Genetically encoded fluorescent indicators for live cell pH imaging

AbstractChronic imaging of neuronal networks in vitro has provided fundamental insights into mechanisms underlying neuronal function. Current labeling and optical imaging methods, however, cannot be used for continuous and long-term recordings of the dynamics and evolution of neuronal networks, as fluorescent indicators can cause phototoxicity. Here, we introduce a versatile platform for label-free, comprehensive and detailed electrophysiological live-cell imaging of various neurogenic cells and tissues over extended time scales. We report on a dual-mode high-density microelectrode array, which can simultaneously record in (i) full-frame mode with 19,584 recording sites and (ii) high-signal-to-noise mode with 246 channels. We set out to demonstrate the capabilities of this platform with recordings from primary and iPSC-derived neuronal cultures and tissue preparations over several weeks, providing detailed morpho-electrical phenotypic parameters at subcellular, cellular and network level. Moreover, we develop reliable analysis tools, which drastically increase the throughput to infer axonal morphology and conduction speed.

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Upconversion nanoparticle-mOrange protein FRET nanoprobes for self-ratiometric/ratiometric determination of intracellular pH, and single cell pH imaging

Biosensors and Bioelectronics ◽

10.1016/j.bios.2020.112115 ◽

2020 ◽

Vol 155 ◽

pp. 112115 ◽

Cited By ~ 1

Author(s):

Sandip Ghosh ◽

Yu-Fen Chang ◽

De-Ming Yang ◽

Surojit Chattopadhyay

Keyword(s):

Single Cell ◽

Intracellular Ph ◽

Cell Ph ◽

Ph Imaging

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Multicoloured fluorescent indicators for live-cell and in vivo imaging of inorganic mercury dynamics

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2018.04.562 ◽

2018 ◽

Vol 121 ◽

pp. 26-37

Author(s):

Rongkun Tao ◽

Mei Shi ◽

Yejun Zou ◽

Di Cheng ◽

Qiaohui Wang ◽

...

Keyword(s):

In Vivo Imaging ◽

Inorganic Mercury ◽

Live Cell ◽

Fluorescent Indicators

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Single-cell pH imaging and detection for pH profiling and label-free rapid identification of cancer-cells

Scientific Reports ◽

10.1038/s41598-017-01956-1 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 20

Author(s):

Hui Hou ◽

Yangyang Zhao ◽

Chuanping Li ◽

Minmin Wang ◽

Xiaolong Xu ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Rapid Identification ◽

Label Free ◽

Cell Ph ◽

Ph Imaging

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Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645981 ◽

1987 ◽

Vol 58 (02) ◽

pp. 737-743 ◽

Cited By ~ 12

Author(s):

Frarnçois Lanza ◽

Alain Beretz ◽

Martial Kubina ◽

Jean-Pierre Cazenave

Keyword(s):

Intracellular Calcium ◽

Shape Change ◽

Calcium Ionophore ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Free Calcium ◽

Membrane Bilayer ◽

Fluorescent Indicators ◽

Fura 2 ◽

Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

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α-Methylene-β-Lactone Probe for Measuring Live-Cell Reactions of Small Molecules

10.26434/chemrxiv.12078582.v2 ◽

2020 ◽

Author(s):

Lei Wang ◽

Louis Riel ◽

Bekim Bajrami ◽

Bin Deng ◽

Amy Howell ◽

...

Keyword(s):

Mass Spectra ◽

Live Cell ◽

Live Cells ◽

The Novel ◽

Proteomics Analysis ◽

Chemical Proteomics ◽

Tandem Mass Spectra ◽

Modified Peptides ◽

Covalent Probes ◽

Ht 29

The novel use of the α-methylene-β-lactone (MeLac) moiety as a warhead of multiple electrophilic sites is reported. In this study, we demonstrate that a MeLac-alkyne is a competent covalent probe and reacts with diverse proteins in live cells. Proteomics analysis of affinity-enriched samples identifies probe-reacted proteins, resolves their modified peptides/residues, and thus characterizes probe-protein reactions. Unique methods are developed to evaluate confidence in the identification of the reacted proteins and modified peptides. Tandem mass spectra of the peptides reveal that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael addition or acyl addition. A peptide-centric proteomics platform, using MeLac-alkyne as the measurement probe, successfully analyzes the Orlistat selectivity in live HT-29 cells. MeLac is a versatile warhead demonstrating enormous potential to expedite the development of covalent probes and inhibitors in interrogating protein (re)activity. MeLac-empowered platforms in chemical proteomics are widely adaptable for measuring the live-cell action of reactive molecules.

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Quantitative Ratiometric pH Imaging Using a 1,2-Dioxetane Chemiluminescence Resonance Energy Transfer Sensor

10.26434/chemrxiv.12235361 ◽

2020 ◽

Author(s):

Lucas S. Ryan ◽

Jeni Gerberich ◽

Uroob Haris ◽

ralph mason ◽

Alexander Lippert

Keyword(s):

Energy Transfer ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Whole Body ◽

Photon Flux ◽

Ph Homeostasis ◽

Ph Imaging ◽

Confounding Variables ◽

Significant Difference

Regulation of physiological pH is integral for proper whole-body and cellular function, and disruptions in pH homeostasis can be both a cause and effect of disease. In light of this, many methods have been developed to monitor pH in cells and animals. In this study, we report a chemiluminescence resonance energy transfer (CRET) probe Ratio-pHCL-1, comprised of an acrylamide 1,2-dioxetane chemiluminescent scaffold with an appended pH-sensitive carbofluorescein fluorophore. The probe provides an accurate measurement of pH between 6.8-8.4, making it viable tool for measuring pH in biological systems. Further, its ratiometric output is independent of confounding variables. Quantification of pH can be accomplished both using common fluorimetry and advanced optical imaging methods. Using an IVIS Spectrum, pH can be quantified through tissue with Ratio-pHCL-1, which has been shown in vitro and precisely calibrated in sacrificed mouse models. Initial studies showed that intraperitoneal injections of Ratio-pHCL-1 into sacrificed mice produce a photon flux of more than 10^10 photons per second, and showed a significant difference in ratio of emission intensities between pH 6.0, 7.0, and 8.0.

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