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

Proceedings of the National Academy of Sciences, Journal Year: 2022, Volume and Issue: 119(52)

Published: Dec. 19, 2022

Capturing mitochondria's intricate and dynamic structure poses a daunting challenge for optical nanoscopy. Different labeling strategies have been demonstrated live-cell stimulated emission depletion (STED) microscopy of mitochondria, but orthogonal are yet to be established, image acquisition has suffered either from photodamage the organelles or rapid photobleaching. Therefore, nanoscopy mitochondria largely restricted two-dimensional (2D) single-color recordings cancer cells. Here, by conjugation cyclooctatetraene (COT) benzo-fused cyanine dye, we report mitochondrial inner membrane (IM) fluorescent marker, PK Mito Orange (PKMO), featuring efficient STED at 775 nm, strong photostability, markedly reduced phototoxicity. PKMO enables super-resolution (SR) IM dynamics extended periods in immortalized mammalian cell lines, primary cells, organoids. Photostability phototoxicity open door three-dimensional (3D) 3D analysis convoluted IM. is optically with green far-red markers, allowing multiplexed using commercial microscopes. Using multi-color microscopy, demonstrate that imaging can capture interactions different cellular components such as endoplasmic reticulum (ER) cytoskeleton, Bcl-2-associated X protein (BAX)-induced apoptotic process, crista phenotypes genetically modified all sub-100 nm resolution. Thereby, this work offers versatile tool studying architecture manner.

Language: Английский

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Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(5), P. 3075 - 3083

Published: Jan. 30, 2023

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, irreversible attachment probe to precludes imaging applications that require transient binding comes risk photobleaching. Here, we introduce exchangeable ligands fluorescence (xHTLs) reversibly bind can be coupled rhodamines different colors. In stimulated emission depletion (STED) microscopy, exchange xHTLs allows reduced photobleaching as compared labeling. Transient fluorogenic fusion proteins enables points accumulation nanoscale topography (PAINT) MINFLUX microscopy. We furthermore pairs mutants dual-color PAINT STED thus open up new possibilities across microscopy platforms widely used approach.

Language: Английский

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Brightness of fluorescent organic nanomaterials

Chemical Society Reviews, Journal Year: 2023, Volume and Issue: 52(14), P. 4525 - 4548

Published: Jan. 1, 2023

Brightness is a fundamental property of fluorescent nanomaterials reflecting their capacity to absorb and emit light. In sensing materials, brightness crucial for high-sensitivity (bio)molecular detection, while in optical bioimaging it ensures high spatial temporal resolution. Fluorescent organic nanoparticles (NPs) are particularly attractive because superior compared dyes. With the ever-growing diversity nanomaterials, important establish universal principles measuring estimating brightness. This tutorial review provides definitions describes major approaches its analysis based on ensemble single-particle techniques. We present current chemical fight Aggregation-Caused Quenching (ACQ) fluorophores, which challenge design bright nanomaterials. The main classes NPs described, including conjugated polymer NPs, aggregation-induced emission neutral ionic Their other properties systematically compared. Some brightest examples bulk solid-state emissive materials also mentioned. Finally, we analyse importance particle biological applications, such as biosensing. will provide guidelines chemists with improved performance help them estimate compare new literature reports. Moreover, biologists select appropriate imaging applications.

Language: Английский

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A general method for the development of multicolor biosensors with large dynamic ranges

Nature Chemical Biology, Journal Year: 2023, Volume and Issue: 19(9), P. 1147 - 1157

Published: June 8, 2023

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most suffer from relatively low dynamic ranges. Here, we introduce a family designed Förster resonance energy transfer (FRET) pairs near-quantitative FRET efficiencies based on reversible interaction fluorescent proteins fluorescently labeled HaloTag. These enabled straightforward design for calcium, ATP and NAD+ unprecedented The color each these can be readily tuned by changing either protein or synthetic fluorophore, which enables simultaneous monitoring free in different subcellular compartments following genotoxic stress. Minimal modifications furthermore allow their readout to switched fluorescence intensity, lifetime bioluminescence. thus establish new concept development highly sensitive tunable biosensors.

