scholarly journals A Software Tool for High-Throughput Real-Time Measurement of Intensity-Based Ratio-Metric FRET

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1541 ◽  
Author(s):  
Masoud Ramuz ◽  
Alveera Hasan ◽  
Lena Gruscheski ◽  
Ivan Diakonov ◽  
Nikoleta Pavlaki ◽  
...  
Keyword(s):  
Real Time ◽  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Induced Pluripotent Stem Cell ◽  
Software Tool ◽  
Live Cells ◽  
Non Invasive ◽  
Significant Difference ◽  
Induced Pluripotent

Förster resonance energy transfer (FRET) is increasingly used for non-invasive measurement of fluorescently tagged molecules in live cells. In this study, we have developed a freely available software tool MultiFRET, which, together with the use of a motorised microscope stage, allows multiple single cells to be studied in one experiment. MultiFRET is a Java plugin for Micro-Manager software, which provides real-time calculations of ratio-metric signals during acquisition and can simultaneously record from multiple cells in the same experiment. It can also make other custom-determined live calculations that can be easily exported to Excel at the end of the experiment. It is flexible and can work with multiple spectral acquisition channels. We validated this software by comparing the output of MultiFRET to that of a previously established and well-documented method for live ratio-metric FRET experiments and found no significant difference between the data produced with the use of the new MultiFRET and other methods. In this validation, we used several cAMP FRET sensors and cell models: i) isolated adult cardiomyocytes from transgenic mice expressing the cytosolic epac1-camps and targeted pmEpac1 and Epac1-PLN sensors, ii) isolated neonatal mouse cardiomyocytes transfected with the AKAP79-CUTie sensor, and iii) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) transfected with the Epac-SH74 sensor. The MultiFRET plugin is an open source freely available package that can be used in a wide area of live cell imaging when live ratio-metric calculations are required.

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 AIE-Driven Fluorescent Polysaccharide Polymersome as Enzyme-responsive FRET Nanoprobe to Study the Real-time Delivery Aspects in Live-Cells

2021 ◽  
Author(s):  
Nilesh Umakant Deshpande ◽  
Mishika Virmani ◽  
Manickam Jayakannan
Keyword(s):  
Energy Transfer ◽  
Confocal Microscopy ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Imaging Techniques ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Intracellular Enzyme ◽  
Bio Imaging

We report aggregation induced emission (AIE) driven polysaccharide polymersome as fluorescence resonance energy transfer (FRET) nanoprobes to study their intracellular enzyme-responsive delivery by real-time live-cell confocal microscopy bio-imaging techniques. AIE...

scholarly journals A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells

2020 ◽  
Vol 21 (7) ◽  
pp. 2572 ◽  
Author(s):  
Urooj Fatima ◽  
Mohammad K. Okla ◽  
Mohd Mohsin ◽  
Ruphi Naz ◽  
Walid Soufan ◽  
...  
Keyword(s):  
Metabolic Network ◽  
Real Time ◽  
Fluorescent Proteins ◽  
Essential Element ◽  
Resonance Energy ◽  
Primary Source ◽  
Essential Amino Acids ◽  
Living Cells ◽  
Live Cells ◽  
Non Invasive

Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N- and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.

scholarly journals Highly multiplexed imaging of biosensors in live cells

2020 ◽  
Author(s):  
Jr-Ming Yang ◽  
Wei-Yu Chi ◽  
Jessica Liang ◽  
Pablo Iglesias ◽  
Chuan-Hsiang Huang
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Spectral Space ◽  
Fluorescent Biosensors ◽  
Multiplexed Imaging ◽  
Mixed Populations

AbstractFluorescent biosensors allow for real-time monitoring of biochemical activities in cells, but their multiplexing capacity is severely limited by the availability of spectral space. We overcome this problem by developing a set of barcoding proteins that are spectrally separable from commonly used FRET (fluorescence resonance energy transfer)-based and single-fluorophore biosensors. Mixed populations of barcoded cells expressing different biosensors can be concurrently imaged and computationally unmixed to achieve highly multiplexed tracking of biochemical activities in live cells.

scholarly journals Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 206
Author(s):  
Irene Carlon-Andres ◽  
Sergi Padilla-Parra
Keyword(s):  
Single Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Novel Approach ◽  
Different Populations ◽  
Hiv Envelope ◽  
Hiv 1

The first steps of human immunodeficiency virus (HIV) infection go through the engagement of HIV envelope (Env) with CD4 and coreceptors (CXCR4 or CCR5) to mediate viral membrane fusion between the virus and the host. New approaches are still needed to better define both the molecular mechanistic underpinnings of this process but also the point of fusion and its kinetics. Here, we have developed a new method able to detect and quantify HIV-1 fusion in single live cells. We present a new approach that employs fluorescence lifetime imaging microscopy (FLIM) to detect Förster resonance energy transfer (FRET) when using the β-lactamase (BlaM) assay. This novel approach allows comparing different populations of single cells regardless the concentration of CCF2-AM FRET reporter in each cell, and more importantly, is able to determine the relative amount of viruses internalized per cell. We have applied this approach in both reporter TZM-bl cells and primary T cell lymphocytes.

scholarly journals cAMP Biosensors Based on Genetically Encoded Fluorescent/Luminescent Proteins

Biosensors ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 39
Author(s):  
Namdoo Kim ◽  
Seunghan Shin ◽  
Se Won Bae
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Intracellular Signaling ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Early Stage ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adenosine Monophosphate ◽  
Non Invasive

Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders. For example, modulation of cAMP-mediated intracellular signaling pathways by anti-tumor drugs could reduce tumor growth. However, most early stage tools used for measuring the cAMP level in living organisms require cell disruption, which is not appropriate for live cell imaging or animal imaging. Thus, in the last decades, tools were developed for real-time monitoring of cAMP distribution or signaling dynamics in a non-invasive manner. Genetically-encoded sensors based on fluorescent proteins and luciferases could be powerful tools to overcome these drawbacks. In this review, we discuss the recent genetically-encoded cAMP sensors advances, based on single fluorescent protein (FP), Föster resonance energy transfer (FRET), single luciferase, and bioluminescence resonance energy transfer (BRET) for real-time non-invasive imaging.

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>

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