Large-Area Fluorescence and Electron Microscopic Correlative Imaging With Multibeam Scanning Electron Microscopy

Abstract

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates.

Cell therapy for spinal cord injury by using human iPSC-derived region-specific neural progenitor cells

“…The detailed procedure of pre-embedding immuno-electron microscopic analysis was described previously [ 44 ]. Briefly, the frozen sections of the spinal cord on the slide glasses were dried up with cool wind dryer and autoclaved for 1 h in pH 6.0 citrate buffer with the setting for 5 min at 105 °C, followed by the incubation with the blocking solution [5.0% Block Ace (DS Pharma Biomedical) solution with 0.01% saponin in 0.1 M PB] for an hour at 25 °C.…”

Long-term selective stimulation of transplanted neural stem/progenitor cells for spinal cord injury improves locomotor function

“…The detailed immunoelectron microscopy procedure was described previously (Shibata et al, 2019). Spinal cord tissues were perfused, postfixed, cryoprotected, and sectioned as mentioned above.…”

Niwaki Instead of Random Forests: Targeted Serial Sectioning Scanning Electron Microscopy With Reimaging Capabilities for Exploring Central Nervous System Cell Biology and Pathology

“…In order to target molecularly defined structures at the post-fixation stage, pre-embedding immune-labeling approaches are the method of choice. Classical immune-gold staining on vibratome sections provides discrete and specific signals with high precision, which is ideally suited for intracellular structures ( Norris et al, 2017 ; Shibata et al, 2019 ; Sun et al, 2020 ). HRP coupled antibodies are typically preferred to render whole cells electron-dense as the spatial resolution of the signal is limited.…”