Fluorescence lifetime optical projection tomography

J Biophotonics. 2008 Oct;1(5):390-4. doi: 10.1002/jbio.200810044.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Embryo, Mammalian / ultrastructure
  • Fluorescence
  • Fluorescent Dyes
  • Imaging, Three-Dimensional / methods
  • Mice
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Tomography, Optical / methods*

Substances

  • Fluorescent Dyes