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。对这些力量的了解对于开发有效的疫苗接种、皮肤移植、组织再生至关重要，并提供了对自身免疫性疾病的更多了解。我们建议开发新的技术，将光学捕获与光学切片显微镜相结合，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，并将允许用户直接捕获和操作感兴趣的细胞，而不需要将异物或‘手柄’引入样品。这些细胞将被激光光束捕获，其轮廓被设计成最大限度地减少细胞滚动，从而提高技术的准确性。细胞的位置将在纳米尺度上被监测，从这里将知道激光施加的光学力，并将量化例如来自另一细胞的任何附加外力。这种专门的光学捕捉系统将与结构光照明相结合--结构光照明是光学切片显微镜的一种形式。光学切片显微镜提供了高质量的图像，与传统的宽视场显微镜相比，轴向分辨率有了很大的提高。这使用户能够建立他们感兴趣的样本的具有亚细胞级别分辨率的三维图像。作为这项提议的一部分，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属性和结构的信息，同时量化细胞相互作用力。所使用的光学切片显微镜类型将是结构光照明，它可以在荧光中工作，并具有与共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，允许实时确定和比较图像中的细胞相互作用和信息。例如，在共焦显微镜中，激光需要扫描图像和信号强度记录中的每个点，在结构光照明中，最少可以拍摄三个图像，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，为了准确和可靠地量化细胞相互作用力，它将被用于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提议，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.