Introduction to the CLEM technique developed in the field of neuroanatomy

Abstract

CLEM, which allows the observation of the same structure using both light and electron microscopes, is becoming increasingly popular in various fields in recent years as molecular and cell biology research deepens and experimental techniques become more sophisticated. Although CLEM is often considered a specialized technique, it has been used for 40 years to observe neuroanatomy since the era of Golgi-EM, so it does not require specialized equipment and can be applied relatively easily to various tissues and even cultured cells at a single cell level. The specific methodology is introduced here.

Keywords: Confocal laser scanning microscopy; Immunoelectron microscopy; Multiple immunofluorescence staining; PAP method; Rapid freezing and thawing method.

Fig. 1

CLEM in single labeling using DAB. A Immunohistochemical staining for parvalbumin (PV) in the mouse hippocampus. m dentate gyrus molecular layer, g granule cell layer, h hilus. B An enlargement of the boxed area shown in (B). B Numbers indicate the parts of the PV-immunopositive dendrites in the hilar region. C The area to be observed by electron microscopy was cut out from the resin film of the tissue section. D The cutout part of the section was embedded in the upper surface of a cylindrical resin block (arrow). E The surface of the block after trimming. The immunopositive structures can be clearly observed. F When the semi-thin section reaches the surface of the sample, some of the immunopositive structures (arrows) appear. G The area corresponding to (B) was found in the serial sections by electron microscopy. H Low-magnification images of structures 2, 3, and 4. I High-magnification image of structure 2. The PV-immunopositive dendrites are surrounded by axon terminals that form asymmetric synapses. The latter are thought to originate from the mossy fiber axons of granule cells. Scale bars, 0.5 mm A; 10 μm B; 100 μm C; 5 mm D; 100 μm E; 10 μm G; 1 μm H, )

Fig. 2

Double-labeled CLEM. A–D Immunofluorescent triple staining of cat primary visual cortex layer 4. Many GAD-positive (red) and vesicular glutamate transporter 2 (VGluT2)-positive (blue) boutons are in contact with the soma of PV-positive neurons (green). (E) PV labeling was visualized with DAB and VGluT2 labeling with DAB Nickel. Numbers shown for individual VGluT2-positive boutons in (D) correspond to those in (E). (F–H) All VGluT2-positive boutons formed asymmetric synapses with PV neurons. GAD-positive boutons in (C) were identified by electron microscopy based on their relative positions in (B-D), and the presence of symmetric synapses was confirmed as shown in (I-Q). Scale bars, 50 μm A; 5 μm B-E; 1 μm F; 0.1 μm (F Inset, G-Q). Reprinted from Fukuda (2017) under Elsevier’s copyright policy for open access article with CC BY-NC-ND

Fig. 3

Schematic illustrations of the procedures of CLEM. Note that only conventional laboratory instruments, which are available in every EM department, are necessary to execute CLEM of high quality and sufficient reliability. Use of CLSM is very helpful but not essential; CSLM can be skipped when DAB-labeled immunostaining is conducted as an initial staining