Propagation of Light in Tissue and Imaging

光在组织中传播的理论模型考虑了光的散射和吸收，预测了再发射光的表面强度的空间分布。 该模型允许所引入的光和由嵌入的发色团生成的荧光的再发射，所述发色团是组织固有的或引入组织中的。 一系列的表面强度分布的测量允许使用逆分析技术的组织的三维结构的重建。 原型仪器已经开发出来，用于捕获表面图像进行分析。 激光扫描系统在感兴趣区域中的一系列位点处将光引入组织中，并且所发射的光通过二向色滤光器进行光学过滤并成像到冷却的电荷耦合检测器上。 使用这种仪器的测量嵌入在一个高度散射混浊介质的荧光标记物产生了极好的重建的理论预测和这些已知的网站的实验测量之间的协议。 通过扩展仪器以捕获光谱的近红外区域中的图像以及通过使用新型红外化合物作为定位在组织内的期望部位处的探针，可以识别组织内的更深结构。 LBPS与NCI和NICHD合作，以前曾探索过将纳米晶体用作血管造影造影剂。 在目前的工作中，我们已经探索了商业生产的上转换纳米晶体-UPNC-（通过正式的材料转移协议- MTA获得）的生物问题的应用。 到目前为止，这些晶体的用途一直受到其大尺寸的限制。 现在可通过MTA获得UPNC，其尺寸低至10 nm，表面经改性以呈现胺或羧酸酯基团作为生物应用的附着剂。 UPNC的一个特别吸引人的方面是它们作为分离科学如毛细管电泳的诊断标记物的潜在用途。 对诊断标志物的最低水平可检测性的主要限制是来自自体荧光和/或底物/包膜的固有荧光背景信号。 UPNC潜在地可以使用例如近红外（例如980 nm）中的激发源和可见光（例如550 nm）中的发射来显著降低背景信号。 作为证明有效性的初始策略，将对UPNC和荧光染料标记的分析物之间的免疫捕获分离进行比较。 偏振光的使用已经与NICHD和NCI一起被探索作为跟踪组织结构变化的方法。 偏振摄影提供了区分皮肤表面下发展的隐藏结构的潜力，例如由X射线辐射引起的纤维化。 可变角度偏振照明系统将激光施加到无胸腺小鼠的皮肤表面。 使用偏振敏感检测器分析散射光（以使直接反射光最小化的角度捕获），以确定由光和组织的相互作用引起的偏振变化。 在正常无胸腺小鼠和X射线照射的无胸腺小鼠的皮肤之间，观察到明显的差异的偏振度。 从皮肤和组织样体模对照漫反射的线偏振650 nm探测光的等强度分布拟合为椭圆。 半长轴的取向倾向于垂直于靠近探测光束的进入点的胶原纤维取向，但在更大的距离处，偏心率变得平行于纤维。 此外，偏振度图案的傅里叶变换滤波允许确定在诸如由X辐射引起的纤维化的条件下在皮肤表面下形成的隐藏结构的取向和特征尺寸。 偏振度图案的傅立叶变换分析和从皮肤反向散射的类锯齿状偏振光束的等强度分布的测量可以允许纤维化疾病的表征，并且可以提供用于更安全的放射治疗的手段。 与NICHD合作制造和组装了原型仪器。 目前正在评估其用于研究宫颈组织结构的变化。 一个关键特征是将偏振照明光集成到偏振敏感可视化光轴中。

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.