Visualising apoptosis in live zebrafish using fluorescence lifetime imaging with optical projection tomography to map FRET biosensor activity in space and time.

Natalie Andrews,Douglas J Kelly,Sean C Warren,Sunil Kumar,James Mcginty,Margaret J Dallman,Marie‐Christine Ramel,Laurence Bugeon,Paul M W French,Nicola Lockwood,Louise Kerry,Yuriy Alexandrov,Paul Frankel,Antonina Frolov

doi:10.1002/jbio.201500258

Natalie Andrews, Douglas J Kelly + Show 12 more

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https://doi.org/10.1002/jbio.201500258

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Abstract

Fluorescence lifetime imaging (FLIM) combined with optical projection tomography (OPT) has the potential to map Förster resonant energy transfer (FRET) readouts in space and time in intact transparent or near transparent live organisms such as zebrafish larvae, thereby providing a means to visualise cell signalling processes in their physiological context. Here the first application of FLIM OPT to read out biological function in live transgenic zebrafish larvae using a genetically expressed FRET biosensor is reported. Apoptosis, or programmed cell death, is mapped in 3‐D by imaging the activity of a FRET biosensor that is cleaved by Caspase 3, which is a key effector of apoptosis. Although apoptosis is a naturally occurring process during development, it can also be triggered in a variety of ways, including through gamma irradiation. FLIM OPT is shown here to enable apoptosis to be monitored over time, in live zebrafish larvae via changes in Caspase 3 activation following gamma irradiation at 24 hours post fertilisation. Significant apoptosis was observed at 3.5 hours post irradiation, predominantly in the head region. figureWiley-VCH Verlag & Co.KGaA

Highlights

Microscopy of cells in culture has been invaluable in biology, leading to countless discoveries of cellular function, cell-cell communication, protein-protein interactions and protein structure
We choose to work with optical projection tomography (OPT) but note that light sheet microscopy (LSM) techniques can be applied to image zebrafish embryos [12,13,14] they typically have a sub-mm field of view and require multiple acquisitions for larger specimens
In this paper we demonstrate, we believe for the first time, that FLIM OPT can map changes in the Förster resonant energy transfer (FRET) signal of a Caspase 3 biosensor following gamma irradiation, used as a means to induce and follow apoptosis, in whole live Tg(Ubi : Caspase3bios) zebrafish embryos

Summary

Introduction

Microscopy of cells in culture has been invaluable in biology, leading to countless discoveries of cellular function, cell-cell communication, protein-protein interactions and protein structure. Micron-scale computed tomography (Micro-CT) is a powerful tool for imaging small specimens and is being used as the primary tool in the Zebrafish Phenome project [6] It can provide cellular resolution through the use of highly coherent, synchrotron generated X-rays, either alone for imaging of bone or in conjunction with contrast agents for soft tissue imaging. This approach requires samples to be fixed so cannot be applied in vivo and probe development is not yet at the stage to permit monitoring of functional protein activity [6, 7]. We choose to work with OPT but note that LSM techniques can be applied to image zebrafish embryos [12,13,14] they typically have a sub-mm field of view and require multiple acquisitions for larger specimens

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