To test out the dual-tracer background subtraction approach in phantom and animal studies, fluorescence from targeted and untargeted fluorescent tracers was imaged on a micro-computed tomography-guided time-domain FT system. Much of the particulars of the system have been covered in depth previously,27,28 however, some significant changes were made to the system to permit the simultaneous imaging of two fluorophores emitting at two different wavelengths. A schematic of the dual-wavelength version of the system is presented in Fig. 3(a). In brief, the FT system is a noncontact fan-beam geometry system using two pulsed-diode lasers (Picoquant, Berlin, Germany): one centered at 635 nm and the other centered at 755 nm. For the experiments carried out in this study, the lasers were pulsed at 40 MHz, 180 deg out of phase from each other. Each laser beam was passed through a 10-nm band-pass filter (Chroma Technologies, Bellows Falls, Vermont) centered at the respective wavelengths of the lasers, before both beams were coupled into separate 50-μm multimode optical fibers (Thorlabs, Newton, New Jersey). Tests of the laser demonstrated that the 755 nm laser was more powerful than the 635 nm laser, so as a means of having control over the relative strengths of the two lasers, the output of the 755-nm laser fiber was passed through an in-line motorized variable neutral density filter (OZ Optics, Ottawa, Ontario), before the optical fibers from both lasers were coupled together into a single 100-μm fiber using a custom-made fiber combiner (OZ Optics, Ottawa, Ontario). The output of the fiber combiner was then passed through a second variable neutral density filter to control the overall excitation power of the system, and then separated into two fibers using a in-line beamsplitter (OZ Optics, Ottawa, Ontario). The 4% output was projected onto a reference photomultiplier tube (PMT, Hamamatsu Photonics, Japan) controlled by a time-correlated single photon counting (TCSPC) card (Becker & Hickl GmbH, Berlin, Germany) set to measure photon arrival times in a 25-ns window at a resolution of approximately 24 ps. This allowed the temporal pulse spread functions (TPSFs) of both laser pulses to be monitored during the experiment simultaneously [Fig. 3(b)]. The 96% output of the beamsplitter was coupled to the FT imaging gantry and focused onto the center of the gantry where the imaging specimen was placed.