Use of a-SiC:H multilayer transducers for detection of fluorescence signals from reactive cyan and yellow fluorophores

2011 ◽  
Vol 1321 ◽  
Author(s):  
P. Louro ◽  
M. Vieira ◽  
M. A. Vieira ◽  
J. Costa ◽  
M. Fernandes ◽  
...  
Keyword(s):  
Spectral Response ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optical Filters ◽  
Electrical Model ◽  
Excitation Light ◽  
Independent Test ◽  
Opposite Behavior ◽  
Blue Background ◽  
Insight Into

ABSTRACTThe transducer consists of a p-i’(a-SiC:H)-n/p-i(a-Si:H)-n heterostructures produced by PECVD and optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet(400 nm) and emissions in the cyan (470 nm) and yellow (588 nm) range of the spectrum. The thickness and the absorption coefficient of the i’- and i- layers were tailored for cyan and yellow optical confinement, respectively in the front and back photodiodes acting both as optical filters. The devices were characterized through transmittance and spectral response measurements and under different electrical.To simulate the FRET pairs and the excitation light a chromatic time dependent combination of violet, cyan and yellow wavelengths was applied to the device. The generated photocurrent was measured under negative and positive bias to readout the combined spectra. The independent test signals were chosen in order to sample all the possible chromatic. Different wavelength backgrounds were also superimposed.Results show that under negative bias the phorocurrent signal presents eight separate levels each one assigned to the different polychromatic mixtures. If a blue background is superimposed the yellow channel is enhanced and the cyan suppressed while under red irradiation the opposite behavior occurs. So under appropriated steady state optical bias the sensor will detect separately the cyan and yellow fluorescence pairs. An electrical model, supported by a numerical simulation, gives insight into the transduction mechanism.

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

A Small Molecule – Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence

2020 ◽  
Author(s):  
Brittany Benlian ◽  
Pavel Klier ◽  
Kayli Martinez ◽  
Marie Schwinn ◽  
Thomas Kirkland ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Small Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Potential Oscillations ◽  
Excitation Light ◽  
Voltage Imaging ◽  
Induced Pluripotent

<p>We report a small molecule enzyme pair for optical voltage sensing via quenching of bioluminescence. This <u>Q</u>uenching <u>B</u>ioluminescent V<u>olt</u>age Indicator, or Q-BOLT, pairs the dark absorbing, voltage-sensitive dipicrylamine with membrane-localized bioluminescence from the luciferase NanoLuc (NLuc). As a result, bioluminescence is quenched through resonance energy transfer (QRET) as a function of membrane potential. Fusion of HaloTag to NLuc creates a two-acceptor bioluminescence resonance energy transfer (BRET) system when a tetramethylrhodamine (TMR) HaloTag ligand is ligated to HaloTag. In this mode, Q-BOLT is capable of providing direct visualization of changes in membrane potential in live cells via three distinct readouts: change in QRET, BRET, and the ratio between bioluminescence emission and BRET. Q-BOLT can provide up to a 29% change in bioluminescence (ΔBL/BL) and >100% ΔBRET/BRET per 100 mV change in HEK 293T cells, without the need for excitation light. In cardiac monolayers derived from human induced pluripotent stem cells (hiPSC), Q-BOLT readily reports on membrane potential oscillations. Q-BOLT is the first example of a hybrid small molecule – protein voltage indicator that does not require excitation light and may be useful in contexts where excitation light is limiting.</p> <p> </p>

scholarly journals Elucidation and refinement of synthetic receptor mechanisms

2020 ◽  
Author(s):  
Hailey I. Edelstein ◽  
Patrick S. Donahue ◽  
Joseph J. Muldoon ◽  
Anthony K. Kang ◽  
Taylor B. Dolberg ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Functional Performance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Performance Characteristics ◽  
Therapeutic Gene ◽  
High Performing ◽  
Synthetic Receptor ◽  
And Performance ◽  
Insight Into

ABSTRACTSynthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer (FRET)-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.

