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Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of orga...

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Autores principales: Rinnenthal, Jan Leo, Börnchen, Christian, Radbruch, Helena, Andresen, Volker, Mossakowski, Agata, Siffrin, Volker, Seelemann, Thomas, Spiecker, Heinrich, Moll, Ingrid, Herz, Josephine, Hauser, Anja E., Zipp, Frauke, Behne, Martin J., Niesner, Raluca
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629055/
https://www.ncbi.nlm.nih.gov/pubmed/23613717
http://dx.doi.org/10.1371/journal.pone.0060100
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author Rinnenthal, Jan Leo
Börnchen, Christian
Radbruch, Helena
Andresen, Volker
Mossakowski, Agata
Siffrin, Volker
Seelemann, Thomas
Spiecker, Heinrich
Moll, Ingrid
Herz, Josephine
Hauser, Anja E.
Zipp, Frauke
Behne, Martin J.
Niesner, Raluca
author_facet Rinnenthal, Jan Leo
Börnchen, Christian
Radbruch, Helena
Andresen, Volker
Mossakowski, Agata
Siffrin, Volker
Seelemann, Thomas
Spiecker, Heinrich
Moll, Ingrid
Herz, Josephine
Hauser, Anja E.
Zipp, Frauke
Behne, Martin J.
Niesner, Raluca
author_sort Rinnenthal, Jan Leo
collection PubMed
description Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.
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spelling pubmed-36290552013-04-23 Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation Rinnenthal, Jan Leo Börnchen, Christian Radbruch, Helena Andresen, Volker Mossakowski, Agata Siffrin, Volker Seelemann, Thomas Spiecker, Heinrich Moll, Ingrid Herz, Josephine Hauser, Anja E. Zipp, Frauke Behne, Martin J. Niesner, Raluca PLoS One Research Article Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation. Public Library of Science 2013-04-16 /pmc/articles/PMC3629055/ /pubmed/23613717 http://dx.doi.org/10.1371/journal.pone.0060100 Text en © 2013 Rinnenthal et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Rinnenthal, Jan Leo
Börnchen, Christian
Radbruch, Helena
Andresen, Volker
Mossakowski, Agata
Siffrin, Volker
Seelemann, Thomas
Spiecker, Heinrich
Moll, Ingrid
Herz, Josephine
Hauser, Anja E.
Zipp, Frauke
Behne, Martin J.
Niesner, Raluca
Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation
title Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation
title_full Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation
title_fullStr Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation
title_full_unstemmed Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation
title_short Parallelized TCSPC for Dynamic Intravital Fluorescence Lifetime Imaging: Quantifying Neuronal Dysfunction in Neuroinflammation
title_sort parallelized tcspc for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629055/
https://www.ncbi.nlm.nih.gov/pubmed/23613717
http://dx.doi.org/10.1371/journal.pone.0060100
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