biological system , materials science , chemistry , biology , physics , computer science , artificial intelligence , paleontology , biochemistry , gene
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.