Quantitative Biophotonics for Tissue Characterization and Function

用于组织表征和功能的定量生物光子学

AMIR GANDJBAKHCHE (? to ?) $7,360,731

Project ID: ZIAHD000261 (NICHD)

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Abstract

Transition from normal tissue function to diseased tissue can be detected by quantifying irregular patterns. The degree of statistical similarities in a region of interest can carry valuable comparative information about the structural features of the tissue and can help to characterize tissue, i.e., analyze disease localization and progression. To visualize subsurface structural features of biological tissues, we have developed a user-friendly polarization imaging system that simultaneously images cross- and co-polarized light. We have developed a quantitative statistical tool, based on Pearson correlation coefficient analysis to enhance the image quality and reveal regions of high statistical similarities within the noisy tissue images. We have shown that under certain conditions, such maps of the correlation coefficient are determined by the textural character of tissues and not the choice of the reference image region, providing information on tissue structure. As an example, the subsurface texture of a demineralized tooth sample was enhanced from a noisy polarized light image. We have recently adapted the polarized camera to be used in a colposcopeto study tissue structures in the cervix. Fluorophore lifetime imaging is a promising tool for studying tissue environment such as tumors. The lifetime (time for an electron to return from excited state to initial state) of a fluorophore can vary in response to changes in the immediate environment such as temperature, pH, tissue oxygen content, nutrient supply, and bioenergetic status. Mapping the lifetime and location of a fluorophore in tissue at different depths can be used to monitor such parameters. Toward this goal, we have developed a time-resolved lifetime imaging system for in vivo small animal studies that maps fluorophores lifetimes. The system consists of a single source-multiple detector array that scans the surface of the tissue. Using several source-detector separations, one is able to probe different depths of the medium. We have used a pH sensitive dye in the near-infrared region in order to study the tumor environment below the skin. We have demonstrated that by using simplified back projections we are able to map near surface fluorescent lifetime in vivo. Combining this with the pre-calibrated lifetime response to pH, we have shown that biologically plausible, non-invasive, quantification of pH in mouse tumors can be determined. In collaboration with Dr. Capala in the Radiation Oncology Branch project of NCI who has developed probe for in vivo monitoring of HER2-positive cancer (for example breast cancer) non invasively and HER2 specific delivery of therapeutic agents. We have been able to quantify the bio-distribution of the probe and, HER2 receptors on tumor cells, showing the potentila of this method for monitoring non-invasively and quantitatively the teatment response. To further this work to include deeper tissue imaging we have examined the use of novel data-types for reconstructing location and lifetime of fluorophores embedded deeper in a turbid medium. We have developed a set of local data-types which provide enhanced noise tolerance over the standard global data-types for imaging purposes. Analysis of deeply embedded tissue abnormalities, using time-resolved fluorescence, should take into account highly scattering nature of biological tissues. To address corresponding complications we investigated the limitations of previously developed analytical model of photon migration for localized fluorophores. It was shown that to better describe experimental results the model should incorporate more realistic distribution of fluorescence lifetimes thus providing more flexibility to the inverse model to converge. This analytic model has been used as a forward model to reconstruct the lifetime