Abstract
Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. Conventional fluorescence microscopy can only provide ~300 nm resolution, which is insufficient to visualize mitochondrial cristae. Here, we developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells with a superresolution technique. The low saturation intensity and high photostability of MitoESq-635 make it ideal for long-term, high-resolution (stimulated emission depletion) STED nanoscopy. We performed time-lapse imaging of the mitochondrial inner membrane over 50 min (3.9 s per frame, with 71.5 s dark recovery) in living HeLa cells with a resolution of 35.2 nm. The forms of the cristae during mitochondrial fusion and fission can be clearly observed. Our study demonstrates the emerging capability of optical STED nanoscopy to investigate intracellular physiological processes with nanoscale resolution for an extended period of time.
Highlights
Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality
A hexylamidophenylarsenicate moiety is conjugated to the sulfide atom at the central position of the fourmembrane ring in the squaraine dye (Supplementary Note 1), which can potentially be used as a protein label for the mitochondrial membrane or other organelles in live cells (Supplementary Note 2)
Due to the fast binding of phenylarsenicate to vicinal dithiols and the prioritised targeting of mitochondria by the dye molecules, incubating live cells with MitoESq-635 for a few minutes is sufficient to label the mitochondrial membrane with high density
Summary
Mitochondria play a critical role in generating energy to support the entire lifecycle of biological cells, yet it is still unclear how their morphological structures evolve to regulate their functionality. We developed an enhanced squaraine variant dye (MitoESq-635) to study the dynamic structures of mitochondrial cristae in live cells with a superresolution technique. The term mitochondria comes from the Greek words mito (particle) and chondria (lines), which describe their various structural characteristics in live cells. Limited by the diffraction of light, conventional optical microscopy techniques are insufficient to visualise the sub-mitochondrial structures (cristae) and their dynamics[2]. Ishigaki et al.[8] reported the rich dynamics of mitochondria with a rhodamine derivative Because they are limited by spatial resolution, the cristae can only be visualised as lamellar curtain-like structures. We reported the use of Hessian structured illumination microscopy (SIM) to investigate mitochondrial dynamics, in which the cristae can be clearly visualised[10]. The Testa group demonstrated 3D nanoscale imaging of the mitochondrial outer membrane labelled with rsEGFP2-Omp[25] (8 vol for 26 s)[20]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
