OPT is the direct optical equivalent of X-ray computed tomography (CT), in which parallel ray projection is assumed and 3-D image stacks can be reconstructed using the FBP algorithm as long as the sample is contained within the depth of field of the imaging lens (i.e. in focus) such that the illuminating light approximates to parallel ray projection. It can be directly applied to samples that have been rendered transparent by a chemical clearing process³ or which are inherently transparent.  Important examples of the latter include live organisms that can be genetically manipulated to serve as disease models such as d. melanogaster⁴, c. elegans⁵ and danio rerio (zebrafish) larvae⁶. Thus OPT complements other mesoscopic imaging techniques such as LSFM. OPT can be applied to reconstruct the absorption as well as the fluorescence distributions in optically transparent samples and can be applied over sub-mm to ~cm scales, although the requirement to approximate to parallel projection means that the achievable optical resolution decreases with increasing sample size as the depth of field must be increased proportionately. This trade-off between sample size and resolution can be mitigated by “focal scanning”, which entails translating the focus of a high NA imaging lens such that all parts of the sample are in focus for some time during the acquisition of each projection image⁷. The FBP reconstruction process suppress the out of focus light although it can impact the signal to noise ratio of the OPT image stacks. An alternative approach to address this trade-off is to use multiple imaging set-ups focussed to different depths in the sample such that different sub-volumes of the sample are simultaneously imaged in focus during a single OPT acquisition⁸. This significantly increases the light collection efficiency and therefore the imaging speed, which is important when imaging live organisms. OPT also offers the opportunity to decrease image acquisition times through the use of compressive sensing techniques originally developed for X-ray CT, where fewer angularly resolved projection images are acquired than is necessary to meet the Nyquist sampling criterion set by the spatial resolution of the detectors. Providing the data is sparse, 3-D images can be reconstructed without loss of information using iterative algorithms. We have implemented this for OPT⁹ of live zebrafish in order to reduce the time that they need to be maintained under anaesthetic.

³ Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher-Sørensen, J., Baldock, R. and Davidson, D., Science, 296 (2002) 541
⁴ Vinegoni, C., Pitsouli, C., Razansky, D., Perrimon, N. and Ntziachristos, V., Nature Methods, 5 (2008) 45
⁵ Birk, U. J., Rieckher, M., Konstantinides, N., Darrell, A., Sarasa-Renedo, A., Meyer, H., Tavernarakis, N. and Ripoll, J., Biomedical Optics Express, 1 (2010) 87
⁶ McGinty, J., Taylor, H. B., Chen, L., Bugeon, L., Lamb, J. R., Dallman, M. J. and French, P. M. W., Biomedical Optics Express, 2 (2011) 1340
⁷ L Chen, S. Kumar, D. Kelly, N. Andrews, M.J. Dallman,  P. M W French and J. McGinty, Biomed Optical Express, 5 (2014) 3367-3375 DOI:10.1364/BOE.5.003367g
⁸ L. Chen. J. McGinty and P. M. W. French, Opt Lett 38, (2013) 851
⁹ Correia et al. PLoS ONE 10(8): e0136213, doi:10.1371/journal.pone.0136213,

When applying optical tomography to larger and more highly scattering samples, the filtered back-projection algorithm for tomographic image reconstruction becomes less valid, although it still can provide useful 3-D images in the presence of moderate scattering, as is encountered in live adult zebrafish.  For strongly scattering samples, it is beneficial to take into account the multiple scattering of the detected photons and to use statistical diffuse light propagation algorithms to reconstruct the 3-D fluorescence quantum efficiency and lifetime distributions based on the probable trajectories of the detected photons.  Diffuse fluorescence tomography (DFT), also described as fluorescence molecular tomography (FMT)¹⁰ presents significantly decreased spatial resolution compared to OPT of transparent samples but, nevertheless, it is able to reconstruct fluorescence lifetime distributions in scattering phantoms^{11, 12} and in ex vivo¹³ and live mice^{14, 15} and recently we reported the first FLIM FRET tomography of genetically expressed fluorescent proteins in a live mouse¹⁶. We note that the precision of fluorophore localization is still compromised by the absorption and scattering of excitation and fluorescence radiation and by the background autofluorescence of the biological tissue. The impact of absorption and scattering could be mitigated in future by engineering new FRET probes with donor-acceptor combinations utilizing fluorophores that are both excited and emit at longer wavelengths, such as red and near infrared fluorescent proteins. These advances towards the practical application of FLIM FRET to read out molecular interactions and biosensors in vivo would facilitate longitudinal in vivo studies for a variety of research fields, including drug discovery, and could lead to a significant reduction in the number of animals needed for biomedical research.

¹⁰ Ntziachristos, V., Annu Rev Biomed Eng, 8 (2006) 1
¹¹ Kumar, A. T. N., Raymond, S. B., Bacskai, B. J. and Boas, D. A., Optics Letters, 33 (2008) 470
¹² McGinty, J., Soloviev, V. Y., Tahir, K. B., Laine, R., Stuckey, D. W., Hajnal, J. V., Sardini, A., French, P. M. W. and Arridge, S. R., Optics Letters, 34 (2009) 2772
¹³ Venugopal, V., Chen, J., Lesage, F. and Intes, X., Optics Letters, 35 (2010) 3189
¹⁴ Nothdurft, R. E., Patwardhan, S. V., Akers, W., Ye, Y., Achilefu, S. and Culver, J. P., Journal of Biomedical Optics, 14 (2009) 024004
¹⁵ Rusanov, A. L., Ivashina, T. V., Vinokurov, L. M., Fiks, I. I., Orlova, A. G., Turchin, I. V., Meerovich, I. G., Zherdeva, V. V. and Savitsky, A. P., Journal of Biophotonics, 3 (2010) 774
¹⁶ McGinty, J., Stuckey, D. W., Soloviev, V. Y., Laine, R., Wylezinska-Arridge, M., Wells, D. J., Arridge, S. R., French, P. M. W., Hajnal, J. V. and Sardini, A., Biomedical Optics Express, 2 (2011) 1907