High-speed three-dimensional cross-sectional measurement of cultured neurons by scatterometry that improves resolution by an order of magnitude

In conventional three-dimensional (3D) measurements using lens imaging or holography, the practical sample size in visible light is as large as 50 µm, and the resolution is limited to about 5 µm due to image distortion caused by artifacts. Rigorous calculations of neuronal cell models confirm these limitations in the sample size and resolution for lens imaging. Scatterometry, on the other hand, is a technique to obtain 3D cross-sectional structures by measuring the diffraction patterns of a sample. It is commonly used to rigorously analyze symmetric and periodic structures of semiconductors at high resolution and speed. Despite its high resolution, it has not been applied to isolated, less symmetric structures such as cells. In this study we developed an optical measurement system that enables "one-shot" observation of targeted areas of neurons and a semi-automatic analysis of their asymmetric shape. For sample sizes as small as 1.8 μm, we have achieved measurements without fluorescent labeling, a resolution of 0.2 μm, and high speed.