Cellular Force Microscope.

在过去的十年里，由于人们越来越意识到机械性能和生物化学之间的耦合在生物学中发挥着核心作用，并可能在许多疾病中发挥核心作用，机械生物学领域已经呈指数级增长。这种扩展是由于采用了许多在细胞和亚细胞尺度上测量机械性能的技术，其中可以说最广泛使用的是原子力显微镜(AFM)。通过在样品表面上压入一个连接在力传感悬臂梁上的尖锐探头，可以测量压痕与力的关系曲线，并由此计算出样品的弹性系数。AFM仪器制造商使这一过程变得相对简单，使分析自动化，并催生了一个活跃的科学界的增长。所获得的数字现在被用来为模型提供信息，并改善对基本生物过程的理解，如细胞运动、内吞作用和组织发育，以及推动翻译方面，如组织修复和干预。不幸的是，有一个基本的问题，因为所获得的模数测量包含通常10s到100s%的系统误差-力传感器，AFM悬臂，根本不够灵敏，无法准确地测量要求它的微小的力。为了解决这个问题，我们将使用具有所需灵敏度的新一代小而软的AFM悬臂，我们将建立一个检测系统，能够准确地测量它们的硬度和挠度，消除测量误差的另一个主要来源，并将其放置在定制设计的仪器中，能够在10s微米范围内准确地测量压痕。由此产生的细胞力显微镜(CFM)将具有测量软细胞和组织特性所需的灵敏度和准确性。我们将使用该仪器查看两个我们拥有丰富经验的示例系统。周围神经系统(PNS)中的神经包含许多不同类型的细胞，包括神经元和雪旺细胞，即使对动物细胞来说，这两种细胞也格外柔软。三叉神经核的创伤性损伤很常见，会导致感觉和功能的丧失。神经修复仍然是一个相当大的挑战，更好地了解神经的力学特性对于选择合适的组织工程支架进行神经再生具有重要的作用。我们将使用我们的新仪器从PNS获得细胞力学性能的初步数据，目的是为机械性能匹配材料的选择提供信息。我们要研究的第二个系统是骨内部软组织的力学。这是一种机械异质的组织，但包含非常柔软的区域，因此是AFM开始用于的那种先进的、更活体的机械生物学研究的极好例子。作为乳腺癌转移项目的一部分，我们目前正在使用传统的AFM表征骨骼的特性，因此它为应用这项新技术提供了一个极好的组织模型，利用多余的骨组织。我们的新数据将有助于了解骨骼的力学机制是否影响癌症向该部位的继发性扩散。

Over the past decade the field of mechanobiology has expanded exponentially due to a growing realisation that the coupling between mechanical properties and biochemistry plays a central role in biology and potentially in a number of diseases. This expansion has been enabled by the adoption of a number of techniques for measuring mechanical properties at a cellular and sub-cellular scale, arguably the most widely used of which is atomic force microscopy (AFM). By indenting a sharp probe attached to a force sensing cantilever into a sample surface, a curve of indentation against force can be measured, and from this the sample's elastic modulus can be calculated. AFM instrument manufacturers have made the process relatively straightforward, automated the analysis, and have spawned the growth of an active scientific community. The numbers obtained are now used to inform models and improve understanding of fundamental biological processes such as cellular motility, endocytosis and tissue development, as well as to drive translational aspects such as tissue repair and intervention. Unfortunately there is a fundamental problem in that the measurements of modulus obtained contain systematic errors typically of 10s to 100s of percent - the force sensor, the AFM cantilever, is simply not sensitive enough to accurately measure the small forces asked of it.To address this problem we will use a new generation of small and soft AFM cantilevers that have the required sensitivity, we will build a detection system that enables accurate measurement of their stiffness and deflection, removing another major source of measurement error, and house it within a custom designed instrument able to measure indentation accurately over 10s of micrometres. The resultant Cellular Force Microscope (CFM) will have the sensitivity and accuracy necessary to measure the properties of soft cells and tissue.We will use the instrument to look at two example systems in which we have considerable experience. Nerves in the peripheral nervous system (PNS) contain a number of different cell types, including neurones and Schwann cells, both of which are exceptionally soft even for animal cells. Traumatic damage to the PNS is common and can result in loss of feeling and function. Nerve repair remains a considerable challenge, and better understanding of nerve mechanical properties has an important role to play in the selection of suitable tissue engineering scaffolds for nerve regeneration. We will use our new instrument to obtain preliminary data on the mechanical properties of cells from the PNS, with the aim of informing the selection of mechanical property matching materials. The second system we will study is the mechanics of the soft tissue in the interior of bone. This is tissue that is mechanically heterogeneous but contains regions that are very soft and as such is an excellent example of the kind of advanced, more in vivo, mechanobiology study that AFM is starting to be used for. As part of a project on breast cancer metastasis we are currently characterising the properties of bone using conventional AFM so it provides an excellent tissue model for applying the new technology, using surplus bone tissue. Our new data will contribute to that work on understanding whether the mechanics of the bone influences the secondary spread of cancer to this site.