and location of a point source fluorophore. Other advances have been made in studying the noise sensitivity of different data-types in time-resolved fluorescence imaging, we suggested a new local set of data-types that is likely toprovide more stability to noise than classical statistical (global) data-types used in diffuse optical tomography. We have pursued also another approach to quantification of fluorescence lifetimes of deeply embedded fluorophores that can work when intrinsic lifetime of the fluorophore is comparable to photon migration time in the medium, using general scaling relations to correct observed time-resolved intensity distributions from fluorescent targets at a given depth z inside turbid medium to an expected surface distribution (from the same fluorophore), revealing the intrinsic fluorescence lifetime without the need for full-scale reconstruction. Similar corrections could be applied when comparing the time-resolved data obtained from the same deeply embedded fluorophore by several detectors, positioned at different distances from the source (excitation photon entry point into the medium). . We experimentally verified these relations, usingtissue-like phantoms. Developed random walk model of time-resolved fluorescence imaging substantiates these scaling relations. Another optical imaging modality of interest to the group is Two-Photon Microscopy. Colleagues at NHLBI have recently developed a new system for Two-Photon imaging based on the Total Emission Detection (TED) principle. Here instead of using a standard TED system where only transmitted or reflected light is collected the TED Two Photon system captures all light emitted from the sample. Researchers in this group were contacted to model Two Photon emission and evaluate the systems performance. Using our Monte- Carlo simulation code we were able to evaluate different aspects of Two-Photon Imaging and the related field of Fluorescence Correlation Spectroscopy in collaboration with Section on Cell Biophysics at NICHD. The oncology community is testing a number of novel targeted approaches for use against a variety of cancers. With regard to monitoring vasculature, it is desirable to develop and assess noninvasive and quantitative techniques that can not only monitor structural changes, but can also assess the functional characteristics or the metabolic status of the tumor. We are testing three potential noninvasive imaging techniques to monitor patients undergoing an experimental therapy: infrared thermal imaging (thermography), laser Doppler imaging (LDI) and multi-spectral imaging. These imaging techniques are being tested on subjects with Kaposi s sarcoma (KS), a highly vascular tumor that occurs frequently among people infected with acquired immunodeficiency syndrome (AIDS). Cutaneous KS lesions are easily accessible for noninvasive techniques that involve imaging of tumor vasculature, and they thus represent a tumor model in which to assess certain parameters of angiogenesis. The KS studies are ongoing clinical trials under four different NCI protocols. We have shown that our multi-modality techniques can non-invasively monitor the functional properties of the tumor and surrounding tissues and has the potential to predict treatment outcomes. To better quantify the blood volume and oxygenation we have developed a frequency domain Optical Coherence Tomography system to better correlate the strctural information with functional activities of the tumor. Integration of this technique is underway. High-resolution confocal laser microscopy is an intensively active field in modern bioimaging technologies because this technique provides sharp, high-magnification, three dimensional imaging with submicron resolution by non-invasive optical sectioning and rejection of out-of-focus information. We have developed a simple fiber-optic confocal microscope with nanoscale depth resolution beyond the diffraction barrier. We are extensively working to increase the acquisition time for data collection in 3D.

