Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

Halabi, Elias A.; Pinotsi, Dorothea; Rivera‐Fuentes, Pablo

doi:10.26434/chemrxiv.7165745.v1

Cited by 5 publications

(6 citation statements)

References 11 publications

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“…Interestingly, there is some suggestion that resPAINT may have been utilised in several previous studies, without formalising or fully realising the concept, where it is typically referred to as no-wash labelling. 33,[38][39][40][41] We demonstrate that resPAINT can improve D R A F T the localisation rate, and therefore the speed of acquisition, for a given binder up to 50-fold. We implement resPAINT using the DHPSF 2,3 a common volumetric imaging technique.…”

Section: Introductionmentioning

confidence: 85%

“…These implementations have typically been referred to as no-wash labelling protocols. 33,[38][39][40][41] We provide the first detailed description of the kinetic requirements of resPAINT and explore the space over which the technique is useful for bioimaging. We generalise this concept by using a selection of probes (photoactivation and spontaneously blinking), various imaging modalities (DHPSF, tetrapod PSF, SMLFM) and apply the technique to multiple systems (whole-cell, membrane topography and membrane proteins).…”

Section: Discussionmentioning

confidence: 99%

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resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Sanders

Carr

Bruggeman

et al. 2022

Preprint

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Points for accumulation in nanoscale topography (PAINT) allows the acquisition of practically unlimited measurements in localisation microscopy. However, PAINT is inherently limited by unwanted background fluorescence at high probe concentrations, especially in large depth-of-field volumetric imaging techniques. Here we present reservoir-PAINT (resPAINT), in which we combine PAINT with active control of probe photophysics. In resPAINT, a "reservoir" of non-fluorescent activatable probes accumulate on the target, which makes it possible to drastically improve the localisation rate (by up to 50-fold) compared to conventional PAINT, without any compromise in contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate volumetric super-resolution imaging of entire cell surfaces. We then generalise the approach by implementing multiple switching strategies, including photoactivation and spontaneous blinking. We also implement alternative volumetric imaging modalities including the double-helix point-spread function, the tetrapod point-spread function and single-molecule light field microscopy. Finally, we show that resPAINT can be used with a Fab to image membrane proteins, effectively extending the operating regime of conventional PAINT to encompass a larger range of biological interactions.

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Section: Introductionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Sanders

Carr

Bruggeman

et al. 2022

Preprint

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“…22 To assess whether reductive stress induced by probe 1 triggers mitophagy, we transfected cells with either parkin or the autophagosomal marker LC3 fused to the bright fluorescent protein mTurquoise2 (Figure S9 and Table S2). 23 No recruitment of parkin to the mitochondrial membrane was observed in cells treated with probe 1. In contrast, cells treated with CCCP recruited parkin efficiently (Figure 3B).…”

Section: Confocal Microscopy Experiments Confocal Microscopy Experiments Employingmentioning

confidence: 98%

Disruption of Redox Homeostasis by Enzymatic Activation of a Triakylphosphine Probe in Mitochondria

