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Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca sensing.
Yang J, Cumberbatch D, Centanni S, Shi SQ, Winder D, Webb D, Johnson CH
(2016) Nat Commun 7: 13268
MeSH Terms: Animals, Batrachoidiformes, Calcium, Fluorescence Resonance Energy Transfer, HEK293 Cells, HeLa Cells, Humans, Luciferases, Luminescence, Luminescent Measurements, Microscopy, Fluorescence, Optogenetics
Show Abstract · Added March 26, 2019
Optogenetic techniques allow intracellular manipulation of Ca by illumination of light-absorbing probe molecules such as channelrhodopsins and melanopsins. The consequences of optogenetic stimulation would optimally be recorded by non-invasive optical methods. However, most current optical methods for monitoring Ca levels are based on fluorescence excitation that can cause unwanted stimulation of the optogenetic probe and other undesirable effects such as tissue autofluorescence. Luminescence is an alternate optical technology that avoids the problems associated with fluorescence. Using a new bright luciferase, we here develop a genetically encoded Ca sensor that is ratiometric by virtue of bioluminescence resonance energy transfer (BRET). This sensor has a large dynamic range and partners optimally with optogenetic probes. Ca fluxes that are elicited by brief pulses of light to cultured cells expressing melanopsin and to neurons-expressing channelrhodopsin are quantified and imaged with the BRET Ca sensor in darkness, thereby avoiding undesirable consequences of fluorescence irradiation.
0 Communities
1 Members
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MeSH Terms
Non-invasive bioluminescence imaging of β-cell function in obese-hyperglycemic [ob/ob] mice.
Patel M, Gleason A, O'Malley S, Connolly B, Suresch D, Virostko J, Phillips N, Lin SA, Chen TB, Klimas M, Hargreaves RJ, Sur C, Williams DL, Powers AC, Bednar B
(2014) PLoS One 9: e106693
MeSH Terms: Animals, Diabetes Mellitus, Type 2, Hyperglycemia, Islets of Langerhans, Luminescence, Mice, Obesity
Show Abstract · Added January 20, 2015
BACKGROUND - Type 2 diabetes results from failure of the β-cells to compensate for increased insulin demand due to abnormal levels of metabolic factors. The ob/ob(lep-/-) mouse has been extensively studied as an animal model of type 2 diabetes. Previous studies have shown a correlation between β-cell function and bioluminescent imaging in lean genetically engineered mice. The ability to noninvasively monitor β-cell function in ob/ob mice could provide new information on β-cell regulation in type 2 diabetes.
METHODS - To create the B6 Albino ob/ob MIP-luc mice (ob/ob-luc), the ob/ob mouse was crossed with the CD1 MIP-luc mouse. All mice were backcrossed over multiple generations to ensure the genetic background of the transgenic mice was over 96% similar to the background of the original ob/ob mouse. Animal weight, blood glucose levels, insulin in plasma, and in vivo bioluminescence (BLI) were monitored weekly or biweekly for up to 70 weeks of age. BL imaging was performed using IVIS Spectrum (Perkin Elmer) and calculated by integrating the bioluminescence signal between 5 and 10 min after i.v. injection of D-luciferin. Insulin immunohistochemistry determined islet beta cell count and insulin secretion assay determined islet insulin function.
RESULTS - There were significant increases in BLI and insulin levels as the ob/ob-luc mice aged while glucose levels gradually decreased. Ob/ob-luc were sacrificed at different time points to determine ex vivo BLI, islet function and total β-cell numbers using a cell counting training algorithm developed for the Vectra image analysis system (Perkin Elmer). The number of β-cells increased as the mice aged and all three ex vivo measurements correlated with BLI.
CONCLUSIONS - The ob/ob-luc mice can serve as a model of metabolic stress, similar to human type 2 diabetes using BLI as a surrogate marker for β-cell function.
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7 MeSH Terms
Secreted Gaussia princeps luciferase as a reporter of Escherichia coli replication in a mouse tissue cage model of infection.
Liu M, Blinn C, McLeod SM, Wiseman JW, Newman JV, Fisher SL, Walkup GK
(2014) PLoS One 9: e90382
MeSH Terms: Animals, Biocatalysis, Chromosomes, Bacterial, Colony Count, Microbial, Copepoda, Disease Models, Animal, Erwinia, Escherichia coli, Escherichia coli Infections, Genes, Reporter, Genetic Loci, Imaging, Three-Dimensional, Luciferases, Luminescence, Mice, Polysaccharide-Lyases
Show Abstract · Added March 20, 2014
Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.
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16 MeSH Terms
Synthesis of brightly PEGylated luminescent magnetic upconversion nanophosphors for deep tissue and dual MRI imaging.
