Correlative microscopy and block-face imaging (CoMBI) method for both paraffin-embedded and frozen specimens

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.

Visualising the effect of freezing on the vascular system of wheat in three dimensions by in‐block imaging of dye‐infiltrated plants

“…A disadvantage is that in vivo staining is required which can be limited by the ability of the dye to uniformly infiltrate tissue of interest prior to embedding. This issue was discussed in detail 12 in a description of block-face imaging.…”

“…A disadvantage is that in vivo staining is required which can be limited by the ability of the dye to uniformly infiltrate tissue of interest prior to embedding. This issue was discussed in detail 12 in a description of block‐face imaging. In our case, the vascular system was ideally suited for in‐block imaging since dye solution was readily drawn into water conducting vessels.…”

“…In this paper, we describe a method for building a 3D volume of plants that simplifies a previously described 3D reconstruction technique. 10,11 A variation of block-face imaging 12 with several important differences allowed us to reconstruct the vascular system of dye-infiltrated winter cereals before and after freezing.…”

Label-free imaging of large samples: 3D rendering and morphological analysis within histological workflows using serial block face imaging

“…The rotary microtome ( 1) is placed on an optical table to which is attached an aluminum frame (2) that holds the camera (3) orthogonally to the surface of the mounted sample (4). The microtome is connected to the control unit (5) which itself is connected to a laptop computer (6). The acquisitions are controlled by the control software (7) that triggers the microtome through the control unit (5).…”

“…Different home-made SBFI systems have proposed effective 3D reconstruction of wax embedded samples [6][7][8][9] . With regard to these systems, it seems that the critical point to obtain a quality 3D volume is to improve the contrast between the embedded sample and the surrounding paraffin.…”