A general, highly efficient and facile synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy

Bucevičius, Jonas; Gerasimaitė, Rūta; Kiszka, Kamila A.; Pradhan, Shalini; Kostiuk, Georgij; Koenen, Tanja; Lukinavičius, Gražvydas

doi:10.1101/2022.06.13.495683

Cited by 2 publications

(2 citation statements)

References 47 publications

(97 reference statements)

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“…This work shows that a cyanine dye, once equipped with triplet-state quenchers and spectroscopically tailored, offers superior performance in live-cell STED nanoscopy. Thereby, it adds itself to the ever-growing mitochondrial imaging options ( 12 , 14 , 69 , 70 ) and also sheds light on potential development strategies of future live-cell stains for nanoscopy.…”

Section: Discussionmentioning

confidence: 96%

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Liu

Stephan

Chen

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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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 two-dimensional (2D) single-color recordings of cancer cells. Here, by conjugation of cyclooctatetraene (COT) 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 (SR) recordings of IM 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 three-dimensional (3D) STED nanoscopy of mitochondria for 3D 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 microscopy, we demonstrate that imaging with PKMO can capture interactions of mitochondria with different cellular components such as the endoplasmic reticulum (ER) or the cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, or crista phenotypes in genetically modified cells, all at sub-100 nm resolution. Thereby, this work offers a versatile tool for studying mitochondrial IM architecture and dynamics in a multiplexed manner.

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Section: Discussionmentioning

confidence: 96%

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Liu

Stephan

Chen

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…However, this feature is achieved only with one of the three possible chemical isomers. The different isomers of these orange and red emitting dyes have been tested on living cells and tissues by staining actin, microtubules, DNA, mitochondria and lysosomes as well as SNAP-tag and HaloTag proteins [26,28,29]. Due to a neighboring group effect in the isomer 4, cell permeability is increased with respect to the other two possible isomers.…”

Section: Photophysical Properties Of the Dyesmentioning

confidence: 99%

Two-color live-cell STED nanoscopy by click labeling with cell-permeable fluorophores

Gregor

Grimm²,

Rehman³

et al. 2022

Preprint

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STED nanoscopy allows for the direct observation of dynamic processes in living cells and tissues with diffraction-unlimited resolution. Although fluorescent proteins can be used for STED imaging, these labels are often outperformed in photostability by organic fluorescent dyes. This feature is especially crucial for time-lapse imaging. Unlike fluorescent proteins, organic fluorophores cannot be genetically fused to a target protein but require different labeling strategies. To achieve simultaneous imaging of more than one protein in the interior of the cell with organic fluorophores, bioorthogonal labeling techniques and cell-permeable dyes are required. In addition, the fluorophores should preferentially emit in the red spectral range to reduce potential phototoxic effects that can be induced by the STED light, which further restricts the choice of suitable markers. Here we demonstrate two-color STED imaging of living cells using various pairs of dyes that fulfill all of the above requirements. To this end, we combine click-chemistry-based protein labeling with other orthogonal and highly specific labeling methods, enabling long-term STED imaging of different target structures in living specimens.

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A general, highly efficient and facile synthesis of biocompatible rhodamine dyes and probes for live-cell multicolor nanoscopy

Cited by 2 publications

References 47 publications

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Multi-color live-cell STED nanoscopy of mitochondria with a gentle inner membrane stain

Two-color live-cell STED nanoscopy by click labeling with cell-permeable fluorophores

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