DYNAMICS OF CELL MIGRATION IN 3D USING MULTI-PHOTON EXCITATION, SHG AND OCT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 细胞迁移是创伤愈合和胚胎发育等生理过程中的重要环节。此外，它还可能导致炎症和癌症转移等病理性疾病。1 -3细胞迁移的过程是一个复杂的过程，其中数百种蛋白质参与细胞粘附到细胞外基质（ECM）的形成以及与细胞骨架的相互作用。已发现ECM对细胞施加的机械刺激是细胞分化的决定因素之一。4 我们的研究小组在2D中使用相关性分析进行了关于Paxilin（一种“锚”蛋白）作用的研究，Paxilin对于细胞粘附的形成至关重要。5，6下一个合乎逻辑的步骤是研究3D中的细胞相互作用，这代表了更真实的生物系统。因此，我们的模型组织由包埋在3D胶原基质凝胶中的细胞组成，其中胶原基质用于模拟ECM。使用LAMMP研究人员建立的双光子激发荧光（TPEF），二次谐波产生（SHG）和光学相干断层扫描（OCT）的多模式系统，我们能够对细胞与ECM的相互作用进行成像，例如细胞的运动以及由此产生的ECM变形。 我们能够根据从OCT和MPM通道获得的信号，使用时空图像相关光谱（STICS）7确定图像中每个点的位移和速度。本研究的新奇在于，这是我们第一次可以使用来自OCT通道的信号检测速度图（与OCT-多普勒效应无关）。从两个图像获得的速度图是互补的。我们建议开发互相关速度图，以获得有关影响细胞和胶原基质运动的常见因素的信息。 我们还计划计算一个真实的3D速度图，我们将从理论上对系统进行建模，以量化运动及其与ECM拉伸强度的关系。 特别是，我们想知道胶原基质聚合度和细胞运动之间的关系。 此外，我们还想研究粘着斑蛋白如Paxilin的积累与细胞运动之间的关系。最终，当我们测量ECM中的变形时，我们希望提取施加在各种细胞粘附上的力。

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. Cell migration is an essential process in several physiological events such as wound healing and embryonic development. Additionally, it can contribute to pathological disorders such as inflammation and cancer metastasis.1-3 The process by which cells migrate is a complicated procedure where hundreds of proteins participate in the formation of the cell adhesions to the extra cellular matrix (ECM) and the interactions with the cytoskeleton. The mechanical stimulation that is exerted by the ECM on the cell has been found to be one of the determining factors for cell differentiation.4 Previous studies using correlation analyses in 2D have been performed by our group about the role of Paxilin, an "anchor" protein, that is essential for the formation of cellular adhesions.5,6 The next logical step is to study the cellular interactions in 3D which represents a more realistic biological system. Hence, our model tissue is comprised of cells embedded in a 3D collagen matrix gel where the collagen matrix is used to simulate the ECM. Using the multi-modal system of two-photon excited fluorescence (TPEF), second harmonic generation (SHG) and optical coherence tomography (OCT) built by LAMMP researchers, we are able to image the interactions of the cell with the ECM such as the movement of the cell and the resulting deformation in the ECM. We are able to determine the displacement and the velocities at every point in the image using Spatio-Temporal Image Correlation Spectroscopy STICS)7 from the signals obtained from the OCT and MPM channels. The novelty of this study is that this is the first time that we can detect velocity maps (not related to OCT- Doppler effect) using the signals from OCT channel. The velocity maps obtained from the two images complement each other. We propose to develop cross correlation velocity maps to obtain information about common factors affecting the motion of the cell and of the collagen matrix. We also plan to calculate a true 3D velocity map and we will theoretically model the system to quantify the motion and its relation to the tensile strength of the ECM. In particular, we would like to know the relationship between the collagen matrix degree of polymerization and the movement of the cell. Furthermore we would like to study the relationship between the accumulation of the focal adhesion proteins such as Paxilin and the movement of the cell. Ultimately, as we measure the deformation in the ECM, we would like to extract the force applied at the various cell adhesions.