Fluorescence lifetime optical projection tomography

• Published in: Journal of biophotonics 2008-10, Vol.1 (5), p.390-394
• Main Authors: McGinty, James, Tahir, Khadija B., Laine, Romain, Talbot, Clifford B., Dunsby, Christopher, Neil, Mark A. A., Quintana, Laura, Swoger, James, Sharpe, James, French, Paul M. W.
• Format: Article
• Language: English
• Subjects: 3-D imaging; Animals; Embryo, Mammalian - ultrastructure; Fluorescence; Fluorescence lifetime imaging; fluorescence tomography; Fluorescent Dyes; Imaging, Three-Dimensional - methods; Mice; Microscopy, Confocal; Microscopy, Fluorescence; optical projection tomography; time-resolved imaging; Tomography, Optical - methods
• ISSN: 1864-063X; 1864-0648
• DOI: 10.1002/jbio.200810044

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. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)