Language: Английский

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Optimized Red-Absorbing Dyes for Imaging and Sensing

Journal of the American Chemical Society, Journal Year: 2023, Volume and Issue: 145(42), P. 23000 - 23013

Published: Oct. 16, 2023

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property rhodamines is their equilibrium between colorless lactone and zwitterion. Tuning the lactone–zwitterion constant (KL–Z) can optimize dye properties specific biological applications. Here, we use known novel organic chemistry to prepare comprehensive collection rhodamine elucidate structure–activity relationships that govern KL–Z. We discovered auxochrome substituent strongly affects equilibrium, providing roadmap rational design improved dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings yield bright, red-shifted fluorophores live-cell single-particle tracking (SPT) multicolor imaging. The N-aryl combined fluorination yields Förster resonance energy transfer (FRET) quencher useful creating new semisynthetic indicator sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this expands synthetic methods available synthesis, generates reagents advanced experiments, describes will guide future

Language: Английский

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Ultra-photostable small-molecule dyes facilitate near-infrared biophotonics

Nature Communications, Journal Year: 2024, Volume and Issue: 15(1)

Published: March 22, 2024

Abstract Long-wavelength, near-infrared small-molecule dyes are attractive in biophotonics. Conventionally, they rely on expanded aromatic structures for redshift, which comes at the cost of application performance such as photostability, cell permeability, and functionality. Here, we report a ground-state antiaromatic strategy showcase concise synthesis 14 cationic aminofluorene with mini (molecular weights: 299–504 Da) distinct spectra covering 700–1600 nm. Aminofluorene cell-permeable achieve rapid renal clearance via simple 44 Da carboxylation. This accelerates optical diagnostics injury by 50 min compared to existing macromolecular approaches. We develop compact molecular sensing platform vivo intracellular sensing, demonstrate versatile applications these multispectral fluorescence optoacoustic imaging. find that aromaticity reversal upon electronic excitation, indicated magnetic descriptors, not only reduces energy bandgap but also induces strong vibronic coupling, resulting ultrafast excited-state dynamics unparalleled photostability. These results support argument antiaromaticity useful design rule dye development, enabling performances essential modern

Language: Английский

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An All‐Rounder for NIR‐II Phototheranostics: Well‐Tailored 1064 nm‐Excitable Molecule for Photothermal Combating of Orthotopic Breast Cancer

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(26)

Published: April 19, 2024

The second near-infrared (NIR-II, 1000-1700 nm) light-activated organic photothermal agent that synchronously enables satisfying NIR-II fluorescence imaging is highly warranted yet rather challenging on the basis of overwhelming nonradiative decay. Herein, such an agent, namely TPABT-TD, was tactfully designed and constructed via employing benzo[c]thiophene moiety as bulky electron donor/π-bridge tailoring peripheral molecular rotors. Benefitting from its high donor-acceptor strength finely modulated intramolecular motion, TPABT-TD simultaneously exhibits ultralong absorption in region, intense emission NIR-IIa (1300-1500 region nanoaggregates, conversion upon 1064 nm laser irradiation. Those intrinsic advantages endow nanoparticles with prominent fluorescence/photoacoustic/photothermal trimodal imaging-guided therapy against orthotopic 4T1 breast tumor negligible adverse effect.

Language: Английский

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Organic Fluorophores with Large Stokes Shift for the Visualization of Rapid Protein and Nucleic Acid Assays

Angewandte Chemie International Edition, Journal Year: 2024, Volume and Issue: 63(17)

Published: March 6, 2024

Organic small-molecule fluorophores, characterized by flexible chemical structure and adjustable optical performance, have shown tremendous potential in biosensing. However, classical organic fluorophore motifs feature large overlap between excitation emission spectra, leading to the requirement of advanced set up filter desired signal, which limits their application scenarios with simple settings. Here, a series wavelength-tunable fluorescent dyes (PTs) bearing moieties been developed, exhibit Stokes shift 262 nm, molar extinction coefficients ranged 30,000-100,000 M

Language: Английский

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Event-driven acquisition for content-enriched microscopy

Nature Methods, Journal Year: 2022, Volume and Issue: 19(10), P. 1262 - 1267

Published: Sept. 8, 2022

Language: Английский

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Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

Angewandte Chemie International Edition, Journal Year: 2022, Volume and Issue: 61(41)

Published: July 19, 2022

Abstract For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize detection biochemical activities. Dark quenchers relax from excited state ground non‐radiatively, and are promising alternatives fluorescent FRET acceptors. Small‐molecule (dark) have been as acceptors in FRET‐based probes monitor various physiological processes with minimum background signal. Herein, we summarize relevant advances small‐molecule that probes. This Review intended provide suggestions regarding rationale design selection appropriate fluorophore–quencher pairs, which fully compatible challenging analytical applications biological systems. Finally, an outlook future biomedical developments this field presented.

Language: Английский

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