Nguyen¹,

Tirla²,

Rivera‐Fuentes

2019

Preprint

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Redox homeostasis is essential for cell function and its disruption is associated with cancer, metabolic and neurodegenerative diseases.Redox balance is largely regulated by the relative concentrations of reduced and oxidized glutathione. In eukaryotes, this ratio is different in each cell compartment, and disruption of the mitochondrial redox balance has been specifically linked to pathologies such as obesity and type II diabetes.Although reduced glutathione can be scavenged using electrophiles, there is a lack of probes that can produce it. In this study, we report an organellespecific reducing agent based on tributylphosphine that selectively reduces oxidized glutathione in mitochondria. This probe is activated by endogenous nitroreductases and subsequently releases tributylphosphine, as well as a fluorescent reporter, within the organelle. Confocal imaging and biological assays in human cells revealed that, counterintuitively, increased reduced glutathione induced oxidative stress through accumulation of superoxide.Transcriptomic analysis was used to establish that mitochondrial redox stress activates a cellular response orchestrated by transcription factor ATF4, which upregulates genes involved in glutathione metabolism.the activity of enzymes that are present only in these organelles to trigger the release of tributylphosphine from a masked precursor. Here, we report the development of such probe, its validation in live human cells, and its application to characterize the cellular response to mitochondrial reductive stress. RESULTS AND DISCUSSIONDesign, Synthesis and Enzymatic Activation. To release tributylphosphine exclusively in mitochondria, we selected endogenous nitroreductases (NTRs) as the activating agent. Although the enzymes that are responsible for this reactivity have not been fully identified in mammalian cells, they are capable of reducing nitroaromatic compounds to amines under normoxic conditions. 12,13 Based on this knowledge, we developed the tributylphosphonium probe 1 with a nitro group as the enzymatically activatable trigger (Scheme 1A). Upon enzymatic reaction, the strong electron-withdrawing nitro group is converted to 1 for Imaging and Detection of Endogenous Superoxide in Live Cells and in Vivo. J. Am. Chem. Soc. 2015, 137, 6837-6843. https://doi.org/10.1021/jacs.5b01881. (18) Lange, P. S.; Chavez, J. C.; Pinto, J. T.; Coppola, G.; Sun, C.-W.; Townes, T. M.; Geschwind, D. H.; Ratan, R. R. ATF4 Is an Oxidative Stress-Inducible, Prodeath Transcription Factor in Neurons in Vitro and in Vivo. J. Exp. Med. 2008, 205, 1227-1242. https://doi.org/10.1084/jem.20071460. (19) Liberman, E. A.; Topaly, V. P.; Tsofina, L. M.; Jasaitis, A. A.; Skulachev, V. P. Mechanism of Coupling of Oxidative Phosphorylation and the Membrane Potential of Mitochondria. Nature 1969, 222, 1076-1078. https://doi.org/10.1038/2221076a0. https://doi.org/10.1093/bioinformatics/btu170. (36) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T. R. STAR: Ultraf...

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“…Acidic solvents favor the formation of the open form of RSLs 14,[16][17] , while the effect of solvent polarity is more variable 18 . RSL photoactivation has been used in a variety of applications, most notably in super-resolution microscopy 15,[19][20][21] . Other applications of RSL photoswitching include volumetric displays 14,17 , and optoelectronics 22 .…”

Section: Photoswitching Is Another Mechanism Of Rsl Control Between C...mentioning

confidence: 99%

Synthesis and Optical Characterization of a Rhodamine B Spirolactam Dimer

Stratton

Pierre

Riser

et al. 2022

Preprint

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Amide derivatives of xanthene dyes such as rhodamine B are useful in a variety of sensing applications due to their colorimetric responses to stimuli such as acidity changes and UV light. The optical properties of these molecules can be influenced by intermolecular associations into dimeric structures, but the exact impact can be hard to predict. We have designed a covalently linked intramolecular dimer of the dye rhodamine B utilizing p-phenylene diamine to link the two dyes via amide bonds. The doubly closed spirolactam version of this dimer, RSL2, is isolated as a colorless solid. Under acidic conditions or UV exposure, RSL2 solutions develop a pink color that is expected for the ring-opened form of the molecule. However, NMR and single crystal diffraction data show that the equilibrium still prefers the closed dimer state. Interestingly, the emission profile of RSL2 shows solvatochromic blue fluorescence. Control studies of model compounds with similar structural motifs do not display similar blue fluorescence, indicating that this optical behavior is unique to the dimeric form. This behavior may lend itself to applications of such xanthene dimers to more sophisticated sensors beyond those with traditional binary on/off fluorescence profiles.

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Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

Cited by 5 publications

References 11 publications

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

resPAINT: Accelerating volumetric super-resolution localisation microscopy by active control of probe emission

Disruption of Redox Homeostasis by Enzymatic Activation of a Triakylphosphine Probe in Mitochondria

Synthesis and Optical Characterization of a Rhodamine B Spirolactam Dimer

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