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Functional in vivo imaging using fluorescence lifetime light sheet microscopy (Conference Presentation)
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Paper Abstract

Light sheet microscopy has become an indispensable tool for fast, low phototoxicity volumetric imaging of biological samples; predominantly providing structural or analyte concentration data in its standard format. Fluorescence lifetime imaging microscopy (FLIM) provides functional contrast, but often at limited acquisition speeds and with complex implementation. We have developed a digitally scanned light sheet microscope for fast 2-colour volumetric imaging with imaging performed in the frequency domain at 20MHz using the PCO.FLIM camera. The camera enables rapid acquisition of two phases simultaneously at 0 and 180 degrees and with a phase shift relative to the modulated square-wave excitation. Whilst this frequency domain method has been well known for decades, application to light-sheet imaging is novel and provides straightforward functional read-out of fluorescence resonance energy transfer for protein interaction studies deep inside intact specimens such as Zebrafish. We demonstrate application of fluorescence lifetime contrast using the fluorescent protein biosensors in both live Zebrafish and organoids in digitally scanned light sheet FLIM. We apply signal processing techniques to improve data analysis and visualization and discuss this alongside practical application of real-time volumetric imaging of live biological specimens.

Paper Details

Date Published: 4 March 2019
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Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820J (4 March 2019); doi: 10.1117/12.2509727
Show Author Affiliations
Simon M. Ameer-Beg, King's College London (United Kingdom)
Claire A. Mitchell, King's College London (United Kingdom)
Simon P. Poland, King's College London (United Kingdom)
Robert D. Knight, King's College London (United Kingdom)
Guoqing Wang, King's College London (United Kingdom)
Gerhard Holst, PCO AG (Germany)


Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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