Signal/noise analysis of FRET-based sensors.

Abstract:

:Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Woehler A,Wlodarczyk J,Neher E

doi

10.1016/j.bpj.2010.07.053

subject

Has Abstract

pub_date

2010-10-06 00:00:00

pages

2344-54

issue

7

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(10)00925-2

journal_volume

99

pub_type

杂志文章
  • Characteristics of a simple, high-resolution flow cytometer based o a new flow configuration.

    abstract::A new flow configuration allows a flow cytometer of high resolution and stability to be assembled from an inverted fluorescence microscope with incident illumination, a pulse photometer, and a multichannel pulse height analyzer. A nozzle produces a hydrodynamically focused sample stream in a liquid jet that id directe...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(79)85157-7

    authors: Lindmo T,Steen HB

    更新日期:1979-10-01 00:00:00

  • A physical model of ATP-induced actin-myosin movement in vitro.

    abstract::The nature of the mechanism limiting the velocity of ATP-induced unidirectional movements of actin-myosin filaments in vitro is considered. In the sliding process two types of "cyclic" interactions between myosin heads and actin are involved, i.e., productive and nonproductive. In the productive interaction, myosin he...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(91)82228-7

    authors: Tawada K,Sekimoto K

    更新日期:1991-02-01 00:00:00

  • Carbohydrate conformation and lipid condensation in monolayers containing glycosphingolipid Gb3: influence of acyl chain structure.

    abstract::Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2014.07.023

    authors: Watkins EB,Gao H,Dennison AJC,Chopin N,Struth B,Arnold T,Florent JC,Johannes L

    更新日期:2014-09-02 00:00:00

  • Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles.

    abstract::Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid dis...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2019.01.016

    authors: Doktorova M,Heberle FA,Marquardt D,Rusinova R,Sanford RL,Peyear TA,Katsaras J,Feigenson GW,Weinstein H,Andersen OS

    更新日期:2019-03-05 00:00:00

  • KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels.

    abstract::Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 pot...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(03)74585-8

    authors: Grunnet M,Rasmussen HB,Hay-Schmidt A,Rosenstierne M,Klaerke DA,Olesen SP,Jespersen T

    更新日期:2003-09-01 00:00:00

  • Characterization of the effect of the mitochondrial protein Hint2 on intracellular Ca(2+) dynamics.

    abstract::Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca(2+) handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affec...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2013.06.048

    authors: Ndiaye D,Collado-Hilly M,Martin J,Prigent S,Dufour JF,Combettes L,Dupont G

    更新日期:2013-09-03 00:00:00

  • X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

    abstract::We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms....

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(98)77856-7

    authors: Tregear RT,Edwards RJ,Irving TC,Poole KJ,Reedy MC,Schmitz H,Towns-Andrews E,Reedy MK

    更新日期:1998-03-01 00:00:00

  • Contrasting factors on the kinetic path to protein complex formation diminish the effects of crowding agents.

    abstract::The crowded environment of cells poses a challenge for rapid protein-protein association. Yet, it has been established that the rates of association are similar in crowded and in dilute solutions. Here we probe the pathway leading to fast association between TEM1 β-lactamase and its inhibitor protein BLIP in crowded s...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2012.08.009

    authors: Phillip Y,Harel M,Khait R,Qin S,Zhou HX,Schreiber G

    更新日期:2012-09-05 00:00:00

  • The alignment of a voltage-sensing peptide in dodecylphosphocholine micelles and in oriented lipid bilayers by nuclear magnetic resonance and molecular modeling.

    abstract::The S4 segments of voltage-gated sodium channels are important parts of the voltage-sensing elements of these proteins. Furthermore, the addition of the isolated S4 polypeptide to planar lipid bilayers results in stepwise increases of ion conductivity. In order to gain insight into the mechanisms of pore formation by ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(99)77051-7

    authors: Mattila K,Kinder R,Bechinger B

    更新日期:1999-10-01 00:00:00

  • Large Scale Global Structural Changes of the Purple Membrane during the Photocycle.

    abstract::Both the solution and the oriented film absorption and circular dichroic spectra of the bacteriorhodopsin (bR(568)) and M(412) intermediate of the purple membrane photocycle were compared over the wavelength region 800-183 nm to assess structural changes during this photocycle. The main findings are (a) loss of the ex...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(85)83943-6

    authors: Draheim JE,Cassim JY

    更新日期:1985-04-01 00:00:00

  • Orientation and dynamics of synthetic transbilayer polypeptides containing GpATM dimerization motifs.

