Reactive oxygen species (ROS) are believed to be involved in many diseases and injuries to the
brain, but the molecular processes are not well understood due to a lack of in vivo imaging
techniques to evaluate ROS. The fluorescent oxidation products of dihydroethidium (DHE) can
monitor ROS production in vivo. Here we demonstrate the novel optical imaging of brain in live
mice to measure ROS production via generation of fluorescent DHE oxidation products (ox-DHE)
by ROS. We show that in Sod2+/- mice, which have partial loss of a key antioxidant enzyme,
superoxide dismutase-2, that ox-DHE fluorescence intensity was significantly higher than in hSOD1
mice, which have four-fold overexpression of superoxide dismutase-1 activity, which had almost no
ox-DHE fluorescence, confirming specificity of ox-DHE to ROS production. The DHE oxidation
products were also confirmed by detecting a characteristic fluorescence lifetime of the oxidation
product, which was validated with ex vivo measurements.
In vivo optical molecular imaging of fluorescent probes predominantly employs continuous wave
techniques to measure fluorescence intensity. Alternatively, time domain techniques permit
measurement of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime
allows discrimination of fluorescent probes with contrasting lifetime or inference of a probe's
environment due to lifetime sensitivity. Here, we present the use of fluorescence lifetime contrast to
evaluate the relative concentrations of a mixture of fluorophores in a scattering medium. This
approach offers the potential to perform dual-probe in vivo optical molecular imaging at a single
wavelength employing lifetime contrast rather than via spectral intensity contrast.
A time-domain optical method to evaluate the concentration (n), lifetime (), and depth (d) of a fluorescent inclusion is described by the complete analysis of the fluorescence temporal point-spread function (TPSF). The behavior of parameters in the fluorescence TPSF is explored, and we demonstrate the method with experimental data from a localized fluorescent inclusion in scattering media to recover images of n, , and d. The method has potential application for in vivo fluorescence imaging.
In the last few years there has been a growing interest in the use of small animal optical molecular imaging systems to
conduct preclinical studies. Most of these imaging systems are based on continuous wave (CW) technology to measure
the bioluminescence or fluorescence light intensity from optical probes in small animals. The eXplore Opti<sup>TM</sup> is
currently the only commercially available imaging system based on time domain (TD) technology. In addition to
measuring the light intensity, the TD approach provides extra information to help determine the depth and concentration
of optical probes in small animals. Furthermore, the TD approach uniquely allows the fluorescence lifetime of a
fluorophore-based optical probe to be measured. Recently, our single wavelength eXplore-Opti<sup>TM</sup> system has been
upgraded to a multi-wavelength (eXplore Optix<sup>TM</sup>-MX) system with the addition of 3 laser wavelengths and
corresponding filters. This has enabled us to image a variety of fluorophores for different preclinical applications.
Preliminary results evaluating the performance of the eXplore-Opti<sup>TM</sup>-MX are presented employing fluorophores with
different spectral and lifetime characteristics.