A theoretical model of light propagation in tissue, which accounts for scattering and absorption of light, predicts the spatial profile of the surface intensity of re-emitted light. The model allows for both re-emission of the introduced light and fluorescent light generated by an embedded chromophore, either inherent to or introduced in the tissue. Measurement of a series of surface intensity profiles allows reconstruction of the three-dimensional structure of the tissue using inverse analytical techniques. Prototype instrumentation has been developed to capture surface images for analysis. A laser scanning system introduces light into the tissue at a series of sites in the region of interest and the emitted light is optically filtered through a dichroic filter and imaged onto a cooled charge coupled detector. Measurements using this instrumentation of fluorescent markers embedded in a highly scattering turbid medium yielded excellent agreement between the reconstructed theoretical prediction and the experimental measurement of these known sites. Identification of deeper structures within the tissue is possible by extending the instrumentation to capture images in the near infra-red region of the spectrum and by the use of novel infrared compounds to act as probes localized at desired sites within the tissue.? ? LBPS, in collaboration with NCI and NICHD, has previously explored the use of nanocrystals as angiographic contrast agents. In the current work, we have explored application of commercially produced upconverting nanocrystals - UPNCs - (obtained through a formal Material Transfer Agreement - MTA) to biological problems. Until present, the utility of these crystals has been limited by their large size. UPNCs are now available, via the MTA, with sizes down to 10nm with surfaces modified to present amine or carboxylate groups as attachment agents for biological applications. A particularly appealing aspect of UPNCs is their potential use as diagnostic markers for the separation sciences such as capillary electrophoresis. A major limitation to the lowest level of detectability of diagnostic markers is the inherent fluorescent background signal from autofluorescence and/or the substrate/envelope. UPNCs potentially can reduce the background signal significantly using for example excitation source in the near ir, for example 980nm, and an emission in the visible, for example 550nm. As an initial strategy to demonstrate effectiveness, a comparison will be made for an immuno-capture separation between UPNC and fluorescent dye tagged analytes.? ? The use of polarized light has been explored in conjunction with NICHD and NCI as a method to track changes in tissue structure. Polarized photography offers the potential of distinguishing hidden structures developed below the skin surface, such as fibrosis resulting from X-ray radiation. A variable-angle polarized illumination system applied laser light to the surface of the skin of an athymic mouse. The scattered light (captured at an angle to minimize directly reflected light) was analyzed using a polarization sensitive detector for changes in polarization resulting from the interaction of the light and the tissue. Clear differences were observed for the degree of polarization between the skin of normal athymic mice and athymic mice irradiated with X-rays. ? ? Equi-intensity profiles of linearly polarized 650nm probe light diffusely reflected from skin and tissue-like phantom controls were fitted to ellipses. The orientation of the semi-major axis has a tendency to be perpendicular to collagen fiber orientation close to the entry point of the probe beam, but at larger distances the eccentricity becomes parallel to the fibers. Further, Fourier transform filtering of the polarization degree pattern allows the determination of the orientation and characteristic size of hidden structures developed under the skins surface under conditions such as fibrosis resulting from x-radiation. Fourier transform analysis of the polarization degree pattern and measurement of the equi-intensity profiles of a pencil-like polarized beam, backscattered from the skin, may allow characterization of fibrotic diseases, and may offer a means for safer radiation treatment.? ? Prototype instrumentation, in collaboration with NICHD, has been fabricated and assembled. It is currently being evaluated for studying changes in the structure of cervical tissue. A key feature is the integration of the polarized illumination light into the polarization sensitive visualization optical axis.
| Hassan, Moinuddin; Riley, Jason; Chernomordik, Victor et al. (2007) Fluorescence lifetime imaging system for in vivo studies. Mol Imaging 6:229-36 |
