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Edited by Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, and approved June 24, 2019 (received for review April 6, 2019)
Significance
Stimulated emission depletion (STED) microscopy is one of the most appealing tools to visualize nanoscale cellular structures and dynamics in living cells. However, its practical utility is significantly limited by the rapid photobleaching of fluorescent dyes under the ultrastrong depletion laser. In this context, superphotostable fluorescent probes, which enable repeated recording of the STED images, are crucial chemical tools to extend the application of STED microscopy in live-cell imaging. Herein, we report a developed fluorescent probe, MitoPB Yellow, which has the characters of outstanding photostability, long fluorescence lifetime, and mitochondrial inner membrane selectivity for staining. In combination with a time-gated-STED microscopy, the mitochondrial inner-membrane structures and dynamics can be impressively visualized in living cells.
Abstract
Stimulation emission depletion (STED) microscopy enables ultrastructural imaging of organelle dynamics with a high spatiotemporal resolution in living cells. For the visualization of the mitochondrial membrane dynamics in STED microscopy, rationally designed mitochondrial fluorescent markers with enhanced photostability are required. Herein, we report the development of a superphotostable fluorescent labeling reagent with long fluorescence lifetime, whose design is based on a structurally reinforced naphthophosphole fluorophore that is conjugated with an electron-donating diphenylamino group. The combination of long-lived fluorescence and superphotostable features of the fluorophore allowed us to selectively capture the ultrastructures of the mitochondrial cristae with a resolution of ∼60 nm when depleted at 660 nm. This chemical tool provides morphological information of the cristae, which has so far only been observed in fixed cells using electron microscopy. Moreover, this method gives information about the dynamic ultrastructures such as the intermembrane fusion in different mitochondria as well as the intercristae mergence in a single mitochondrion during the apoptosis-like mitochondrial swelling process.
Footnotes
- ↵1To whom correspondence may be addressed. Email: taki{at}itbm.nagoya-u.ac.jp, y.okada{at}riken.jp, or yamaguchi{at}chem.nagoya-u.ac.jp.
Author contributions: C.W., M.T., and S.Y. designed research; C.W., Y.S., H.Y., and Y.O. performed research; M.T., Y.T., H.Y., Y.O., and S.Y. contributed new reagents/analytic tools; C.W., M.T., Y.S., Y.T., H.Y., Y.O., and S.Y. analyzed data; and C.W., M.T., Y.T., Y.O., and S.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1905924116/-/DCSupplemental.
Published under the PNAS license.
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A photostable fluorescent marker for the superresolution live imaging of the dynamic structure of the mitochondrial cristae
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