IN SITU TWO PHOTON IMAGE CORRELATION STUDIES OF ADHESION RECEPTORS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。对图像相关光谱学（ICS）和图像互相关光谱学（ICCS）的应用研究正在继续进行。黏附受体的聚集状态和转运特性的调节被认为在迁移细胞中控制细胞黏附的潜在机制中起着重要作用。我们将结合双光子显微镜和ICS方法对活细胞进行测量，继续研究细胞粘附、结构形成和分解的动力学。ICS的使用提供了一种同时监测荧光标记生物分子在质膜内的聚集状态及其运输特性的方法。此外，双色图像相互关联光谱（ICCS）允许通过在两个波长通道中成像非相同的物种来直接测量活细胞中的动态共定位现象。然后通过空间和时间的相互关系对这些进行分析。ICS/ICCS和双光子显微镜的结合使得这些测量能够以微创的方式对活细胞标本进行，使用破坏性较小的红外波长激光激发。为了研究焦点黏附成分的动力学和聚集性，我们制备了a5整合素亚基、paxillin和a-actin的GFP融合体。当在CHO或WI-38细胞中异位表达时，它们定位于局灶性粘连，不干扰局灶性粘连的迁移、扩散或形成。我们采用双光子ICS和ICCS两种独特的方法，研究了在多种激活和非激活细胞外基质（ECM）包被基质上培养的活成纤维细胞粘附结构的动态调控。此外，我们将ICS技术应用于光和电子显微镜图像，以确定实验动物脑组织中的脊柱密度。NCMIR使用图像相关光谱学的初步进展产生了以下出版物：Wiseman PW， Squier JA, Ellisman MH, Wilson KR.（2000）双光子图像相关光谱学和图像相互关联光谱学。中国生物医学工程学报，2009(1):14-25。Wiseman PW, Capani F, Squier JA, Martone ME。（2002）用图像相关光谱分析计算脑组织切片中的树突棘。中国生物医学工程学报，2009(2):379 - 379。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Continued efforts are being made the study the use of image correlation spectroscopy(ICS) and image cross-correlation spectroscopy (ICCS). The regulation of the aggregation state and transport properties of adhesion receptors is believed to play a significant role in the underlying mechanisms that control cellular adhesion in migratory cells. We continue to study the dynamics of cellular adhesion, structure formation, and disassembly by combing two-photon microscopy and ICS methods for live cell measurements. The use of ICS offers a way to simultaneously monitor the aggregation state of fluorescently labeled bio-molecules within the plasma membrane as well as their transport properties. Additionally, two-color image cross correlation spectroscopy (ICCS) permits the direct measurement of dynamic co-localization phenomena in live cells by imaging non-identical species in two wavelength channels. These are then analyzed by spatial and temporal cross-correlation. The combination of ICS/ICCS and two-photon microscopy allows these measurements to be performed in a minimally invasive way on living cell specimens using less damaging infrared wavelength laser excitation. To study the dynamics and aggregation of focal adhesion components, we have prepared GFP fusions of the a5 integrin subunit, paxillin and a-actinin. When expressed ectopically in CHO or WI-38 cells, they localize to focal adhesions and do not perturb migration, spreading or formation of focal adhesions. We have studied the dynamic regulation of adhesion structures in living fibroblasts cultured on various activating and non-activating extra cellular matrix (ECM) coated substrates by the unique approaches offered by two-photon ICS and ICCS. Furthermore, we have applied ICS techniques to light and electron microscopy images to determine spine densities in brain tissue of laboratory animals. Initial progress using image correlation spectroscopy at NCMIR has produced the following publications: Wiseman PW, Squier JA, Ellisman MH, Wilson KR. (2000) Two-photon image correlation spectroscopy and image cross-correlation spectroscopy. J Microsc. 200 ( Pt 1):14-25. Wiseman PW, Capani F, Squier JA, Martone ME. (2002) Counting dendritic spines in brain tissue slices by image correlation spectroscopy analysis. J Microsc. 205(Pt 2):177-86.