Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructural dynamics of cells. However, the correlation of subcellular structures observed by live cell imaging with the EM ultrastructure remains technically challenging. In order to optimise the workflow for live-cell CLEM, we have designed a modifiable imaging surface for cellular optical imaging [1]. We first investigated the suitability of polymer films as a support film for live cell imaging. In contrast to glass coverslips, the use of polymer films not only allows for subsequent cryo-preservation, but also allowed us to directly adapt the surface of the film for correlative imaging. We made use of an office printer and laminator to mark the polymer films with a toner-based reference grid, which is already visible by eye. The toner also forms part of a multiscalar fiducial reference system which enables subcellular spatial alignment. We have used this CLEM-strategy to investigate the fate of mitochondria during induced mitochondrial degradation by mitophagy [2]. Mitophagy is a selective pathway that targets and delivers mitochondria to the lysosomes for degradation. The protonophore CCCP causes depolarization of mitochondria and induces their degradation by mitophagy. When mammalian cells overexpress the ubiquitin ligase Parkin, treatment with CCCP for more than 24 hours has been reported to trigger the clearance of all mitochondria. However, using CLEM in Parkin-expressing HeLa cells, we show that mitochondrial remnants remain present in the cell. The mitochondria were no longer easily identifiable as such due to morphological alterations, providing a possible explanation why earlier EM studies may have missed these structures. Further investigation by live-cell microscopy showed that CCCP inhibits mitophagy at both the initiation and lysosomal degradation stages. In summary, we have developed an inexpensive and robust CLEM procedure that simplifies optical imaging without limiting the choice of optical microscope. We have verified the technique by providing novel biological insights into the mechanism of mitochondrial degradation.
[1] B. S. Padman, M. Bach and G. Ramm. 2014 An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM. PloS one 9 (4), e95967. [2] B. S. Padman, M.Bach, G. Lucarelli, M. Prescott and G.Ramm. 2013 The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells. Autophagy 9(11), 1862-1875.
This work has been supported by the National Health and Medical Research Council under project grant 596849. We would also like to thank Stephen Firth, Alex Fulcher and July Callaghan from Monash Micro Imaging for help with optical microscopy.