Underwater atomic force microscopy for in situ observation of microorganisms in the deep sea

We present a novel underwater atomic force microscope system (underwater AFM system), which is mountable on underwater vehicles or submersible seafloor platforms. The mission of the system is to observe microorganisms and microparticulates in situ, which are suspended and dispersed in deepwater, with high spatial resolution down to nanometer scale. The system is composed of three major technological elements: a main unit of the underwater AFM, fluidic devices for the sample collection from deepwater (for example, pumps and a filtration unit using membrane filters), and robust mounting mechanisms for the underwater vehicles or the submersible seafloor platforms. Since we use a commercially available self-sensitive cantilever as the AFM probe, the deflection of the cantilever is measured by the integrated piezoresistive gauges. For insulation of the self-sensitive cantilever from the seawater under high pressure in the deep sea, we applied a thin layer coating of poly(p-xylylene) polymers (Parylene) onto the cantilever. We have successfully balanced the imaging quality and the insulation performance by optimizing the conditions of the layer formation, for example dimmer types of Parylene and the final layer thickness. We also have invented a novel sample stage for the underwater AFM equipped with a sample filtration mechanism using membrane filters. As a test result of the sample stage with the membrane filters in the deep sea exploration, the microorganisms suspended and dispersed in the deepwater have been successfully collected and fixed on the membrane filter. The present underwater AFM system would be a useful tool for in situ observation of the living microorganisms and microparticulates with nanoscale spatial resolution leading to future new findings in the deep sea.