Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes.

ABSTRACT: Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (V_m) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: frequency- and spectrally-tuned multiplexing (FAST^M). Different from present multiplexing approaches, FAST^M uses phase-sensitive signal detection, which renders various combinations of common probes for V_m and ions accessible for multiplexing. Using kinetic stopped-flow fluorimetry, we show that FAST^M allows simultaneous recording of rapid changes in Ca²⁺, pH, Na⁺, and V_m with high sensitivity and minimal crosstalk. FAST^M is also suited for multiplexing using single-cell microscopy and genetically encoded FRET biosensors. Moreover, FAST^M is compatible with optochemical tools to study signaling using light. Finally, we show that the exceptional time resolution of FAST^M also allows resolving rapid chemical reactions. Altogether, FAST^M opens new opportunities for interrogating cellular signaling.

SUBMITTER: Kierzek M

PROVIDER: S-EPMC8700268 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes.

Project description:Fluorescence lifetime multiplexing requires fluorescent probes with distinct fluorescence lifetimes but similar spectral properties. Even though synthetic probes for many cellular targets are available for multicolor live-cell fluorescence microscopy, few of them have been characterized for their use in fluorescence lifetime multiplexing. Here, we demonstrate that, from a panel of 18 synthetic probes, eight pairwise combinations are suitable for fluorescence lifetime multiplexing in living mammalian cell lines. Moreover, combining multiple pairs in different spectral channels enables us to image four and with the help of self-labeling protein tags up to eight different biological targets, effectively doubling the number of observable targets. The combination of synthetic probes with fluorescence lifetime multiplexing is thus a powerful approach for live-cell imaging.

| S-EPMC9207807 | biostudies-literature

Electronically tuned 1,3,5-triarylpyrazolines as Cu(I)-selective fluorescent probes.

Project description:We have prepared and characterized a Cu(i)-responsive fluorescent probe, constructed using a large tetradentate, 16-membered thiazacrown ligand ([16]aneNS(3)) and 1,3,5-triaryl-substituted pyrazoline fluorophores. The fluorescence contrast ratio upon analyte binding, which is mainly governed by changes of the photoinduced electron transfer (PET) driving force between the ligand and fluorophore, was systematically optimized by increasing the electron withdrawing character of the 1-aryl-ring, yielding a maximum 50-fold fluorescence enhancement upon saturation with Cu(i) in methanol and a greater than 300-fold enhancement upon protonation with trifluoroacetic acid. The observed fluorescence increase was selective towards Cu(i) over a broad range of mono- and divalent transition metal cations. Previously established Hammett LFERs proved to be a valuable tool to predict two of the PET key parameters, the acceptor potential (E(A/A(-)) and the excited state energy DeltaE(00), and thus to identify a set of pyrazolines that would best match the thermodynamic requirements imposed by the donor potential E(D(+)/D) of the thiazacrown receptor. The described approach should be applicable for rationally designing high-contrast pyrazoline-based PET probes selective towards other metal cations.

| S-EPMC3164304 | biostudies-literature

Spectrally-resolved response properties of the three most advanced FRET based fluorescent protein voltage probes.

Project description:Genetically-encoded optical probes for membrane potential hold the promise of monitoring electrical signaling of electrically active cells such as specific neuronal populations in intact brain tissue. The most advanced class of these probes was generated by molecular fusion of the voltage sensing domain (VSD) of Ci-VSP with a fluorescent protein (FP) pair. We quantitatively compared the three most advanced versions of these probes (two previously reported and one new variant), each involving a spectrally distinct tandem of FPs. Despite these different FP tandems and dissimilarities within the amino acid sequence linking the VSD to the FPs, the amplitude and kinetics of voltage dependent fluorescence changes were surprisingly similar. However, each of these fluorescent probes has specific merits when considering different potential applications.

| S-EPMC2641041 | biostudies-literature

The interaction of fluorescent nanodiamond probes with cellular media.