Chen H, Qi B, Moore T, Colvin DC, Crawford T, Gore JC, Alexis F, Mefford OT, Anker JN
(2014) Small 10: 160-8
MeSH Terms: Contrast Media, Diagnostic Imaging, Luminescence, Magnetic Resonance Imaging, Nanoparticles, Polyethylene Glycols
Show Abstract · Added March 7, 2014
A method is developed to fabricate monodispersed biocompatible Yb/Er or Yb/Tm doped β-NaGdF4 upconversion phosphors using polyelectrolytes to prevent irreversible particle aggregation during conversion of the precursor, Gd2 O(CO3 )2.H2 O:Yb/Er or Yb/Tm, to β-NaGdF4 :Yb/Er or Yb/Tm. The polyelectrolyte on the outer surface of nanophosphors also provided an amine tag for PEGylation. This method is also employed to fabricate PEGylated magnetic upconversion phosphors with Fe3 O4 as the core and β-NaGdF4 as a shell. These magnetic upconversion nanophosphors have relatively high saturation magnetization (7.0 emu g(-1) ) and magnetic susceptibility (1.7 × 10(-2) emu g(-1) Oe(-1) ), providing them with large magnetophoretic mobilities. The magnetic properties for separation and controlled release in flow, their optical properties for cell labeling, deep tissue imaging, and their T1 - and T2 -weighted magnetic resonance imaging (MRI) relaxivities are studied. The magnetic upconversion phosphors display both strong magnetophoresis, dual MRI imaging (r1 = 2.9 mM(-1) s(-1) , r2 = 204 mM(-1) s(-1) ), and bright luminescence under 1 cm chicken breast tissue.
Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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1 Members
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6 MeSH Terms
pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.
Zhang Y, Xie Q, Robertson JB, Johnson CH
(2012) PLoS One 7: e43072
MeSH Terms: Cell Line, Cytoplasm, Cytosol, Energy Transfer, Fluorescence Resonance Energy Transfer, Fluorescent Dyes, HeLa Cells, Humans, Hydrogen-Ion Concentration, Luminescence, Luminescent Proteins, Saccharomyces cerevisiae, Sodium Fluoride, Time Factors
Show Abstract · Added February 12, 2015
We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.
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14 MeSH Terms
Electrochemical detection of catecholamine release using planar iridium oxide electrodes in nanoliter microfluidic cell culture volumes.
Ges IA, Currie KP, Baudenbacher F
(2012) Biosens Bioelectron 34: 30-6
MeSH Terms: Aptamers, Nucleotide, Biosensing Techniques, Catecholamines, Chromaffin Cells, Electrochemical Techniques, Equipment Design, Exocytosis, Humans, Iridium, Luminescence, Microelectrodes, Microfluidics, Thrombin
Show Abstract · Added March 30, 2013
Release of neurotransmitters and hormones by calcium regulated exocytosis is a fundamental cellular/molecular process that is disrupted in a variety of psychiatric, neurological, and endocrine disorders. Therefore, this area represents a relevant target for drug and therapeutic development, efforts that will be aided by novel analytical tools and devices that provide mechanistically rich data with increased throughput. Toward this goal, we have electrochemically deposited iridium oxide (IrOx) films onto planar thin film platinum electrodes (20 μm×300 μm) and utilized these for quantitative detection of catecholamine release from adrenal chromaffin cells trapped in a microfluidic network. The IrOx electrodes show a linear response to norepinephrine in the range of 0-400 μM, with a sensitivity of 23.1±0.5 mA/M mm(2). The sensitivity of the IrOx electrodes does not change in the presence of ascorbic acid, a substance commonly found in biological samples. A replica molded polydimethylsiloxane (PDMS) microfluidic device with nanoliter sensing volumes was aligned and sealed to a glass substrate with the sensing electrodes. Small populations of chromaffin cells were trapped in the microfluidic device and stimulated by rapid perfusion with high potassium (50mM) containing Tyrode's solution at a flow rate of 1 nL/s. Stimulation of the cells produced a rapid increase in current due to oxidation of the released catecholamines, with an estimated maximum concentration in the cell culture volume of ~52 μM. Thus, we demonstrate the utility of an integrated microfluidic network with IrOx electrodes for real-time quantitative detection of catecholamines released from small populations of chromaffin cells.
Copyright © 2011 Elsevier B.V. All rights reserved.
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13 MeSH Terms
Multimodal image coregistration and inducible selective cell ablation to evaluate imaging ligands.
