BioAFM: Integrated atomic force and light microscopy for mechanochemical cell biology

我们通过多种感官的组合来探索我们周围的环境，即视觉、听觉、嗅觉、味觉和触觉。用光感应是非常强大的，因为它允许我们在没有直接接触的情况下检测物体。然而，有一些类型的信息不是单靠视觉就能获得的。例如，触摸告诉我们一个物体的机械特性。通过用手指触摸某人的手，你可以感觉到皮肤的质地以及皮肤的柔软或坚硬程度。你还可以知道那只手有多少根手指，甚至皮肤和肉下面的骨头。在分子机械、细胞和组织范围内的生命科学一直严重依赖光学显微镜，特别是当我们试图观察它们的生存状态时。通过“触摸”测量机械性能应该会为我们对生物物体的感知增加另一个维度，并提供光学显微镜无法获得的信息。原子力显微镜(AFM)是一种实现这种测量的仪器。我们将原子力显微镜与光学显微镜相结合，它具有两种不同的观察模式：1)全内荧光显微镜(TIRFM)，它可以通过荧光检测单分子；2)高分辨率3D荧光显微镜(3DFM)，它适合观察细胞和组织等厚样品。AFM和TIRFM的结合将使我们能够可视化生物分子机器组装的实时动态，这些生物分子机器在细胞内的运输/交通中发挥关键作用。AFM+3DFM使我们能够通过使用荧光标记作为向导来测量复杂的多细胞环境中感兴趣的单个细胞的各种力学性质。我们提出的和未来的生物原子力显微镜实验将为细胞和组织形态发生的细胞和分子机制以及其他生物过程提供新的见解，其中机械力起着关键作用。

We explore the environment surrounding us through the combination of multiple senses, i.e., sight, hearing, smell, taste, and touch. Sensing with light is very powerful as it allows us to detect objects without direct contact. However, there are types of information that can't be obtained by sight alone. For example, touch tells us the mechanical properties of an object. By touching someone's hand with your finger, you can feel the texture of the skin and how soft or hard the skin is. You can also find how many fingers exist on that hand and even the bones beneath the skin and flesh. Life science at the scale of molecular machinery, cells and tissues has been heavily relying on light microscopy especially when we try to observe them in their living state. Measurement of the mechanical properties by 'touching' should add another dimension to our sensing of the biological object and provides information that can't be obtained by light microscopy. An atomic force microscope (AFM) is an instrument that achieves such measurements.We integrate an AFM with a light microscope that has two different modes of observation, 1) total internal fluorescence microscopy (TIRFM), which can detect single molecules by fluorescence, and 2) high-resolution 3D fluorescence microscopy (3DFM), which is suitable for observation of thick samples such as cell and tissues. The combination of AFM and TIRFM will allow us to visualize the live dynamics of the assembly of biomolecular machines that play key roles in the transport/traffic inside the cell. AFM+3DFM enables us to measure various mechanical properties of a single cell of interest in a complex multicellular environment by using a fluorescent marker as a guide. Our proposed and future BioAFM experiments will give new insights into cellular and molecular mechanisms of cell and tissue morphogenesis and other biological processes, in which mechanical force plays a key role.