Abstract
Correlative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.
Highlights
Morphological analyses are carried out in various ways in today’s biological laboratories for the purpose of understanding the anatomical structure and molecular distribution in specimens
We previously developed a 3D imaging method based on the block-face imaging technique and named it “correlative microscopy and block-face imaging” (CoMBI)[15]
Through these collaborative studies and ongoing collaborations, we found that users of the conventional Correlative microscopy and block-face imaging (CoMBI)-C system desired compatibility with frozen specimens and paraffin-embedded specimens
Summary
Morphological analyses are carried out in various ways in today’s biological laboratories for the purpose of understanding the anatomical structure and molecular distribution in specimens. The correlation between the section and corresponding block-face tells us the location where the section originated in the specimen, which facilitates the interpretation of 2D microscopic images This correlation can add molecular data to the 3D morphological data, the molecular data are limited to 2D distribution. The CoMBI-C system has been used in various research fields, such as 3D analysis of primordia of beetle horn16, 3D distribution analysis of myelinated fibers of human facial n erves17, 3D morphological analysis of polycystic kidney of knockout mice[18], and 3D visualization of regenerating bones and cartilage of the axolotl jaw[19] Through these collaborative studies and ongoing collaborations, we found that users of the conventional CoMBI-C system desired compatibility with frozen specimens and paraffin-embedded specimens. We describe our new CoMBI-S system and sample preparation methods, and present correlative 2D microscopic images and 3D datasets of various biological specimens
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