Photoelastic Gel Microscopy (PGM): towards beacon-free direct imaging of cellular traction forces

在过去的二十年中，已经证明机械力在生理和病理条件下决定细胞过程中起着至关重要的作用，这导致了机械生物学这一新兴领域的兴起。已经证明，细胞以一种双向的方式与它们的环境进行机械相互作用，在这种方式下，它们能够施加力，并以类似的方式破译机械线索，如体积刚度、刚度的时空变化、应力松弛、纳米形貌或剪切力b[2]的存在。尽管控制这种双向相互作用的确切分子途径尚不清楚，但已知细胞内和细胞外隔室之间的机械力交换是通过局灶粘连发生的。这些是超分子复合物，通常面积为几平方微米，通过大量蛋白质连接细胞内和细胞外环境。因此，细胞能够通过众所周知的肌动肌不收缩机制在细胞内产生的力来拉动周围环境，并通过局灶粘连传导到外部。然后，细胞通过相同的传导机制感知周围环境的机械反应，这些机械反应是内源性的（即细胞外基质或ECM）或人造的（如水凝胶），这反过来又可以产生和影响大量的细胞过程[1]。因此，为了充分利用机械生物学在诊断应用中的潜力，人们越来越需要专门的工具，以提供高精度和可重复性的单细胞水平的揭示和测量力的能力。在这种情况下，细胞对其基底施加的收缩力是特别感兴趣的-产生通常称为牵引力的收缩应力。在生理条件下，这些力量强烈地参与了组织和器官三维组织的发展和建立。相反，观察牵引力生理模式的损伤，可能是一种推断异常病理发病的方法，例如癌症，已知与细胞机械行为的变化有关，细胞从原始位置分离，挤压，侵入并最终呈现不同的三维组织，即转移[5,6]。尽管揭示机械生物学的丰富含义的道路仍处于起步阶段，但在生物医学研究的背景下，已经迈出了第一步，解决了单细胞水平上牵引力的组织，并评估了具体事件-生物化学，遗传或机械-如何反过来影响这种模式。最常用的方法是测量细胞-基质界面的细胞力。自15年前首次出现以来，这一领域发展迅速，导致了现在所知的牵引力显微镜（TFM）[7,8]。最常见的TFM方法是在含有参考荧光珠的已知硬度的底物（水凝胶）上播种细胞。当细胞在衬底上施加牵引力时，这些珠子的位移被光学监测，并从位移中获得施加的力。力的间接测量既需要基材机械响应的本构模型，也需要对其物理特性的精确了解。线性弹性弹簧的例子很容易理解，其本构模型由胡克定律（F = k(x - x0)）给出，其中F是力，k是弹簧的刚度，x - x0是弹簧相对于其参考位置x0的位移。如果不测量x（及其参考状态x0），不知道k和整体本构律，原则上就无法获得力[7,8]。值得注意的是，这不是一件小事

Over the last two decades, it has been shown that mechanical forces play a crucial role in determining cellularprocesses - both in physiological and pathological conditions - which led to the emerging field of mechanobiology[1]. It has been demonstrated that cells mechanically interact with their environment in a bidirectional fashion,where they are able to exert forces and, in like manner, decipher mechanical cues, such as bulk stiffness, spatiotemporal changes in stiffness, stress relaxation, nanotopography or the presence of shear forces [2]. Whereasthe exact molecular pathways governing this bidirectional interplay remain unclear, the mechanical exchange offorces between the intracellular and extracellular compartments is known to occur via focal adhesions. Theseare supramolecular complexes, often of several square micrometres in area, bridging the intracellular and extracellular environments through a multitude of proteins [3]. As a result, cells are able to pull on their surroundingenvironment - via forces that are intracellularly generated through the well known mechanism of acto-myosincontractility - and transduced externally via focal adhesions. Cells then sense the mechanical response of theirsurrounding, being endogenous (i.e. the Extracellular Matrix or ECM) or man-made (e.g. hydrogels) via thesame transducing machinery, which, in turn, can give rise and influence a large number of cellular processes [1].As a consequence, in order to fully exploit the potential of mechanobiology for diagnostic applications, there isan increasing need for specialised tools providing the ability to reveal and measure forces down to the singlecell level with high accuracy and reproducibility.In this context, the contractile forces that cells exert on their substrate are of particular interest - resultingin contractile stresses commonly referred to as traction forces. These forces have been known to stronglyparticipate in the development and establishment of the three-dimensional organisation of tissues and organs inphysiological conditions [4]. Conversely, looking at the impairment of the physiological pattern of traction forces,might be a way to infer about the onset of an aberrant pathology, such as a cancer, known to be associated toa change in the mechanical behaviour of cells, which detach from their original location, extrude, invade andfinally assume a different three dimensional organisation, the metastasis [5, 6].Whereas the path to disclose the wealth of implications of mechanobiology is still in its infancy, the firststeps have been taken in the context of biomedical research addressing the organisation of traction forces at thelevel of the single cell, and evaluating how specific events - either biochemical, genetic or mechanical - in turninfluence this pattern. The most common approach is to measure cellular forces at the cell-matrix interface.This field has grown rapidly since it first emerged 15 years ago, leading to what is now known as Traction ForceMicroscopy (TFM) [7, 8]. The most common TFM approach is to seed cells on a substrate of known stiffness(a hydrogel) containing reference fluorescent beads. As cells apply tractions on the substrate, the displacementof such beads is optically monitored and the exerted forces retrieved from the displacement. The indirectmeasurement of forces requires both a constitutive model of the substrate's mechanical response and preciseknowledge of its physical properties. This is easily understood with the example of a linear elastic spring, whoseconstitutive model is given by Hooke's law (F = k(x - x0)), where F is the force, k is the spring's stiffnessand x - x0 is the spring's displacement with respect to its reference position, x0. Without a measurement ofx (and its reference state x0), knowledge of k and the overall constitutive law, the force, in principle, cannotbe retrieved [7, 8]. Notably, this is not a triv