Advanced optical manipulation and imaging techniques for the accurate quantification of cellular interaction forces

目前还没有一种有效的技术可以准确、可靠地量化细胞相互作用力。了解这些力量对于有效的疫苗接种、皮肤移植、组织再生的发展至关重要，并提供了对自身免疫性疾病的更好理解。我们建议开发将光学捕获与光学切片显微镜相结合的新技术，以充分了解和探测细胞相互作用。光学捕获系统将专门为此目的而设计，并允许用户直接捕获和操纵感兴趣的细胞，而无需向样品引入异物或“手柄”。这些细胞将被一束激光捕获，其轮廓设计是为了最小化细胞卷，从而提高技术的准确性。细胞的位置将在纳米尺度上进行监测，从这里可以知道激光束施加的光力，任何额外的外力，例如来自另一个细胞的外力，都将被量化。这种专门的光学捕获系统将与结构光照明相结合-光学切片显微镜的一种形式。光学切片显微镜提供高质量的图像，与传统的宽视场显微镜相比，轴向分辨率大大提高。这使用户能够建立一个三维图像，与亚细胞水平的分辨率，他们感兴趣的样本。作为该提议的一部分，光学切片显微镜将与光学捕获系统相结合，以获得尽可能多的关于细胞特性和配置的信息，同时量化细胞相互作用力。使用的光学切片显微镜的类型将是结构光照明，它可以在荧光中操作，并且具有与共聚焦显微镜相似的轴向分辨率。结构光照明的主要好处是它提供了感兴趣的样品的快速成像，允许细胞相互作用和图像中的信息被确定和实时比较。例如，在共聚焦显微镜中，激光束需要扫描图像中的每个点并记录信号强度，而在结构光照明中，至少可以拍摄三张图像，然后进行处理以产生光学切片图像。随着技术挑战的解决和系统的完善，以准确可靠地量化细胞相互作用力，它将被用来回答一些基本的生命科学问题。目前有几位科学家支持这一建议，他们对免疫细胞之间的相互作用水平以及如何修改这种相互作用以及用于三维细胞培养的新型水凝胶基质的粘附特性感兴趣。

At present no effective technique exists for accurately and reliably quantifying cellular interaction forces. An understanding of these forces is crucial to the development of effective vaccinations, skin grafts, tissue regeneration and provides a greater understanding of auto-immune diseases. We propose to develop novel techniques that combine optical trapping with optical sectioning microscopy in order to fully understand and probe cellular interactions. The optical trapping system will be designed specifically for this purpose and will allow the user to directly capture and manipulate the cells of interest without the need to introduce foreign bodies or 'handles' to the sample. The cells will be trapped with a laser beam whose profile has been designed in order to minimize cell roll and hence improve the accuracy of the technique. The position of the cell will be monitored on a nanometer scale and from here the optical force applied by the laser beam will be known and any additional external force, for example from another cell, will be quantified. This specialized optical trapping system will be combined with structured light illumination - a form of optical sectioning microscopy. An optical sectioning microscope provides high quality images with greatly improved axial resolution over conventional widefield microscopy. This enables the user to build up a three-dimensional image, with sub-cellular level resolution, of their sample of interest. As part of this proposal an optical sectioning microscope will be combined with and optical trapping system to gain as much information as possible about the cell properties and configuration whilst quantifying the cellular interaction forces. The type of optical sectioning microscope used will be structured light illumination which can operate in fluorescence and has a similar axial resolution as confocal microscopy. The main benefit of structured light illumination is that it provides rapid imaging of the sample of interest allowing the cellular interactions and information from the image to be determined and compared in real-time. In a confocal microscope, for example, a laser beam would need to be scanned over every point in the image and the signal intensity record, where as in structured light illumination a minimum of three images can be taken and then processed to produce an optically sectioned image. With the technical challenges tackled and the system perfected in order to accurately and reliably quantify cellular interaction forces, it will be employed to answer some fundamental life science questions. There are several scientists currently supporting this proposal and they are interested in the level of interactions between immune cells and how this interaction can be modified and the adhesion properties of a new hydrogel matrix for three-dimensional cell culture.