荧光标记的细胞表面标志物的开发已导致疾病的特定分子诊断的快速进展。这些分子可以靶向特定的细胞受体，对诊断和治疗选择很重要。这种受体的一个例子是众所周知的生物标志物HER2（人类表皮生长因子受体2型），其在侵袭性肿瘤行为中起重要作用并且与不良的临床结果相关。 HER2特异性荧光探针的应用不限于体外/离体测定。这些探针可潜在地用于旨在表征/监测HER2的体内成像，因为在注射后特异性荧光探针优先浓缩在感兴趣的位点（例如，在肿瘤中），提供关于体内HER2表达的定量信息。肿瘤细胞中的探针积聚可以与一些广泛使用的HER2特异性药物（例如曲妥珠单抗）递送至患病部位平行。因此，它开启了“治疗和图像范例”的新时代。个体患者HER2表达的评估将有助于选择最佳治疗策略（例如，使用单克隆抗体MAB），同时持续监测这些生物标记物的状态。治疗将提供早期评估治疗干预效果的手段。目前估计HER2受体表达的技术使用需要组织活检的离体检测。它们与连续监测肿瘤受体对治疗的反应不相容。相反，非侵入性体内方法将是优先的，特别是在疾病的早期阶段。我们开发了一种新的非侵入性方法来表征HER2在体内的表达，使用光学成像和可同时使用的HER2特异性探针HER2靶向治疗.HER2特异性近红外荧光探针，基于小的Affibody分子，in与内部时间分辨近红外荧光成像系统的组合用于表征在癌细胞中具有不同水平的HER2过表达的体内肿瘤。我们测试了与荧光团DyLight 750（乳腺癌的小鼠模型）缀合的这种新型HER2 Affibody，并且表明如果通过考虑探针药代动力学，可以通过分析一系列肿瘤荧光图像来估计HER2过表达。配体 - 受体动力学模型。新探针对HER2受体具有高亲和力（KD = 3.660.26）。将探针（高达0.5mg / kg）注射到小鼠中没有观察到急性毒性。我们的测量结果显示，1分钟后，血液循环中的探针浓度随时间呈指数下降，血液平均半衰期为37分钟，比ABD-半衰期快约65倍（ZHER2：342））2-AlexaFluor750（40小时），用于我们之前研究过的。该探针从血液循环中的短暂冲洗时间使该探针成为潜在临床研究的良好候选者。应用配体 - 受体动力学模型来表征三种乳腺癌异种移植模型中HER2的表达，表达不同水平的HER2。将与DyLight750缀合的HER2特异性Affibody静脉内注射到携带HER2阳性肿瘤的小鼠中，并在几个预定时间点对ROI成像。基于药代动力学研究，计算每种肿瘤类型的归一化累积速率（NRA）的平均值。将它们与通过流式细胞术（FACS）测量的Affibody-DyLight-488保留进行比较。结果表明，两种参数在癌细胞HER2过表达的广泛范围内具有良好的线性相关性，证实了我们的观点，即体内荧光成像可提供HER2过表达的定量信息。时间分辨荧光成像系统可提供有关状态的其他信息。如果荧光寿命与荧光强度一起评估，则对肿瘤进行评估。前一个参数不依赖于荧光团浓度，但它可能对探针的局部生化环境敏感，例如温度，pH或分子相互作用，提供潜在有用的临床信息。特别是，我们在活动物中证明了荧光寿命可用于检测靶向光学探针与体内肿瘤细胞上的细胞外受体的结合。对于该研究，将与Dylight 750缀合的HER2特异性Affibody探针静脉内注射到携带HER2阳性和HER2阴性肿瘤的小鼠中，并用我们的时间分辨成像系统成像。我们比较了活体小鼠中不同肿瘤类型的结合（对肿瘤细胞）和未结合状态的光学探针的荧光寿命。我们的结果表明，我们的模型系统中的荧光寿命变化描绘了体内未结合探针结合的HER2受体。因此，寿命测量可用作受体结合过程的特定指示剂，尤其是用于早期评估疗法的功效。我们与华盛顿大学（Samuel Achilefu博士）和NIH成像探针开发中心合作，将两种pH敏感染料与马来酰胺接头连接起来。以前这些染料的灵敏度是用体模测试的，它们在不同的pH值范围内显示出不同的光学特性，范围为5.5到7.0（正常组织的pH范围是7.0-7.5但是，在许多恶性病变中，它可以低至5.6）。这些染料将与HER2特异性Affibody探针缀合，以进一步研究它们在细胞（体外）和动物（体内）中的敏感性。我们还继续使用极化作为结构变化的标记。极化成像允许人们分离组织的表面镜面反射和来自较深层的背散射光的贡献。因此，隐藏的地下结构可以被可视化以评估组织从健康状态到患病状态的转变。已经设计了相关和滤波算法以在存在噪声的情况下改善图像质量并且揭示具有高统计相似性的区域，例如与组织胶原结构相关的区域。为了实现该方法的潜力，我们将用户友好的偏振成像系统结合到传统的阴道镜中。根据我们对8名受试者的初步研究，我们发现子宫颈表面不够平坦，无法在多个焦距处收集数据，无法获得高质量的子宫颈图像。出于这个原因，升级了偏振成像系统以使焦距自动变化（现在正在收集不同焦平面上的10个共同和交叉偏振图像的堆叠）。我们认为更新的系统比原型更适合临床环境，并计划继续我们的可行性研究，以使用偏振成像评估月经周期的不同时间点的宫颈结构的年龄相关变化。我们继续评估弥漫性多光谱成像作为卡波西肉瘤现有反应评估的潜在有用补充，提供治疗效果的早期和非侵入性标记。在治疗过程中采集卡波西肉瘤皮损的多光谱图像，并通过主成分分析（PCA）获得血容量和氧合浓度图。将这些图像与通过常规手段确定的临床和病理反应进行比较。我们发现氧化血红蛋白有可能成为肿瘤反应的定量标志物。