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.
Highlights
Comprehensive investigation of neural circuits in relatively large and complex brains such as those of humans and marmosets requires simultaneous low- and high-magnification observations within each layer of the cerebral cortex
At the step in which the sample is exposed to absolute ethanol (100% EtOH), the lines from the liquid blocker should be removed from the tops of the slides using a razor blade while the slides are immersed in a 10- or 15-cm plastic dish filled with absolute ethanol to facilitate smooth removal of the section after polymerization (Step #29 in Table 1 and Figure 5E)
For large-area imaging with scanning electron microscopy (SEM), ultrathin sections with a thickness of 50–80 nm were prepared, and the sections were transferred to the silicon wafer from the diamond knife boat using a ring transfer (Figure 6J)
Summary
Comprehensive investigation of neural circuits in relatively large and complex brains such as those of humans and marmosets requires simultaneous low- and high-magnification observations within each layer of the cerebral cortex. The brain tissue block embedded in the plastic is sectioned at a thickness of approximately 50–80 nm using a diamond knife, and the sections are collected on an EM grid This procedure remains in common use for the observation of synaptic structure. For large-area imaging with SEM, ultrathin sections with a thickness of 50–80 nm were prepared, and the sections were transferred to the silicon wafer from the diamond knife boat using a ring transfer (microstar, Tokyo, Japan) (Figure 6J). The silicon wafers containing the sections were attached to the specimen holder with silver DAG and imaged using an optical microscope, Imager Vario (Carl Zeiss, Oberkochen, Germany), to confirm the precise position at low magnification as a reference position from which the fluorescence images were obtained (Step #15 in Table 1 and Figures 7A–C). Identifying better conditions for improving the quality of the images for large sample observation using the LA-CLEM procedure remains an important challenge
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