    abstract::Deuterium NMR spectroscopy was used to study how the positioning of a dimerization motif within a transbilayer polypeptide influences its orientation and dynamics in bilayers. Three polypeptide variants comprising glycophorin A transmembrane (GpATM) dimerization motifs incorporated into lysine-terminated poly-leucine-...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2010.12.3725

    authors: McDonald MC,Booth V,Morrow MR

    更新日期:2011-02-02 00:00:00

  • Models of electrical activity: calibration and prediction testing on the same cell.

    abstract::Mathematical models are increasingly important in biology, and testability is becoming a critical issue. One limitation is that one model simulation tests a parameter set representing one instance of the biological counterpart, whereas biological systems are heterogeneous in their properties and behavior, and a model ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2012.09.034

    authors: Tomaiuolo M,Bertram R,Leng G,Tabak J

    更新日期:2012-11-07 00:00:00

  • The persistence length of DNA is reached from the persistence length of its null isomer through an internal electrostatic stretching force.

    abstract::To understand better the effect of electrostatics on the rigidity of the DNA double helix, we define DNA*, the null isomer of DNA, as the hypothetical structure that would result from DNA if its phosphate groups were not ionized. For the purposes of theoretical analysis, we model DNA* as identical to ordinary DNA but ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.106.089029

    authors: Manning GS

    更新日期:2006-11-15 00:00:00

  • Molecular packing of cord factor and its interaction with phosphatidylinositol in mixed monolayers.

    abstract::Cord factor (trehalose 6,6'-dimycolate, CF) is a glycolipid located in the outer mycobacterial cell wall that is implicated in the pathogenesis of mycobacteria. Furthermore, CF is a convenient model for studying mycolic acid residues, the major lipid constituents of the mycobacterial cell wall that are believed to for...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(96)79523-1

    authors: Almog R,Mannella CA

    更新日期:1996-12-01 00:00:00

  • The conformation of substance P in lipid environments.

    abstract::NMR and CD studies have been used to analyze the model membrane-bound structure of the neuropeptide substance P (RPKPQQFFGLM-NH2, SP), which has previously been proposed as the NK1 receptor active form. Conformations were determined for the SP in the presence of aqueous solutions of zwitterionic dodecylphosphocholine ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(96)79734-5

    authors: Keire DA,Fletcher TG

    更新日期:1996-04-01 00:00:00

  • Temperature dependence of IP3-mediated local and global Ca2+ signals.

    abstract::We examined the effect of temperature (12-40°C) on local and global Ca2+ signals mediated by inositol trisphosphate receptor/channels (IP3R) in human neuroblastoma (SH-SY5Y) cells. The amplitudes and spatial spread of local signals arising from single IP3R (blips) and clusters of IP3R (puffs) showed little temperature...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2012.12.024

    authors: Dickinson GD,Parker I

    更新日期:2013-01-22 00:00:00

  • Nucleation and decay initiation are the stiffness-sensitive phases of focal adhesion maturation.

    abstract::A cell plated on a two-dimensional substrate forms adhesions with that surface. These adhesions, which consist of aggregates of various proteins, are thought to be important in mechanosensation, the process by which the cell senses and responds to the mechanical properties of the substrate (e.g., stiffness). On the ba...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2011.11.010

    authors: Walcott S,Kim DH,Wirtz D,Sun SX

    更新日期:2011-12-21 00:00:00

  • DNA multiphoton absorption generates localized damage for studying repair dynamics in live cells.

    abstract::Investigations into the spatiotemporal dynamics of DNA repair using live-cell imaging are aided by the ability to generate well defined regions of ultravioletlike photolesions in an optical microscope. We demonstrate that multiphoton excitation of DNA in live cells with visible femtosecond pulses produces thymine cycl...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2011.09.031

    authors: Daddysman MK,Fecko CJ

    更新日期:2011-11-02 00:00:00

  • Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester polyethyleneglycol lipid.

    abstract::The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. Th...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/s0006-3495(03)74986-8

    authors: Guo X,MacKay JA,Szoka FC Jr

    更新日期:2003-03-01 00:00:00

  • Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop.

    abstract::The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Pe...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(01)76147-4

    authors: Gallant EM,Curtis S,Pace SM,Dulhunty AF

    更新日期:2001-04-01 00:00:00

  • Oligomerization and aggregation of bovine pancreatic ribonuclease A: characteristic events observed by FTIR spectroscopy.

