Keisuke Ohta (1,2), Shingo Hirashima (2), Yoshihiro Miyazono (2), Akinobu Togo (1), Kei-ichiro Nakamura (2)
Microscopy, 70, Issue 2, April 2021: 161–170. DOI: 10.1093/jmicro/dfaa071

Keywords

CLEM; focused ion beam; mitochondria; dynamics; fragmentation; live imaging

Abstract

Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

How Our Software Was Used

Dragonfly was used for merging the data obtained from light microscopy and electron microscopy.

Author Affiliation

(1) Advanced Imaging Research Center, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.
(2) Department of Anatomy, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.