Applications of subsurface microscopy.

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.