Optical Projection Tomography (OPT), the optical equivalent of x-ray computed tomography, reconstructs the 3D structure of a sample from a series of wide-field 2D projections acquired at different angles . OPT is used to map the optical attenuation and/or fluorescence distributions of intact transparent samples without the need for mechanical sectioning. While it is typically applied to chemically cleared samples, it can also be used to image inherently transparent or weakly scattering live organisms including adult zebrafish up to ~1cm in diameter .
When applying OPT to live samples it is important to minimise the data acquisition time while maximising the image quality in the presence of scattering. The former issue can be addressed using compressive sensing to reduce the number of projections required . Scattered light can be rejected using structured illumination , but this removes emission from regions the excitation modulation does not reach and reduces the available dynamic range. To address this, we have explored the rejection of scattered light by acquiring projections with parallel semi-confocal line illumination and detection in an approach we describe as slice-OPT (sl-OPT).
The impact of optical scattering can also be reduced by imaging at longer wavelengths . We are exploring OPT in the NIR 1&2 spectral windows. However, exotic array detectors, e.g. for short wave infrared light, are costly and so we are also developing a single pixel camera  approach. We will present our progress applying these techniques to 3D imaging of vasculature and tumour burden in live adult zebrafish.
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Current microscopy techniques are not optimal to image fluorescence in whole live animals. We present fluorescence lifetime optical projection tomography (FLIM OPT) applied to imaging enzyme activity in live transgenic zebrafish expressing Förster Resonance Energy Transfer (FRET) biosensors.
OPT can be considered the optical equivalent to x-ray CT. Samples are rotated through 360 with images acquired at set intervals, and a back projection technique is applied to reconstruct the 3D image. It can be performed in transmission or fluorescence modes, allowing a wide range of visualisation techniques, including FLIM. Combination of OPT with FRET FLIM can therefore provide functional information in 3D. The optimal size range for OPT is mm-cm, which fills the size gap between confocal and MRI and is also the size range for zebrafish, making them an ideal model for imaging. Transgenic zebrafish expressing a Caspase 3 FRET biosensor were generated on the TraNac background (a transparent mutant) to provide live readouts of apoptosis.
We have shown that using FLIM OPT we can detect changes in Caspase 3 activity in both embryo and adult Tg(Ubi:Caspase3biosensor) zebrafish. Apoptosis was induced using 25 Gy from a 137Cs source and post irradiation an increase in fluorescence lifetime was quantified in the head region indicative of biosensor cleavage and Caspase 3 activity. Though development of compressive sensing and multiplexed imaging with two imaging arms we have applied OPT and FLIM OPT to adult zebrafish, enabling us to quickly acquire datasets so the fish can be recovered and imaged longitudinally.