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

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

<p>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.</p> <b></b><i></i><u></u><sub></sub><sup></sup><br>

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

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

<p>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.</p> <b></b><i></i><u></u><sub></sub><sup></sup><br>

scholarly journals Förster Resonance Energy Transfer-Based Stability Assessment of PLGA Nanoparticles in Vitro and in Vivo

2019 ◽  
Vol 2 (3) ◽  
pp. 1131-1140 ◽  
Author(s):  
Edyta Swider ◽  
Sanish Maharjan ◽  
Karlijne Houkes ◽  
Nicolaas Koen van Riessen ◽  
Carl Figdor ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Plga Nanoparticles ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Stability Assessment ◽  
Förster Resonance

scholarly journals Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

2006 ◽  
Vol 20 (6) ◽  
pp. 1218-1230 ◽  
Author(s):  
Alicja J. Copik ◽  
M. Scott Webb ◽  
Aaron L. Miller ◽  
Yongxin Wang ◽  
Raj Kumar ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Glucocorticoid Receptor ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Function ◽  
Tata Binding Protein

Abstract The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.

scholarly journals In Vivo Biosensing Using Resonance Energy Transfer

Biosensors ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 76 ◽  
Author(s):  
Shashi Bhuckory ◽  
Joshua C. Kays ◽  
Allison M. Dennis
Keyword(s):  
Energy Transfer ◽  
Fluorescent Proteins ◽  
Low Cost ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optical Methods ◽  
Great Promise ◽  
Molecular Processes

Solution-phase and intracellular biosensing has substantially enhanced our understanding of molecular processes foundational to biology and pathology. Optical methods are favored because of the low cost of probes and instrumentation. While chromatographic methods are helpful, fluorescent biosensing further increases sensitivity and can be more effective in complex media. Resonance energy transfer (RET)-based sensors have been developed to use fluorescence, bioluminescence, or chemiluminescence (FRET, BRET, or CRET, respectively) as an energy donor, yielding changes in emission spectra, lifetime, or intensity in response to a molecular or environmental change. These methods hold great promise for expanding our understanding of molecular processes not just in solution and in vitro studies, but also in vivo, generating information about complex activities in a natural, organismal setting. In this review, we focus on dyes, fluorescent proteins, and nanoparticles used as energy transfer-based optical transducers in vivo in mice; there are examples of optical sensing using FRET, BRET, and in this mammalian model system. After a description of the energy transfer mechanisms and their contribution to in vivo imaging, we give a short perspective of RET-based in vivo sensors and the importance of imaging in the infrared for reduced tissue autofluorescence and improved sensitivity.

Analisis of A-Kinase Anchoring Protein Interactions With PKA Using Fluorescence Resonance Energy Transfer

2000 ◽  
Vol 6 (S2) ◽  
pp. 828-829
Author(s):  
M. L. Ruehr ◽  
D. S. Damron ◽  
M. Bond
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Regulatory Subunit ◽  
Live Cells ◽  
Human Thyroid ◽  
Local Concentration ◽  
Fluorescence Resonance

The clustering of components of a signaling pathway at a specific subcellular location raises the local concentration of the appropriate messengers and serves to amplify the signal. The cAMP dependent-protein kinase (PKA) pathway is regulated by compartmentalization of its components. A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular sites, thus presumably increasing substrate specificity. Phosphorylation of the type II regulatory subunit of PKA (RII) increases its affinity for AKAPs in vitro (1). The purpose of this study was to investigate whether altering the phosphorylation state of RII in live cells changes its affinity for an AKAP. Specifically, we investigated the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid (2), and RII, in response to PKA activators or inhibitors.Fluorescence resonance energy transfer (FRET) was used to monitor binding events between RII and the catalytic subunit (C) of PKA, Ht31, or Ht31P, a mutated form of Ht31 which does not bind RII.

Sign in / Sign up

Export Citation Format

Share Document