Virostko J, Henske J, Vinet L, Lamprianou S, Dai C, Radhika A, Baldwin RM, Ansari MS, Hefti F, Skovronsky D, Kung HF, Herrera PL, Peterson TE, Meda P, Powers AC
(2011) Proc Natl Acad Sci U S A 108: 20719-24
MeSH Terms: Animals, Diabetes Mellitus, Diagnostic Imaging, Female, Humans, Insulin, Insulin-Secreting Cells, Islets of Langerhans, Ligands, Luminescence, Male, Mice, Mice, Inbred NOD, Positron-Emission Tomography, Rats, Tissue Distribution, Tomography, X-Ray Computed
Show Abstract · Added December 5, 2013
We combined multimodal imaging (bioluminescence, X-ray computed tomography, and PET), tomographic reconstruction of bioluminescent sources, and two unique, complementary models to evaluate three previously synthesized PET radiotracers thought to target pancreatic beta cells. The three radiotracers {[(18)F]fluoropropyl-(+)-dihydrotetrabenazine ([(18)F]FP-DTBZ), [(18)F](+)-2-oxiranyl-3-isobutyl-9-(3-fluoropropoxy)-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinoline ((18)F-AV-266), and (2S,3R,11bR)-9-(3-fluoropropoxy)-2-(hydroxymethyl)-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol ((18)F-AV-300)} bind vesicular monoamine transporter 2. Tomographic reconstruction of the bioluminescent signal in mice expressing luciferase only in pancreatic beta cells was used to delineate the pancreas and was coregistered with PET and X-ray computed tomography images. This strategy enabled unambiguous identification of the pancreas on PET images, permitting accurate quantification of the pancreatic PET signal. We show here that, after conditional, specific, and rapid mouse beta-cell ablation, beta-cell loss was detected by bioluminescence imaging but not by PET imaging, given that the pancreatic signal provided by three PET radiotracers was not altered. To determine whether these ligands bound human beta cells in vivo, we imaged mice transplanted with luciferase-expressing human islets. The human islets were imaged by bioluminescence but not with the PET ligands, indicating that these vesicular monoamine transporter 2-directed ligands did not specifically bind beta cells. These data demonstrate the utility of coregistered multimodal imaging as a platform for evaluation and validation of candidate ligands for imaging islets.
2 Communities
3 Members
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17 MeSH Terms
Luminescence as a continuous real-time reporter of promoter activity in yeast undergoing respiratory oscillations or cell division rhythms.
Robertson JB, Johnson CH
(2011) Methods Mol Biol 734: 63-79
MeSH Terms: Aerobiosis, Bioreactors, Cell Culture Techniques, Cell Division, Cells, Cultured, Genes, Reporter, Luminescence, Promoter Regions, Genetic, Saccharomyces cerevisiae, Time Factors
Show Abstract · Added February 12, 2015
This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces cerevisiae that has been genetically modified to express firefly luciferase under the control of a promoter of interest and a continuous culture bioreactor that incorporates a photomultiplier apparatus for detecting light emission. Additionally, this chapter describes a method for observing rhythmic (cell cycle-related) promoter activity in small batch cultures of yeast through luminescence monitoring.
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10 MeSH Terms
White light-emitting diodes based on ultrasmall CdSe nanocrystal electroluminescence.
Schreuder MA, Xiao K, Ivanov IN, Weiss SM, Rosenthal SJ
(2010) Nano Lett 10: 573-6
MeSH Terms: Cadmium Compounds, Crystallization, Electrochemistry, Equipment Design, Light, Luminescence, Nanoparticles, Nanotechnology, Selenium Compounds, Temperature
Show Abstract · Added May 27, 2014
We report white light-emitting diodes fabricated with ultrasmall CdSe nanocrystals, which demonstrate electroluminescence from a size of nanocrystals (<2 nm) previously thought to be unattainable. These LEDs have excellent color characteristics, defined by their pure white CIE color coordinates (0.333, 0.333), correlated color temperatures of 5461-6007 K, and color rendering indexes as high as 96.6. The effect of high voltage on the trap states responsible for the white emission is also described.
0 Communities
2 Members
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10 MeSH Terms
Validation of bioluminescent imaging techniques.
Virostko J, Jansen ED
(2009) Methods Mol Biol 574: 15-23
MeSH Terms: Animals, Biological Availability, Fireflies, Luciferases, Luminescence, Pharmacokinetics, Substrate Specificity
Show Abstract · Added January 20, 2015
Bioluminescence imaging (BLI) is frequently cited for its ease of quantification. This fundamental strength of BLI has led to applications in cancer research, cell transplantation, and monitoring of infectious disease in which bioluminescence intensity is correlated with other metrics. However, bioluminescence measurements can be influenced by a number of factors, among them source location, tissue optical properties, and substrate availability and pharmacokinetics. Accounting for these many factors is crucial for accurate BLI quantification. A number of methods can be employed to ensure correct interpretation of BLI results and validate BLI techniques. This chapter summarizes the use of calibrated light-emitting standards, bioluminescence tomography, and post-mortem validation of luciferase expression for validating quantitative BLI measurements.
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7 MeSH Terms