IN SITU TWO PHOTON IMAGE CORRELATION STUDIES OF ADHESION RECEPTORS

粘附受体的原位两个光子图像相关性研究

• 批准号: 7358041
• 负责人: ALAN F. Rick HORWITZ
• 金额: $ 0.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358041
• 关键词: SITU; TWO; PHOTON; IMAGE; CORRELATION

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。继续努力进行研究，使用图像相关光谱（IC）和图像互相关光谱（ICC）。据信，粘附受体的聚集状态和转运特性的调节在控制迁移细胞中细胞粘附的基本机制中起着重要作用。我们继续通过梳理两光子显微镜和实时细胞测量方法来研究细胞粘附，结构形成和拆卸的动力学。 ICS的使用提供了一种方法，可以同时监测质膜内荧光标记的生物分子的聚集状态及其运输特性。此外，两种颜色图像互相关光谱（ICC）允许通过在两个波长通道中成像非相同物种来直接测量活细胞中动态共定位现象。然后通过空间和时间互相关分析这些。 ICS/ICC和两光子显微镜的组合允许使用较小的破坏性红外波长激发激发以微创的方式对活细胞样品进行这些测量。为了研究焦点粘附成分的动力学和聚集，我们准备了A5整合素亚基，帕西林和a-肌动蛋白的GFP融合。当在CHO或WI-38细胞异位表达时，它们将其定位于局灶性粘连，并且不会扰动局灶性粘连的迁移，扩散或形成。我们研究了通过两光子ICS和ICC提供的独特方法，研究了在各种激活和非激活的额外细胞基质（ECM）涂层底物上培养的活的成纤维细胞中粘附结构的动态调节。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的脊柱密度。 NCMIR的图像相关光谱的初步进展已产生以下出版物：Wiseman PW，Squier JA，Ellisman MH，Wilson KR。 （2000）两光子图像相关光谱和图像互相关光谱。 J显微镜。 200（PT 1）：14-25。 Wiseman PW，Capani F，Squier JA，Martone Me。 （2002）通过图像相关光谱分析计算脑组织切片中的树突状刺。 J显微镜。 205（PT 2）：177-86。