BIPLANE FPALM MICROSCOPY 3D VISUALIZATION OF MITOCHONDRIAL
OUTER MEMBRANE AND INTERMEMBRANE SPACE
Petr Ježek, Lukáš Alán, Hana Engstová, Jitka Šantorová, and Tomáš Špaček
Institute of Physiology, Czech Academy of Sciences, Dept.75
Vídeňská 1083, Prague 4, Czech Republic
E-mail: [email protected]
KEY WORDS: 3D mitochondrial morphology, BiplaneFPALM microscopy, hypoxia
Three-dimensional (3D) super-resolution microscopy, using a biplane detection scheme,
termed biplane photo-activated localization microscopy (Biplane FPALM), enables imaging
of volumes as thick as whole cells [1]. We have imaged 3D mitochondrial network of
hepatoma HepG2 cells, visualized either with an Eos-conjugate of genetically altered FIS1tr
protein (so not to induce mitochondrial fission), as a marker of the outer membrane, or via the
intermembrane space with overepressed genetically altered lactamase-beta protein (LactB)
conjugated with Eos. 3D BiplaneFPALM distinguished a hollow character of mitochondrial
reticulum tubules when visualized by Eos-FIS1tr. Upon network fragmentation, hollow max
~2 micrometer spheres occurred. LactB marker was able to stain both peripheral
intermembrane space and intracristal space. Thus the 3D superresolution imaging in
combination with specific mitochondria morphology markers allows distinction of
submitochondrial compartments which is impossible by conventional confocal imaging.
Fig.1. Mitochondrial network tubules as imaged:
A) as outer membrane by Eos2-FIS , B) fragmented;
C) via intermembrane space
Supported by grant P302/10/0346 (GACR).
[1] M.J. Mlodzianoski, J.M. Schreiner, S.P. Callahan, K. Smolková, A. Dlasková, J.
Šantorová, P. Ježek, J. Bewersdorf. "Sample drift correction in 3D fluorescence
photoactivation localization microscopy,". Opt. Express 19, 15009-15019 (2011).