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

Simon M. Ameer-Beg; Claire A. Mitchell; Simon P. Poland; Robert D. Knight; Guoqing Wang; Gerhard Holst

doi:10.1117/12.2509727

4 March 2019 Functional in vivo imaging using fluorescence lifetime light sheet microscopy (Conference Presentation)

Simon M. Ameer-Beg, Claire A. Mitchell, Simon P. Poland, Robert D. Knight, Guoqing Wang, Gerhard Holst

Proceedings Volume 10882, Multiphoton Microscopy in the Biomedical Sciences XIX; 108820J (2019) https://doi.org/10.1117/12.2509727
Event: SPIE BiOS, 2019, San Francisco, California, United States

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.

Conference Presentation

Citation Download Citation

Simon M. Ameer-Beg, Claire A. Mitchell, Simon P. Poland, Robert D. Knight, Guoqing Wang, and Gerhard Holst "Functional in vivo imaging using fluorescence lifetime light sheet microscopy (Conference Presentation)", Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820J (4 March 2019); https://doi.org/10.1117/12.2509727

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KEYWORDS

Microscopy

Luminescence

In vivo imaging

Fluorescence lifetime imaging

Cameras

Biological research

Microscopes

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