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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2013
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oai:doaj.org-article:2fcfc1539a8f485790179f2a6433f28d2021-11-18T07:49:26ZParallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.1932-620310.1371/journal.pone.0060100https://doaj.org/article/2fcfc1539a8f485790179f2a6433f28d2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23613717/?tool=EBIhttps://doaj.org/toc/1932-6203Two-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.Jan Leo RinnenthalChristian BörnchenHelena RadbruchVolker AndresenAgata MossakowskiVolker SiffrinThomas SeelemannHeinrich SpieckerIngrid MollJosephine HerzAnja E HauserFrauke ZippMartin J BehneRaluca NiesnerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 4, p e60100 (2013) |
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Medicine R Science Q |
| spellingShingle |
Medicine R Science Q Jan Leo Rinnenthal Christian Börnchen Helena Radbruch Volker Andresen Agata Mossakowski Volker Siffrin Thomas Seelemann Heinrich Spiecker Ingrid Moll Josephine Herz Anja E Hauser Frauke Zipp Martin J Behne Raluca Niesner Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation. |
| 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. |
| format |
article |
| author |
Jan Leo Rinnenthal Christian Börnchen Helena Radbruch Volker Andresen Agata Mossakowski Volker Siffrin Thomas Seelemann Heinrich Spiecker Ingrid Moll Josephine Herz Anja E Hauser Frauke Zipp Martin J Behne Raluca Niesner |
| author_facet |
Jan Leo Rinnenthal Christian Börnchen Helena Radbruch Volker Andresen Agata Mossakowski Volker Siffrin Thomas Seelemann Heinrich Spiecker Ingrid Moll Josephine Herz Anja E Hauser Frauke Zipp Martin J Behne Raluca Niesner |
| author_sort |
Jan Leo Rinnenthal |
| title |
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_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_sort |
parallelized tcspc for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2013 |
| url |
https://doaj.org/article/2fcfc1539a8f485790179f2a6433f28d |
| work_keys_str_mv |
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