Project description:Fluorescent nanodiamonds (FNDs) are promising tools to image cells, bioanalytes and physical quantities such as temperature, pressure, and electric or magnetic fields with nanometer resolution. To exploit their potential for intracellular applications, the FNDs have to be brought into contact with cell culture media. The interactions between the medium and the diamonds crucially influence sensitivity as well as the ability to enter cells. The authors demonstrate that certain proteins and salts spontaneously adhere to the FNDs and may cause aggregation. This is a first investigation on the fundamental questions on how (a) FNDs interact with the medium, and (b) which proteins and salts are being attracted. A differentiation between strongly binding and weakly binding proteins is made. Not all proteins participate in the formation of FND aggregates. Surprisingly, some main components in the medium seem to play no role in aggregation. Simple strategies to prevent aggregation are discussed. These include adding the proteins, which are naturally present in the cell culture to the diamonds first and then inserting them in the full medium. Graphical abstractSchematic of the interaction of nanodiamonds with cell culture medium. Certain proteins and salts adhere to the diamond surface and lead to aggregation or to formation of a protein corona.

| S-EPMC5346409 | biostudies-literature

Electrospun Flexible Coaxial Nanoribbons Endowed With Tuned and Simultaneous Fluorescent Color-Electricity-Magnetism Trifunctionality.

Project description:In order to develop new-typed multifunctional nanocomposites, fluorescent-electrical-magnetic trifunctional coaxial nanoribbons with tunable fluorescent color, including white-light emission, have been successfully fabricated via coaxial electrospinning technology. Each stripe of coaxial nanoribbon is composed of a Fe3O4/PMMA core and a [Eu(BA)3phen+Dy(BA)3phen]/PANI/PMMA (PMMA = polymethyl methacrylate, BA = benzoic acid, phen = phenanthroline, polyaniline = PANI) shell. X-ray diffractometry (XRD), field emission scanning electron microscopy (FE-SEM), biological microscopy (BM), vibrating sample magnetometry (VSM), energy dispersive spectrometry (EDS), Hall effect measurement system and photoluminescence (PL) spectroscopy were employed to characterize the coaxial nanoribbons. Emitting color of the coaxial nanoribbons can be tuned by adjusting the contents of Dy(BA)3phen, Eu(BA)3phen, PANI and Fe3O4 in a wide color range of blue-white-orange under the excitation of 273-nm single-wavelength ultraviolet light. The coaxial nanoribbons simultaneously possess excellent luminescent performance, electrical conduction and magnetism compared with the counterpart composite nanoribbons. Furthermore, the electrical and magnetic performances of the coaxial nanoribbons also can be tunable by adding different quantities of PANI and Fe3O4 nanoparticles, respectively. The obtained coaxial nanoribbons have promising applications in many areas, such as electromagnetic interference shielding, microwave absorption, molecular electronics, biomedicine, future nanomechanics and display fields.

| S-EPMC4571655 | biostudies-other

Multiplex recording of cellular events over time on CRISPR biological tape.

Project description:Although dynamics underlie many biological processes, our ability to robustly and accurately profile time-varying biological signals and regulatory programs remains limited. Here we describe a framework for storing temporal biological information directly in the genomes of a cell population. We developed a "biological tape recorder" in which biological signals trigger intracellular DNA production that is then recorded by the CRISPR-Cas adaptation system. This approach enables stable recording over multiple days and accurate reconstruction of temporal and lineage information by sequencing CRISPR arrays. We further demonstrate a multiplexing strategy to simultaneously record the temporal availability of three metabolites (copper, trehalose, and fucose) in the environment of a cell population over time. This work enables the temporal measurement of dynamic cellular states and environmental changes and suggests new applications for chronicling biological events on a large scale.

| S-EPMC7869111 | biostudies-literature

Spectrally tuned structural and pigmentary coloration of birdwing butterfly wing scales.