    abstract::Nonnative protein aggregation, which is a common feature in biotechnology, is also a clinical feature in more than 20 serious degenerative diseases. We studied the specific events of bovine pancreatic ribonuclease A thermal aggregation by a combination of second derivative infrared analysis and two-dimensional infrare...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.105.071530

    authors: Yan YB,Zhang J,He HW,Zhou HM

    更新日期:2006-04-01 00:00:00

  • Comparative Molecular Dynamics Analysis of RNase-S Complex Formation.

    abstract::Limited proteolysis of RNase-A yields a short N-terminal S-peptide segment and the larger S-protein. Binding of S-peptide to S-protein results in the formation of an enzymatically active RNase-S protein. S-peptide undergoes a transition from intrinsic disorder to an ordered helical state upon association with S-protei...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2017.08.008

    authors: Luitz MP,Bomblies R,Zacharias M

    更新日期:2017-10-03 00:00:00

  • Structure and activation dynamics of RBL-2H3 cells observed with scanning force microscopy.

    abstract::Surface and subsurface dynamics of Rat Basophilic Leukemia cells, a model system of stimulated secretion, were imaged using Scanning Force Microscopy (SFM) at a rate of 50-60 s/image. Cytoskeletal elements and organelles were tracked within quiescent cells and those activated after IgE receptor crosslinking. In additi...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(94)80964-6

    authors: Braunstein D,Spudich A

    更新日期:1994-05-01 00:00:00

  • Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data.

    abstract::Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(00)76860-3

    authors: Mariani P,Carsughi F,Spinozzi F,Romanzetti S,Meier G,Casadio R,Bergamini CM

    更新日期:2000-06-01 00:00:00

  • How to squeeze a sponge: casein micelles under osmotic stress, a SAXS study.

    abstract::By combining the osmotic stress technique with small-angle x-ray scattering measurements, we followed the structural response of the casein micelle to an overall increase in concentration. When the aqueous phase that separates the micelles is extracted, they behave as polydisperse repelling spheres and their internal ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/j.bpj.2010.10.019

    authors: Bouchoux A,Gésan-Guiziou G,Pérez J,Cabane B

    更新日期:2010-12-01 00:00:00

  • Model-driven designs of an oscillating gene network.

    abstract::The current rapid expansion of biological knowledge offers a great opportunity to rationally engineer biological systems that respond to signals such as light and chemical inducers by producing specific proteins. Turning on and off the production of proteins on demand holds great promise for creating significant biote...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.105.064204

    authors: Tuttle LM,Salis H,Tomshine J,Kaznessis YN

    更新日期:2005-12-01 00:00:00

  • Intramural multisite recording of transmembrane potential in the heart.

    abstract::Heart surface optical mapping of transmembrane potentials has been widely used in studies of normal and pathological heart rhythms and defibrillation. In these studies, three-dimensional spatio-temporal events can only be inferred from two-dimensional surface potential maps. We present a novel optical system that enab...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1016/S0006-3495(01)75910-3

    authors: Hooks DA,LeGrice IJ,Harvey JD,Smaill BH

    更新日期:2001-11-01 00:00:00

  • Depth-resolved measurement of transient structural changes during action potential propagation.

    abstract::We report noncontact optical measurement of fast transient structural changes in the crustacean nerve during action potential propagation without the need for exogenous chemicals or reflection coatings. The technique, spectral domain optical coherence tomography, provides real-time cross-sectional images of the nerve ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.106.091298

    authors: Akkin T,Joo C,de Boer JF

    更新日期:2007-08-15 00:00:00

  • Regular gaits and optimal velocities for motor proteins.

    abstract::It has been observed in numerical experiments that adding a cargo to a motor protein can regularize its gait. Here we explain these results via asymptotic analysis on a general stochastic motor protein model. This analysis permits a computation of various observables (e.g., the mean velocity) of the motor protein and ...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.108.130674

    authors: DeVille RE,Vanden-Eijnden E

    更新日期:2008-09-15 00:00:00

  • Excitation energy-transfer and the relative orientation of retinal and carotenoid in xanthorhodopsin.

    abstract::The cell membrane of Salinibacter ruber contains xanthorhodopsin, a light-driven transmembrane proton pump with two chromophores: a retinal and the carotenoid, salinixanthin. Action spectra for transport had indicated that light absorbed by either is utilized for function. If the carotenoid is an antenna in this prote...

    journal_title:Biophysical journal

    pub_type: 杂志文章

    doi:10.1529/biophysj.108.132175

    authors: Balashov SP,Imasheva ES,Wang JM,Lanyi JK

    更新日期:2008-09-01 00:00:00