The rapid development of molecularly targeted probes for use in vivo has led to growing interest in clinical applications that incorporate information from these probes. In particular, there is a need for techniques to visualize these targeted probes during surgery; the probes could be used, for example, to identify tissue either for removal or preservation. This project focuses on the development of instrumentation for real-time visualization of fluorescent probes. When looking at fluorescent probes in tissue, the major difficulty is usually separating the probe fluorescence from the tissue autofluorescence. Because the autofluorescence has different spectral properties than the fluorophore, it can easily be separated using multispectral imaging, in which several full emission spectra can be taken for each pixel. Although the implementation of acousto-optic tunable filters has greatly increased the speed of these techniques, the minimum time required to acquire an image cube is still several seconds with commercially available systems. This data acquisition rate makes the use of multispectral imaging to provide real-time feedback during a surgical procedure impractical.? ? As an alternative to multispectral imaging, aggressive filtering of both emission and excitation light can help to minimize the effects of tissue autofluorescence. Although the signal level drops as the spectral window is narrowed, this can be overcome by using a sensitive camera, such as a cooled CCD, ICCD, or even an EMCCD. Because a single spectral window is used, the data-taking rate is much faster, and could easily approach the video rate, thirty frames per second. Depending on the concentration, extinction coefficient, and quantum yield of the fluorophore, it may also be necessary to acquire simultaneous fluorescence images at two wavelengths in order to provide a correction for the autofluorescence. This simple autofluorescence correction could also be implemented with a single camera using two emission filters, but the image acquisition rate would be considerably slower.? ? The immediate application for this instrument is the identification of peritoneal metastases in ovarian cancer, using a GSA-Rhodamine Green probe developed in NCI. For preliminary experiments, we assembled a simple instrument comprised of a fiber optic ring light with a narrow bandpass filter between two fiber bundles, a narrow bandpass emission filter, and a single cooled monochrome CCD camera. This instrument has been successfully used for mock cytoreductive surgery in a murine model of ovarian cancer. The sensitivity was sufficient to identify labeled metastases at an image acquisition rate of five frames per second with specificity comparable to that achieved with a multispectral system. ? ? Future development of the hardware will move forward on several fronts. With some fairly straightforward modifications to the instrument, the sensitivity can probably be increased to the point where video rate image acquisition is possible. In addition, the implementation of a simple autofluorescence correction by using a second fluorescence image will be explored, and incorporated into the instrument if necessary. The illumination will be modified and a second camera used to provide a simultaneous pseudo-white light image of the surgical field. Finally, the implementation of this technique for laparoscopic procedures will be explored.
