The ability to detect multiple targets simultaneously can enable visualization of complex functional and molecular processes in-vivo. Possessing the capacity to approach imaging an unknown turbid medium from multiple imaging pathways is rapidly growing in popularity and effectiveness. Here we present a novel quantitative imaging system capable of resolving multiple contrast agents at different emmitance wavelengths. We have used this system to image cancerous tissue in mice in vivo in a murine model.
The dual wavelength FMT system shown in Figure 1. employs two cw diode lasers (one operating at 672 nm and the other at 748 nm) which are time-mulitplexed with a 1x2 fiber optic switch. The selected iluumination light is then sent into a 1x48 fiber optic switch which sequentially illuminates each of the 47 source fibers located at the entrance to the optical chamber. The 48th fiber is used to front-iluminate the sample through the front glass window of the chamber and it is used for fluorescence reflectance imaging (FRI) studies. A CCD camera with appropriate filter combinations is used to capture the excitation and fluorescence signal transmitted through the sample for each wavelength channel.The signal is then sent to the computer for processing.
Building up on the successes and strengths of our second generation continuous wave system, we have come up with an extension which allows for multi-wavelength imaging capabilities. This capability was accomplished by modifying the system to include an opto-mechanical switch that allows the user to select the input wavelength, as well as theaddition of the appropriate filters on the camera for the second fluorochrome channel. This allows us to bind different probes to different fluochromes and analyze their distribution independent of other fluochromes.
Cross talk of the system was evaluated with studies involving phantoms designed to mimic tumors containing amounts of different fluorochromes. Figure 2 details the response from phantoms used in multi-wavelength experiments. In this experiment, quantative data was taken to assist in developing the parameters the system would use when taking multi-wavelength data. Two tubes were inserted into the imaging chamber with known and varying concentrations of fluorescent material. These tubes were then subjected to a standard 'full' imaging session and the data this provided was then used to calibrate our reconstruction procedures.
Two tubes viewed at the two different wavelengths implemented in the absence of diffusive medium using FRI. The leftmost tube (Figure 3) contained 250nm Cy5.5 and 500nM of AF750 fluorochromes, the rightmost (Figure 4) contained the reverse combination, 500nm of Cy5.5 and 250nM of AF750. The differentiation of fluorochromes is captured well and cross talk was found to be of the order of 1% or less using appropiate filters.