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 diffi cult 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 fi sh teeth was clearly distinguished. A novel combination of two channel oblique illumination and z-stack imaging process increased depth of fi eld and optimized contrast, which has a potential to be further applied in the fi eld of neuroscience, hard tissue biology, and analysis of small organic structures such as ear ossicles and zebra fi sh teeth.

Comprehensive Review of Golgi Staining Methods for Nervous Tissue

“…Recent application of pan-focal 3D Edge microscopy yields a finer image by overcoming the limitation of focal depth ( Fig. 2B and E; Rhyu et al, 2016). …”

“…While Golgi staining is continues to be used as a central technique, it has been combined with fluorescent staining (Koyama & Tohyama, 2013;Spiga et al, 2011). In addition, the latest developments in 3D Edge microscopy provide a solution to overcome the limitation of focal depth during the analysis of thick specimens (Rhyu et al, 2016). Golgi staining has contributed in investigating neuronal cytology and the circuitry and connectivity of brain tissues.…”