RT Journal Article
SR Electronic
T1 Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.05.09.491019
DO 10.1101/2022.05.09.491019
A1 Tianyan Liu
A1 Till Stephan
A1 Peng Chen
A1 Jingting Chen
A1 Dietmar Riedel
A1 Zhongtian Yang
A1 Stefan Jakobs
A1 Zhixing Chen
YR 2022
UL http://biorxiv.org/content/early/2022/05/09/2022.05.09.491019.abstract
AB Capturing mitochondria’s intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated for live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal strategies are yet to be established, and image acquisition has suffered either from photodamage to the organelles or from rapid photobleaching. Therefore, live-cell nanoscopy of mitochondria has been largely restricted to 2D single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene to a benzo-fused cyanine dye, we report a mitochondrial inner-membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability and markedly reduced phototoxicity. PKMO enables super-resolution recordings of inner-membrane dynamics for extended periods in immortalized mammalian cell lines, primary cells, and organoids. Photostability and reduced phototoxicity of PKMO open the door to live-cell 3D STED nanoscopy of mitochondria for three-dimensional analysis of the convoluted IM. PKMO is optically orthogonal with green and far-red markers allowing multiplexed recordings of mitochondria using commercial STED microscopes. Using multi-color STED, we demonstrate that imaging with PKMO can capture the sub-mitochondrial localization of proteins, or interactions of mitochondria with different cellular components, such as the ER or the cytoskeleton at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial inner-membrane architecture and dynamics in a multiplexed manner.Competing Interest StatementZ.C. is an inventor of the patent on the mitochondria dye described in this work (CN 202010492298.8). The patent was applied through Peking University and is currently transferred to Genvivo Tech (in which Z.C. is a shareholder) for commercialization.