Journal
JOURNAL OF BIOPHOTONICS
Volume 1, Issue 5, Pages 390-394Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jbio.200810044
Keywords
Fluorescence lifetime imaging; time-resolved imaging; optical projection tomography; 3-D imaging; fluorescence tomography
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Funding
- DTI Beacon Award
- European Community
- Medical Research Council [MC_U127574434] Funding Source: researchfish
- MRC [MC_U127574434] Funding Source: UKRI
- ICREA Funding Source: Custom
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We describe a quantitative fluorescence projection tomography technique which measures the 3-D fluorescence lifetime distribution in optically cleared specimens up 1 cm. in diameter. This is achieved by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3-D, time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a mono-exponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3-D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa-488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta.
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