Molecular Dynamics And Vibrational Characteristics Of Me

工作总结：我们的研究工作包括两个主要领域：(1)双层脂质对整体膜蛋白结构重组的调节作用；(2)仪器开发和应用(a)振动拉曼和红外光谱成像技术；(b)可见反射高光谱成像方法的临床应用。(1)我们利用振动红外和拉曼光谱技术表征生物膜内波动脂质微域的大小和形成，重点关注脂质簇的形成及其对整体膜蛋白内诱导构象变化的调节影响。细菌视紫红质被用作模型系统，以证明这些定量脂质微聚集体的侧向压缩性特性对蛋白质运动特性的影响。S跨膜螺旋。在研究微域内光谱特异性脂链有序/无序效应时，适当的酰基链氘化可以单独监测每个链段的振动动力学。此外，利用红外光谱技术，我们详细研究了细菌紫质m态中间体的光循环活性和蛋白质整体膜α螺旋的构象柔韧性的脂质控制。（2A）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，通过结合步进扫描干涉测量和最先进的红外敏感二维焦平面阵列探测器。高性能数字成像与无创、高分辨率光谱学的集成允许在各种宿主环境中可视化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，我们将该技术应用于有监督和无监督的前列腺组织病理学，涉及组织微阵列形式的大量组织切片。这种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振动光谱特征，用于对照标本、前列腺上皮内肿瘤样本、良性前列腺增生和腺癌组织的活检标本的病理分析。在这种情况下，我们的成像仪器结合了高灵敏度的线性阵列检测，用于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前列腺组织中出现的十种形态实体。举例来说，最大高斯似然法的使用和概率分类模型的单独使用都允许对十种组织学类别进行客观的、自动的描述，达到95%的顺序。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。特别是，我们已经实现并利用了一种广义形式的干涉快速或连续扫描红外光谱成像（与干涉仪步进扫描方法不同），用于任何类型的焦平面阵列探测器，以秒为单位对不可逆的动态事件进行成像。此外，我们是第一个实现时间分辨傅里叶变换红外光谱成像的团队，例如，可以将半衰期在十分之一毫秒量级的重复动态过程可视化。本案例中使用的示例涉及特定的聚合物液晶复合材料。（2B）我们的成像方法也扩展到可见光区域，利用CCD检测和适当的液晶可调滤波器进行波长区分，获得反射光谱。这种特殊的无创反射装置已成功地用于镰状细胞病患者血红蛋白组织氧合饱和度成像的临床场所，我们检查了一氧化氮（NO）刺激、抑制和给药的影响。除了一氧化氮气体的作用外，还研究了乙酰胆碱（一种刺激内皮细胞释放一氧化氮的激动剂）和硝普钠（一种直接一氧化氮供体和内皮非依赖性血管扩张剂）的作用。我们的结论是，镰状细胞病患者表现出组织氧合受损，尽管其静息血流量值比健康的非裔美国人高两倍。此外，当血流量在药理学上增加7倍时，组织氧合得到改善，但仍远低于健康受试者。这种多功能的可见反射成像方法为血管疾病患者提供了一种有用的实时探针，并为评估疾病严重程度和疾病进展提供了一种新方法。

Summary of Work: Our research efforts encompassed two general areas: (1) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (2) the instrumental development and applications of (a) vibrational Raman and infrared spectroscopic imaging techniques and (b) the clinical utilization of visible-reflectance hyperspectral imaging approaches. (1) Our interest in characterizing the sizes and formation of fluctuating lipid microdomains within biomembranes, using vibrational infrared and Raman spectroscopic techniques, were focused on lipid cluster formation and their modulatory influence on induced conformational changes occurring within integral membrane proteins. Bacteriorhodopsin was used as a model system to demonstrate the effects from the lateral compressibility properties of these quantified lipid microaggregates on the motional characteristics of the protein?s transmembrane helices. In studying spectroscopically specific lipid chain order/disorder effects within the microdomains, appropriate acyl chain deuteration allowed the vibrational dynamics of each chain moiety to be monitored separately. Additionally, using infrared spectroscopic techniques, we examined in detail the lipid control of both the photocycle activity of the M-state intermediates of bacteriorhodopsin and the conformational flexibility of the protein's integral membrane alpha helices. (2A) Considerable emphasis was placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared sensitive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical 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 the technique to both supervised and unsupervised prostate histopathology involving large numbers of tissue sections in the form of tissue microarrays. This 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 the vibrational spectral signatures for the pathologic analyses of biopsied representations of control specimens, prostatic intraepithelial neoplastic samples, samples manifesting benign prostatic hyperplasia and adenocarcinoma tissues. In this case, our imaging instrumentation incorporated highly sensitive linear 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 prostatic tissue. As examples, both the use of the Maximum Gaussian Likelihood Method and, separately, a probabilistic classification model allowed the objective, automated delineation of the ten histologic categories to the order of 95%. 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. In particular, we have implemented and utilized a generalized form of interferometric rapid-, or continuous, scan infrared spectroscopic imaging (to be distinguished from interferometer step-scan approaches) for utilization with any type of focal plane array detector to image nonreversible dynamic events in the order of seconds. Further, we are the first group to implement time-resolved Fourier-transform infrared spectroscopic imaging which permits, for example, the visualization of repetitive dynamic processes with half lives on the order of tenths of milliseconds. The examples used in this case involved specific polymer liquid crystal composites. (2B) Our imaging approaches have also been extended to the visible spectral region in which reflectance spectra are obtained using CCD detection and appropriate liquid crystal tunable filters for wavelength discrimination. This particular noninvasive reflectance unit has been used successfully in clinical venues for imaging hemoglobin tissue oxygenation saturation in sickle cell disease patient in which we examined the effects of nitric oxide (NO) stimulation, inhibition and administration. In addition to the effects of NO gas, actetylcholine, an agonist that stimulates the release of NO from the endothelium, and sodium nitroprusside, a direct NO donor and endothelium-independent vasodilator were examined. We concluded that patients with sickle cell disease exhibit impaired tissue oxygenation despite having resting blood flow values that were two-fold higher than healthy African American subjects. Furthermore, when blood flow was pharmacologically increased by seven-fold, tissue oxygenation was improved, but remained well below healthy subjects. This versatile, visible reflectance imaging approach provides a useful, real time probe of patients with vascular disease and allows a novel means for assessing disease severity and disease progression.