Fluorescence lifetime optical projection tomography.
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.
McGinty
James
J
Department of Physics, Imperial College London, Prince Consort Road, South Kensington, London, SW7 2BW, UK. james.mcginty@imperial.ac.uk
Tahir
Khadija B
KB
Laine
Romain
R
Talbot
Clifford B
CB
Dunsby
Christopher
C
Neil
Mark A A
MA
Quintana
Laura
L
Swoger
James
J
Sharpe
James
J
French
Paul M W
PM