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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。人们正在继续努力研究图像相关光谱（ICS）和图像互相关光谱（ICCS）的使用。粘附受体的聚集状态和运输特性的调节被认为在控制迁移细胞中细胞粘附的潜在机制中发挥着重要作用。我们通过结合双光子显微镜和 ICS 方法进行活细胞测量，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质膜内荧光标记生物分子的聚集状态及其传输特性的方法。此外，双色图像互相关光谱 (ICCS) 允许通过对两个波长通道中的不同物种进行成像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互相关对这些进行分析。 ICS/ICCS 和双光子显微镜的结合允许使用破坏性较小的红外波长激光激发以微创方式对活细胞样本进行这些测量。为了研究粘着斑成分的动力学和聚集，我们制备了α5整合素亚基、桩蛋白和α-肌动蛋白的GFP融合物。当在 CHO 或 WI-38 细胞中异位表达时，它们定位于粘着斑并且不干扰粘着斑的迁移、扩散或形成。我们通过双光子 ICS 和 ICCS 提供的独特方法，研究了在各种激活和非激活细胞外基质 (ECM) 涂层基质上培养的活成纤维细胞中粘附结构的动态调节。此外，我们将 ICS 技术应用于光学和电子显微镜图像，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已产生以下出版物：Wiseman PW、Squier JA、Ellisman MH、Wilson KR。 (2000)双光子图像相关光谱和图像互相关光谱。 J 显微镜。 200（第 1 部分）：14-25。 怀斯曼 PW、卡帕尼 F、斯奎尔 JA、马托内 ME。 (2002)通过图像相关光谱分析计算脑组织切片中的树突棘。 J 显微镜。 205（第 2 部分）：177-86。