Application of Three-Dimensional Light Microscopy for Thick Specimen Studies

Abstract

The thickness of specimen is an important factor in microscopic researches. Thicker specimen contains more information, but it is difficult to obtain well focused image with precise details due to optical limit of conventional microscope. Recently, a microscope unit that combines improved illumination system, which allows real time three-dimensional (3D) image and automatic z-stack merging software. In this research, we evaluated the usefulness of this unit in observing thick samples; Golgi stained nervous tissue and ground prepared bone, tooth, and non-transparent small sample; zebra fish teeth. Well focused image in thick samples was obtained by processing z-stack images with Panfocal software. A clear feature of neuronal dendrite branching pattern could be taken. 3D features were clearly observed by oblique illumination. Furthermore, 3D array and shape of zebra fish teeth was clearly distinguished. A novel combination of two channel oblique illumination and z-stack imaging process increased depth of field and optimized contrast, which has a potential to be further applied in the field of neuroscience, hard tissue biology, and analysis of small organic structures such as ear ossicles and zebra fish teeth.