Infrared, Raman and Visible Reflectance Spectroscopic Imaging

(A)相当多的重点放在加强我们的中红外光谱化学成像显微镜技术，通过结合步进扫描和连续扫描干涉测量与最先进的红外敏感二维焦平面阵列和线性阵列探测器。将高性能数字成像与无创、高分辨率红外光谱相结合，可以可视化不同宿主环境中不同化学物质的空间分布。该技术的力量还体现在同时获取每个空间位置的红外光谱。作为红外成像技术在诊断病理学中应用的一个例子，我们将这种方法应用于有监督和无监督（观察者独立）的前列腺组织病理学，涉及以组织微阵列形式检查的大量组织样本。这种红外光谱方法消除了化学染色组织的必要性。此外，使用组织微阵列所固有的高通量方法允许高效和有效地获取样品振动光谱特征，用于对照、前列腺上皮内瘤变、良性前列腺增生和腺癌等活检标本的病理分析。我们特别展示了一系列档案组织样本的自动组织学分割的应用；采用定义良好的统计显著性检验。这种方法表明，组织病理变化现在可以通过基于生物化学的客观光谱标准来定义，而不一定需要病理学家的干预或解释。在这些例子中，我们的成像仪器结合了高灵敏度的线性阵列和焦平面阵列检测，以快速记录超立方体光谱数据。为了在光谱上阐明前列腺组织中存在的各种组织学特征，开发了非常大的光谱训练集和适当的光谱度量来区分在该特定组织中发生的十种形态实体。最大高斯似然法的使用和基于贝叶斯统计的概率分类模型的单独使用，使得对十种组织学类别的客观、自动描述的准确性达到95-99%。对组织分割过程进行了微调。此外，研究人员还利用受试者工作特征曲线来探讨高通量、光谱描绘区分腺癌的敏感性和特异性之间的关系。这些程序与当前的组织处理程序完全兼容。对于我们的普通红外成像仪器，在光学、探测器配置和数据收集范式方面进行了许多增强功能。

(A)Considerable emphasis was placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan and continuous scanning interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array and linear array detectors. The integration of high performance digital imaging with noninvasive, high resolution infrared spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. As one example of the utility of the infrared imaging technique in diagnostic pathology, we applied this approach to both supervised and unsupervised (observer independent) prostate histopathology involving large numbers of tissue samples examined in the form of tissue microarrays. This infrared spectroscopic method eliminates the necessity for chemically stained tissue. Further, the high throughput approach inherent in the use of tissue microarrays allows the efficient and effective acquisition of a samples vibrational spectral signature for pathologic analyses of biopsied specimens representative of controls, prostatic intraepithelial neoplasia, benign prostatic hyperplasia and adenocarcinoma. We specifically demonstrate the application of automated histologic segementation for a series of archival tissue samples; well-defined tests of statistical significance were incorporated. This approach demonstrates that histopathologic changes can now be defined by biochemistry-based, objective spectroscopic criteria that do not necessarily require a pathologist's intervention or interpretation. In these examples, our imaging instrumentation incorporated highly sensitive linear array and focal plane array detection for rapidly recording hypercube spectral data. For spectroscopically elucidating the various histologic features present in prostate tissue, extraordinarily large spectral training sets and appropriate spectroscopic metrics were developed for distinguishing ten morphological entities occurring in this specific tissue. Both the use of the Maximum Gaussian Likelihood Method and, separately, a probabilistic classification model based on Beyesian statistics, allowed an objective, automated delineation of the ten histologic categories to be correct to the order of 95-99%. Fine tuning of the tissue segmentation process was developed. Additionally, receiver operating characteristic curves were used to explore relationships between sensitivity and specificity of the high throughput, spectroscopic delineations for distinguishing adenocarcinoma. These procedures are entirely compatible with current tissue processing procedures. With regard to our general infrared imaging instrumentation, a number of enhancing features were made in the optics, in detector configurations, and in data collection paradigms. (B)Vibrational infrared spectroscopic imaging techniques involving cellular acetylation studies were conducted. Acetylation and deacetylation of nucleosome core histones is important for modulating gene expression. Increased protein acetylation leads to molecular and biological changes that affect the transcriptional activation of certain genes. Histone deacetylase inhibitors (HDACi) are emerging as a promising new class of anticancer drugs. The anticancer activity of HDACi is ascribed to hyperacetylation of both core histones and to a variety of non-histone proteins critical in maintenance of the malignant phenotype. We developed a spectroscopic imaging technique for assessing HDAC inhibitor activity. The method applies vibrational infrared spectroscopy and multivariate data analysis to examine spectral changes that are indicative of specific biochemical alterations associated with acetylation. Peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated with HDAC inhibitor MS-275, and spectral changes in the 2900 cm-1 methyl and methylene carbon-hydrogen stretching mode regions and in the 1650-1500 cm-1 amide I and II banding mode regions were assessed. Concentration-dependent increases in protein acetylation were detected and quantified in PBMCs exposed to HDACi MS-275 in vitro and in PBMCs from patients treated in vivo. The data demonstrate a new approach to the assessment of global acetylation that is sensitive and totally independent of antibodies, requires minimum cell processing, and is easily adapted to high thoughput screening. (C) With respect to our visible reflectance imaging studies, a straightforward algorithm was developed for real time monitoring of oxygenation levels in blood cells and tissue based on the visible spectrum of hemoglobin. Absorbance images obtained from the visible reflection of white light through separate red and blue bandpass filters recorded by monochrome CCDs are combined to create enhanced images that suggest a quantitative correlation to the degree of oxygenated and deoxygenated hemoglobin in red blood cells. The filter bandpass regions are chosen specifically to mimic the color response of commercial 3-CCD cameras, representative of detectors with which the operating room laparoscopic tower systems are equipped. Adaptation of this filter approach is demonstrated for laparoscopic donor nephrectomies in which images are analyzed in terms of real time, in vivo monitoring of tissue oxygenation. (D) We developed our visible reflectance imaging methods for non- invasive monitoring of tissue oxygenation during nephrectomies. Standard methods for the assessment of organ viability during surgery are typically limited to visual cues and tactile feedback in open surgery. However, during laparoscopic surgery, these processes are impaired. This is of particular relevance during laparoscopic renal donation, where the condition of the kidney must be optimized despite considerable manipulation. However, there is no in vivo methodology to monitor renal parenchymal oxygenation during laparoscopic surgery. We have developed a method for the real time, in vivo, whole organ assessment of tissue oxygenation during laparoscopic nephrectomy to convey meaningful biological data to the surgeon during laparoscopic surgery. We apply the 3-CCD (charge coupled device) camera to monitor qualitatively renal parenchymal oxygenation with potential real-time video capability. We have validated this methodology in a porcine model across a range of hypoxic conditions and have then applied the method during clinical laparoscopic donor nephrectomies during clinically relevant pneumoperitoneum. 3-CCD image enhancement produces mean region of interest intensity values that can be directly correlated with blood oxygen saturation measurements (R2 > 0.96). Using the 3-CCD camera to qualitatively monitor tissue oxygenation provides a means of assessing intraoperative tissue oxygenation. This may be a useful method to avoid unintended ischemic injury during laparoscopic surgery. Preliminary results indicate that no significant changes in renal oxygenation occur as a result of pneumoperitoneum.