Meso-Tetrapyrenylporphyrins: Synthesis, structural, spectral, electrochemical properties and Förster energy transfer (FRET) studies

2020 ◽  
Vol 24 (05n07) ◽  
pp. 985-992
Author(s):  
Tawseef Ahmad Dar ◽  
Amir Sohel Bulbul ◽  
Muniappan Sankar ◽  
Karl M. Kadish
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Redox Potentials ◽  
Free Base ◽  
Efficient Energy Transfer ◽  
Förster Energy Transfer ◽  
Porphyrin Core ◽  
Transfer Properties ◽  
Insight Into

Meso-tetrapyrenylporphyrin and its metal (Co[Formula: see text], Cu[Formula: see text], Ni[Formula: see text] and Zn[Formula: see text]) complexes were synthesized, characterized and studied for their spectral, electrochemical and energy transfer properties. DFT optimization was carried out to gain an insight into the interactions between the porphyrin [Formula: see text]-system and the pyrenyl substituents. The pyrenyl substituents and the porphyrin core remain essentially orthogonal to each other in both the free base and the metallated porphyrins. Redox potentials of the pyrenylporphyrins are marginally shifted as compared to their corresponding phenyl derivatives. Förster resonance energy transfer (FRET) studies were carried out in toluene for free-base pyrenylporphyrin and its Zn(II) complex. Since pyrene is a good donor, an efficient energy transfer from pyrene (D) to the porphyrin core (A) on the order of 80–85% was observed for these two compounds. It was observed that energy transfer occurs mainly via ”through-bond” (TB) interaction rather than ”through-space” (TS) interaction.

Mechanosensing machinery for cells under low substratum rigidity

2008 ◽  
Vol 295 (6) ◽  
pp. C1579-C1589 ◽  
Author(s):  
Wei-Chun Wei ◽  
Hsi-Hui Lin ◽  
Meng-Ru Shen ◽  
Ming-Jer Tang
Keyword(s):  
Physical Environment ◽  
Resonance Energy Transfer ◽  
Collagen Gel ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Mechanical Stimuli ◽  
Integrin Activation ◽  
Integrin Clustering ◽  
Dose Dependent ◽  
Insight Into

Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates β1-integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered β1-integrin (V737N), but not constitutively active β1-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess β1-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers β1-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of β1-integrin clustering enhances the interaction between β1-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.

scholarly journals Jump-starting kinesin

2007 ◽  
Vol 176 (1) ◽  
pp. 7-9 ◽  
Author(s):  
David D. Hackney
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Active Species ◽  
Light Chains ◽  
Interacting Protein ◽  
Heavy Chains ◽  
Structural Insight ◽  
Conventional Kinesin ◽  
Related Paper ◽  
Insight Into

When it is not actively transporting cargo, conventional Kinesin-1 is present in the cytoplasm in a folded conformation that cannot interact effectively with microtubules (MTs). Two important and largely unexplored aspects of kinesin regulation are how it is converted to an active species when bound to cargo and the related issue of how kinesin discriminates among its many potential cargo molecules. Blasius et al. (see p. 11 of this issue) report that either binding of the cargo linker c-Jun N-terminal kinase–interacting protein 1 (JIP1) to the light chains (LCs) or binding of fasciculation and elongation protein ζ1 (FEZ1) to the heavy chains (HCs) is insufficient for activation but that activation occurs when both are present simultaneously. A related paper by Cai et al. (see p. 51 of this issue) provides structural insight into the conformation of the folded state in the cell obtained by fluorescence resonance energy transfer analysis.

scholarly journals Oligomeric Architecture of Mouse Activating Nkrp1 Receptors on Living Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 1884 ◽  
Author(s):  
Ljubina Adámková ◽  
Zuzana Kvíčalová ◽  
Daniel Rozbeský ◽  
Zdeněk Kukačka ◽  
David Adámek ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Lymphoid Cells ◽  
Protein Isoforms ◽  
Major Influence ◽  
Protein Dimerization ◽  
Transmembrane Receptors ◽  
Insight Into

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

scholarly journals Protein conformation: through a lens, darkly

2003 ◽  
Vol 372 (1) ◽  
pp. e1-e2
Author(s):  
Robert INSALL
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Protein Conformation ◽  
Fluorescent Proteins ◽  
Protein Kinase B ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Protein ◽  
Insight Into ◽  
Study Offer

This commentary describes the study by Calleja and co-workers, who have used fluorescence resonance energy transfer between two fluorescent proteins fused to one protein kinase (protein kinase B/Akt) to reveal conformational changes in a functional protein. The findings of their study offer an intriguing insight into the behaviour of an enzyme that is essential for multiple aspects of mammalian signalling.

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