Scanning Transmission Electron Microscopy (STEM) in Scanning ElectronMicroscope for the Correlative Light and Electron Microscopy(CLEM)

Hong-Lim Kim1, Tae-Ryong Riew2, Dong Yong Chung3, In-Beom Kim1,2

1Integrative Research Support Center, Laboratory of Electron Microscope, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea2Department of Anatomy, Catholic Neuroscience Institute, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea3 Yonsei Biomedical Research Center, College of Medicine, The Yonsei University of Korea, Seoul 03722, Korea

We have developed a novel CLEM by combining confocal microscopy and

conventional TEM with immunochemistry based on Tokuyasu's method (Kim

et al., 2021). However, because thin section for TEM have spatial limitations

in the lattice grid loading area, it is difficult to observe various regions of

interest. To overcome these limitations, we applied our CLEM technique

to STEM. STEM in the SEM allows observation of larger viewing field

and than TEM. The high contrast of dark-field images also provides clearer

ultrastructural profiles. Therefore, we tested Investigation on large-area section

by using STEM in the SEM from Epon embedding block for CLEM and

showed the advantage of this alternative approach.

Animal

Normal adult male Sprague-Dawley rat (280~300g) brain cortex.

CLEM (Correlative Light & Electron Microscopy)

Semi-thin sections (2um thick) using cryo-ultramicrotomy were

processed for double-label immunohistochemistry (Kim et al., 2021)

and conventional electron microscopy for STEM.

- Confocal microscope (LSM 900, Carl Zeiss)

Double immunofluorescence immunohistochemistry was performed with

antibodies to GFAP (Rabbit polyclonal anti-GFAP) and NeuN (Mouse

monoclonal anti-NeuN.

- STEM (Merlin, Carl Zeiss)

Ultra-thin section (80~100nm thick) on the formvar coated one hole grid

with uranyl acetate staining.

- Little staining with uranyl acetate in the section on the formvar coated one hole grid showed clear ultrastructure in the STEM.

- STEM can be used as an alternative to TEM for CLEM by observing various areas of interests and correlating light microscopic information with ultrastructural details.

INTRODUCTION

MATERIALS & METHODS

RESULTS

SUMMARY & CONCLUSION

2. Correlated fluorescent signal and electron

microscopic image in the rat brain cortex.

Double-labeling of NeuN and GFAP on

confocal microscope (A) and STEM (B)

image in the SEM of the same field in low

magnification. The boxed area is the region

of interest (ROI). Scale bar = 50μm for A, B

3. Higher-magnification views of the boxed areas in A, B.

Confocal microscopy and STEM images of the same

field showing the immuno-localization of NeuN (red) in

pyramidal cells (a-2, b-2, a-3, b-3) and GFAP (green) around the vessels (a-1,b-1, a-3, b-3).

Scale bar = 5μm for A, B; 10μm for C.

1. The section (80nm thickness) mounted on a formvar

coated one hole grid placed on the holder of the SEM

equipped with STEM.

A

B

a-2

a-1 a-2 a-3

b-1 b-2