Demonstrating Improved Multiple Transport‐Mean‐Free‐Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics. Issue 1 (11th December 2017)

• Record Type: Journal Article
• Authors: Rieckher, Matthias; Psycharakis, Stylianos E.; Ancora, Daniele; Liapis, Evangelos; Zacharopoulos, Athanasios; Ripoll, Jorge; Tavernarakis, Nektarios; Zacharakis, Giannis
• Is Part Of: Biotechnology journal. Volume 13:Issue 1(2018)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-12-11
• Subjects: caenorhabditis elegans -- cancer cell spheroids -- light sheet fluorescence microscopy -- optical projection tomography -- phase retrieved tomography; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201700419
• Languages: English
• ISSNs: 1860-6768

Abstract

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. Abstract : The resolution to depth ratio in microscopy is dictated by light scattering in thick/dense tissues, limiting biomedical observations. The implementation of computational methods to modern microscopy that retrieve scrambled coherent properties enables a shift in the resolution to depth ratio. A Phase Retrieval Tomography (PRT) with Light Sheet Fluorescence Microscopy (LSFM) and Optical Projection Tomography (OPT) is combined to overcome light scattering and improve transport mean free path imaging in two scenarios: a) In the ≈1 MFP regime, when imaging the GFP‐tagged ageing and stress response transcription factor DAF‐16/FOXO in C. elegans providing quantitative data of fluorescence dynamics with 3D images of improved quality. b) In the ≈1 TMFP regime, when imaging optically opaque live samples in the form of cancer cell spheroids demonstrating uniform resolution reconstructions.