Observation of biological and emulsion samples by newly developed three-dimensional impedance scanning electron microscopy.

Imaging at nanometre-scale resolution is indispensable for many scientific fields such as biology, chemistry, material science and nanotechnology. Scanning electron microscopes (SEM) are widely used as important tools for the nanometre-scale analysis of various samples. However, because of the vacuum inside the SEM, a typical analysis requires fixation of samples, a drying process, and staining with heavy metals. Therefore, there is a need for convenient and minimally invasive methods of observing samples in solution. Recently, we have developed a new type of impedance microscopy, multi-frequency impedance SEM (IP-SEM), which allows nanoscale imaging of various specimens in water with minimal radiation damage. Here, we report a new IP-SEM system equipped with a linear-array terminal, which allows eight tilted images to be observed in a single capture by applying eight frequencies of input signals to each electrode. Furthermore, we developed a three-dimensional (3D) reconstruction method based on the Simulated Annealing (SA) algorithm, which enables us to construct a high-precision 3D model from the 8 tilted images. The method reported here can be easily used for 3D structural analysis of various biological samples, organic materials, and nanoparticles.