Quantitative Biophotonics for Tissue Characterization and Function

我们进一步开发了我们的项目，使用近红外荧光成像评估乳腺癌异种移植物（小鼠模型）中HER 2的过表达。基于小Affibody分子的新型特异性探针与内部仪器相结合，使我们能够在体内表征癌细胞中具有不同HER 2过表达水平的肿瘤。测试了具有不同结合/洗脱性质的两种荧光染料（AlexaFluor 750和DyLight）。研究表明，如果通过配体-受体模型考虑探针药代动力学，则可以通过分析一系列肿瘤荧光图像来估计HER 2过表达。已经观察到我们的体内方法和常规离体技术的结果之间的良好相关性。我们能够监测由于在小鼠模型中使用已知抗癌药物17-DMAG治疗而引起的肿瘤变化。寿命是荧光的另一个特征，可以通过使用我们的仪器进行的时间分辨成像来估计。使用适当的染料，它可以潜在地提供关于荧光团环境（（pH、温度、组织氧含量、营养供应和生物能量状态））的信息，这些信息可以具有真实的诊断价值。我们最近与华盛顿大学的Dr. Jumefu合作开发了一种新型的pH敏感染料，并在幻影中进行了初步测试。我们还在研究体内肿瘤（小鼠模型）中HER 2特异性探针的荧光寿命变化，以评估可能对优化治疗很重要的癌细胞特性。 双光子成像项目继续作为对体内装置的研究。该装置只能收集落射荧光，并使用离物镜一定距离的收集皿来收集。研究透镜的组织特性和收集器距照明中心的距离对测量结果的影响是很重要的。这两个因素在选择设备配置时至关重要。从模拟中可以看出，关键问题是距离，我们可以确定反射收集器与照明位置的距离，以确保发生总落射收集。 偏振成像是一种很有前途的工具，可视化组织表面下的隐藏结构。这些结构的分析，例如胶原蛋白网络，可用于评估从正常组织功能到患病组织的可能转变。我们已经开发出先进的技术的数据分析，基于皮尔逊相关程序，允许提高图像质量和揭示区域内的高统计相似性的嘈杂的图像，使得可能表征的生物组织的表面下的结构特征。为了实现该方法的潜力，我们设计了一个用户友好的偏振成像系统，同时图像交叉和共偏振光。初步实验表明，设计的偏振适配器与开发的数据分析算法相结合，可以提供胶原蛋白结构的定量信息。我们已经将这种适配器整合到传统的阴道镜中，并开始了研究宫颈组织结构的临床方案。收集了10名健康志愿者在周期的黄体期和卵泡期的数据，并正在进行分析，以优化系统和更新临床方案 肿瘤学界正在测试一些新的靶向方法，用于治疗各种癌症。关于监测脉管系统，期望开发和评估非侵入性和定量技术，其不仅可以监测结构变化，而且还可以评估肿瘤的功能特征或代谢状态。我们正在测试三种潜在的非侵入性成像技术，以监测正在接受实验性治疗的患者：红外热成像（热成像），激光多普勒成像（LDI）和多光谱成像。这些成像技术正在卡波西肉瘤（KS）受试者身上进行测试，卡波西肉瘤是一种高度血管化的肿瘤，经常发生在感染了获得性免疫缺陷综合征（AIDS）的人群中。皮肤KS病变是很容易接近的非侵入性技术，涉及肿瘤血管成像，因此，他们代表了一个肿瘤模型，其中评估某些参数的血管生成。KS研究是根据四种不同的NCI方案进行的临床试验。我们已经证明，我们的多模态技术可以无创地监测肿瘤和周围组织的功能特性，并有可能预测治疗结果。最近的工作已经在真实的时间获得这些血管生成标记物（血容量和氧合）上进行。使用基于主成分分析的新型数据分析工具，我们已经表明KS病变可以通过肿瘤的功能状态进行真实的实时成像和评估。结合OCT，我们最近已经显示了在真实的时间获得的血容量和氧合的定量结果。我们还发现，如果考虑到底层皮肤结构，则可以获得定量结果。为了获得有关这些结构的信息，我们开发了一种谱域光学相干断层扫描（OCT）系统，它给出了皮肤的3D图像。

We have further developed our project to assess HER2 overexpression in breast carcinoma xenografts (mouse model), using near infrared fluorescence imaging. Novel specific probes, based on small Affibody molecules, in combination with in-house instrumentation, allowed us to characterize tumors with different levels of HER2 overexpression in the cancer cells in vivo. Two fluorescent dyes (AlexaFluor 750 and DyLight) with different binding/washout properties were tested. It was shown that HER2 overexpression can be estimated from analysis of a series of the tumor fluorescence images, if the probe pharmacokinetics is taken into account by a ligand-receptor model. Good correlation between results of our in vivo method and conventional ex vivo techniques has been observed. We were able to monitor changes in the tumor due to its treatment with a known anti-cancer drug 17-DMAG in the mouse model. Lifetime is another characteristic of fluorescence that can be estimated from time-resolved imaging, performed with our instrumentation. With a proper dye it can potentially provide information about fluorophore environment ((pH, temperature, tissue oxygen content, nutrient supply, and bioenergetic status)) that can be of real diagnostic value. In collaboration with Dr. Achilefu from Washington University we have recently developed a novel pH-sensitive dye and performed its initial tests in the phantoms. We are also investigating changes in the fluorescence lifetime for HER2 specific probes in the tumor in vivo (mouse model) in order to assess the properties of the cancer cells that maybe important to optimize treatment. The two-photon imaging project has continued as a study on an in-vivo device. The device is only able to collect epifluorescence and does so using a collection dish at some distance from the objective. It was important to study the effects of the tissue properties of the lens and the distance of the collector from the illumination center. These two factors are crucial in choosing the configuration of the device. It transpired from simulations that the key issue was distance and we could determine a distance for the reflection collector to be set at from the illumination location to ensure that the total epi-collection occurred. Polarization imaging is a promising tool to visualize hidden structures below the tissue surface. Analysis of these structures, for example the collagen network, can be used to assess the possible transition from normal tissue function to diseased tissue. We have developed sophisticated techniques of data analysis, based on Pearson correlation procedure, that allow to enhance the image quality and reveal regions of high statistical similarities within the noisy images, making possible characterization of subsurface structural features of biological tissues. To realize the potential of the method we have designed a user-friendly polarization imaging system that simultaneously images cross- and co-polarized light. Preliminary experiments have demonstrated that designed polarization adapter in combination with developed data analysis algorithms can provide quantitative information on collagen structure. We have incorporated this adapter into a conventional colposcope and started a clinical protocol to study tissue structures in the cervix. Data on 10 healthy volunteers have been collected at luteal and follicular phases of the cycle, and are being analyzed to optimize the system and update the clinical protocol 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. Recent work has been performed on obtaining these angiogenic markers (blood volume and oxygenation) in real time. Using novel data analysis tools based on Principal Component Analysis, we have shown that a KS lesion can be imaged and assessed in means of the functional state of the tumor in real time. In combination with OCT, we have recently shown quantitative results of blood volume and oxygenation, which were obtained in real time. We have also found that quantitative results can be obtained if the underlying skin structures are being taken into account. In order to obtain information about these structures, we have developed a spectral domain Optical Coherence Tomography (OCT) system, which gives 3D images of the skin.