Tetard Laurene, Passian Ali, Farahi Rubye H, Voy Brynn H, Thundat Thomas
Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Methods Mol Biol. 2012;926:331-43. doi: 10.1007/978-1-62703-002-1_21.
Exploring the interior of a cell is of tremendous importance in order to assess the effects of nanomaterials on biological systems. Outside of a controlled laboratory environment, nanomaterials will most likely not be conveniently labeled or tagged so that their translocation within a biological system cannot be easily identified and quantified. Ideally, the characterization of nanomaterials within a cell requires a nondestructive, label-free, and subsurface approach. Subsurface nanoscale imaging represents a real challenge for instrumentation. Indeed the tools available for high resolution characterization, including optical, electron or scanning probe microscopies, mainly provide topography images or require taggants that fluoresce. Although the intercellular environment holds a great deal of information, subsurface visualization remains a poorly explored area. Recently, it was discovered that by mechanically perturbing a sample, it was possible to observe its response in time with nanoscale resolution by probing the surface with a micro-resonator such as a microcantilever probe. Microcantilevers are used as the force-sensing probes in atomic force microscopy (AFM), where the nanometer-scale probe tip on the microcantilever interacts with the sample in a highly controlled manner to produce high-resolution raster-scanned information of the sample surface. Taking advantage of the existing capabilities of AFM, we present a novel technique, mode synthesizing atomic force microscopy (MSAFM), which has the ability to probe subsurface structures such as non-labeled nanoparticles embedded in a cell. In MSAFM mechanical actuators (PZTs) excite the probe and the sample at different frequencies as depicted in the first figure of this chapter. The nonlinear nature of the tip-sample interaction, at the point of contact of the probe and the surface of the sample, in the contact mode AFM configuration permits the mixing of the elastic waves. The new dynamic system comprises new synthesized imaging modes, resulting from sum- and difference-frequency generation of the driving frequencies. The specific electronics of MSAFM allows the selection of individual modes and the monitoring of their amplitude and phase. From these quantities of various synthesized modes a series of images can be acquired. The new images contain subsurface information, thus revealing the presence of nanoparticles inside the cells.
为了评估纳米材料对生物系统的影响，探索细胞内部具有极其重要的意义。在受控实验室环境之外，纳米材料很可能无法方便地进行标记或标记，因此其在生物系统内的转运不易被识别和量化。理想情况下，对细胞内纳米材料的表征需要一种无损、无标记且能探测亚表面的方法。亚表面纳米级成像对仪器来说是一项真正的挑战。实际上，可用于高分辨率表征的工具，包括光学、电子或扫描探针显微镜，主要提供表面形貌图像或需要能发出荧光的标记物。尽管细胞间环境包含大量信息，但亚表面可视化仍是一个探索较少的领域。最近发现，通过对样品进行机械扰动，可以用微谐振器（如微悬臂梁探针）探测表面，以纳米级分辨率及时观察其响应。微悬臂梁在原子力显微镜（AFM）中用作力传感探针，微悬臂梁上的纳米级探针尖端以高度可控的方式与样品相互作用，以产生样品表面的高分辨率光栅扫描信息。利用AFM的现有功能，我们提出了一种新技术，即模式合成原子力显微镜（MSAFM），它能够探测亚表面结构，如嵌入细胞中的未标记纳米颗粒。在MSAFM中，机械致动器（PZT）以不同频率激发探针和样品，如本章第一幅图所示。在接触模式AFM配置中，探针与样品表面接触点处的尖端 - 样品相互作用的非线性性质允许弹性波混合。新的动态系统包括新的合成成像模式，这些模式是由驱动频率的和频与差频产生的。MSAFM的特定电子设备允许选择单个模式并监测其幅度和相位。从各种合成模式的这些量中可以获取一系列图像。这些新图像包含亚表面信息，从而揭示细胞内纳米颗粒的存在。