Project description:The colourful wing patterns of butterflies play an important role for enhancing fitness; for instance, by providing camouflage, for interspecific mate recognition, or for aposematic display. Closely related butterfly species can have dramatically different wing patterns. The phenomenon is assumed to be caused by ecological processes with changing conditions, e.g. in the environment, and also by sexual selection. Here, we investigate the birdwing butterflies, Ornithoptera, the largest butterflies of the world, together forming a small genus in the butterfly family Papilionidae. The wings of these butterflies are marked by strongly coloured patches. The colours are caused by specially structured wing scales, which act as a chirped multilayer reflector, but the scales also contain papiliochrome pigments, which act as a spectral filter. The combined structural and pigmentary effects tune the coloration of the wing scales. The tuned colours are presumably important for mate recognition and signalling. By applying electron microscopy, (micro-)spectrophotometry and scatterometry we found that the various mechanisms of scale coloration of the different birdwing species strongly correlate with the taxonomical distribution of Ornithoptera species.

| S-EPMC4614508 | biostudies-literature

Multiplexing Fluorescence Anisotropy Using Frequency Encoding.

Project description:In this report, a method to multiplex fluorescence anisotropy measurements is described using frequency encoding. As a demonstration of the method, simultaneous competitive immunoassays for insulin and glucagon were performed by measuring the ratio of bound and free Cy5-insulin and FITC-glucagon in the presence of their respective antibodies. A vertically polarized 635 nm laser was pulsed at 73 Hz and used to excite Cy5-insulin, while a vertically polarized 488 nm laser pulsed at 137 Hz excited FITC-glucagon. The total emission was split into parallel and perpendicular polarizations and collected onto separate photomultiplier tubes. The signals from each channel were demodulated using a fast Fourier transform, resolving the contributions from each fluorophore. Anisotropy calculations were carried out using the magnitude of the peaks in the frequency domain. The method produced the expected shape of the calibration curves with limits of detection of 0.6 and 5 nM for insulin and glucagon, respectively. This methodology could readily be expanded to other biological systems and further multiplexed to monitor increased numbers of analytes.

| S-EPMC4991543 | biostudies-literature

Fluorescent probes of the apoptolidins and their utility in cellular localization studies.

Project description:Apoptolidin A has been described among the top 0.1% most-cell-selective cytotoxic agents to be evaluated in the NCI 60 cell line panel. The molecular structure of apoptolidin A consists of a 20-membered macrolide with mono- and disaccharide moieties. In contrast to apoptolidin A, the aglycone (apoptolidinone) shows no cytotoxicity (>10 μM) when evaluated against several tumor cell lines. Apoptolidin H, the C27 deglycosylated analogue of apoptolidin A, displayed sub-micromolar activity against H292 lung carcinoma cells. Selective esterification of apoptolidins A and H with 5-azidopentanoic acid afforded azido-functionalized derivatives of potency equal to that of the parent macrolide. They also underwent strain-promoted alkyne-azido cycloaddition reactions to provide access to fluorescent and biotin-functionalized probes. Microscopy studies demonstrate apoptolidins A and H localize in the mitochondria of H292 human lung carcinoma cells.

| S-EPMC4293314 | biostudies-literature

Photobleaching kinetics and time-integrated emission of fluorescent probes in cellular membranes.

Project description:Since the pioneering work of Hirschfeld, it is known that time-integrated emission (TiEm) of a fluorophore is independent of fluorescence quantum yield and illumination intensity. Practical implementation of this important result for determining exact probe distribution in living cells is often hampered by the presence of autofluorescence. Using kinetic modelling of photobleaching combined with pixel-wise bleach rate fitting of decay models with an updated plugin to the ImageJ program, it is shown that the TiEm of a fluorophore in living cells can be determined exactly from the product of bleaching amplitude and time constant. This applies to mono-exponential bleaching from the first excited singlet and/or triplet state and to multi-exponential combinations of such processes. The TiEm can be used to correct for illumination shading and background autofluorescence without the need for fluorescent test layers or separate imaging of non-stained cells. We apply the method to simulated images and to images of cells, whose membranes were labelled with fluorescent sterols and sphingolipids. Our bleaching model can be extended to include a probability density function (PDF) of intrinsic bleach rate constants with a memory kernel. This approach results in a time-dependent bleach rate coefficient and is exemplified for fluorescent sterols in restricted intracellular environments, like lipid droplets. We show that for small deviations from the classical exponential bleaching, the TiEm of decay functions with rate coefficients remains largely independent of fluorescence lifetime and illumination, and thereby represents a faithful measure of probe distribution.

| S-EPMC6271172 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data