Cyclooctatetraene-conjugated cyanine mitochondrial probes minimize phototoxicity in fluorescence and nanoscopic imaging.

Zhongtian Yang,Yun Zhao,Xiaoshuai Huang,Zhixing Chen,Jing Ling,Yuqing Ying,Tianyan Liu,Liuju Li,Yan Zhao,Liangyi Chen,Kai Lei

doi:10.1039/d0sc02837a

Zhongtian Yang, Yun Zhao + Show 9 more

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https://doi.org/10.1039/d0sc02837a

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Abstract

Modern fluorescence-imaging methods promise to unveil organelle dynamics in live cells. Phototoxicity, however, has become a prevailing issue when boosted illumination applies. Mitochondria are representative organelles whose research heavily relies on optical imaging, yet these membranous hubs of bioenergy are exceptionally vulnerable to photodamage. We report that cyclooctatetraene-conjugated cyanine dyes (PK Mito dyes), are ideal mitochondrial probes with remarkably low photodynamic damage for general use in fluorescence cytometry. In contrast, the nitrobenzene conjugate of Cy3 exhibits enhanced photostability but unaffected phototoxicity compared to parental Cy3. PK Mito Red, in conjunction with Hessian-structural illumination microscopy, enables 2000-frame time-lapse imaging with clearly resolvable crista structures, revealing rich mitochondrial dynamics. In a rigorous stem cell sorting and transplantation assay, PK Mito Red maximally retains the stemness of planarian neoblasts, exhibiting excellent multifaceted biocompatibility. Resonating with the ongoing theme of reducing photodamage using optical approaches, this work advocates the evaluation and minimization of phototoxicity when developing imaging probes.

Highlights

Mitochondria are crucial for ATP production, and for other processes such as cell signaling and cell death.[1,2] Mitochondria contain double-membrane structures that delicately compartmentalize biochemical transformations, dynamically interact with other cellular organelles, and adopt various shapes in different cells
Membranous structures are sensitive to photodynamic damage, as unsaturated lipids and proteins are susceptible to reactive oxygen species (ROS) attack
Phototoxicity has long been harnessed for constructive applications, such as photodynamic therapy[22] and chromophore-assisted light inactivation.[23]

Summary

Introduction

Mitochondria are crucial for ATP production, and for other processes such as cell signaling and cell death.[1,2] Mitochondria contain double-membrane structures that delicately compartmentalize biochemical transformations, dynamically interact with other cellular organelles, and adopt various shapes in different cells. State-of-the-art, super-resolution (SR) microscopy has the potential to reveal intricate structures and novel dynamics of mitochondria in live cells in real time.[4] SR imaging of mitochondria has been made possible by high-density, environmentally sensitive probes based on the singlemolecule on-off switching principle.[5] Recently, mitochondrial crista structures in live cells have been visualized with Hessian structured illumination microscopy (Hessian SIM)[6] and stimulated emission depletion (STED) microscopy.[7,8,9] Yet, in contrast to long-term SR imaging of other organelles, such as endoplasmic reticulum and the cytoskeleton, mitochondrial imaging is much more susceptible to phototoxicity. This process has been consistently observed with multiple types of SR microscopy,[7,10] emerging as a major obstacle in preventing the visualization of physiologically relevant mitochondrial dynamics in live cells

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