Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes.

ABSTRACT: 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.

SUBMITTER: Frei MS

PROVIDER: S-EPMC9207807 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Live-Cell Fluorescence Lifetime Multiplexing Using Synthetic Fluorescent Probes.

Frei Michelle S MS Koch Birgit B Hiblot Julien J Johnsson Kai K

ACS chemical biology 20220518 6

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 mamma ...[more]

PMID: 35584304

Similar Datasets

First Clinical Results of Fluorescence Lifetime-enhanced Tumor Imaging Using Receptor-targeted Fluorescent Probes.

Project description:PurposeFluorescence molecular imaging, using cancer-targeted near infrared (NIR) fluorescent probes, offers the promise of accurate tumor delineation during surgeries and the detection of cancer specific molecular expression in vivo. However, nonspecific probe accumulation in normal tissue results in poor tumor fluorescence contrast, precluding widespread clinical adoption of novel imaging agents. Here we present the first clinical evidence that fluorescence lifetime (FLT) imaging can provide tumor specificity at the cellular level in patients systemically injected with panitumumab-IRDye800CW, an EGFR-targeted NIR fluorescent probe.Experimental designWe performed wide-field and microscopic FLT imaging of resection specimens from patients injected with panitumumab-IRDye800CW under an FDA directed clinical trial.ResultsWe show that the FLT within EGFR-overexpressing cancer cells is significantly longer than the FLT of normal tissue, providing high sensitivity (>98%) and specificity (>98%) for tumor versus normal tissue classification, despite the presence of significant nonspecific probe accumulation. We further show microscopic evidence that the mean tissue FLT is spatially correlated (r > 0.85) with tumor-specific EGFR expression in tissue and is consistent across multiple patients. These tumor cell-specific FLT changes can be detected through thick biological tissue, allowing highly specific tumor detection and noninvasive monitoring of tumor EFGR expression in vivo.ConclusionsOur data indicate that FLT imaging is a promising approach for enhancing tumor contrast using an antibody-targeted NIR probe with a proven safety profile in humans, suggesting a strong potential for clinical applications in image guided surgery, cancer diagnostics, and staging.

| S-EPMC9167767 | biostudies-literature

Engineered HaloTag variants for fluorescence lifetime multiplexing.

Project description:Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

| S-EPMC8748199 | biostudies-literature

Fluorescent Metal Nanoshells: Lifetime-Tunable Molecular Probes in Fluorescent Cell Imaging.

Project description:We reported the preparation of lifetime-tunable fluorescent metal nanoshells and used them as lifetime imaging agents for potential detection of multiple target molecules by a single cell imaging scan. These metal nanoshells were generated to have 40 nm silica cores and 10 nm silver shells. Three kinds of metal-ligand complexes tris(5-amino-1,10-phenanthroline)ruthenium(II) (Ru(NH(2)-Phen)(3) (2+)), tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)(3) (2+)), and tris(2,3-bis(2-pyridyl)pyrazine))ruthenium(II) (Ru(dpp)(3) (2+)) that have similar excitation and emission wavelengths but different lifetimes were respectively encapsulated in the cores of metal nanoshells for the purpose of fluorescence. Compared with the metal-free silica spheres, these metal nanoshells were found to display enhanced emission intensities and shortened lifetimes due to near-field interactions of Ru(II) complexes with the metal shells. The shortened lifetimes of these metal nanoshells were definitely unique relevant to the Ru(II) complexes: 10 ns for the Ru(Phen-NH(2))(3) (2+)-Ag nanoshells, 45 ns for the Ru(bpy)(3) (2+)-Ag nanoshells, and 200 ns for the Ru(dpp)(3) (2+)-Ag nanoshells. These lifetimes were longer than the lifetime of cellular autofluorescence (2 - 5 ns), so the emission signals of these metal nanoshells could be distinctly isolated from the cellular background on the lifetime cell images. Moreover, these lifetimes were also different from one another, resulting in the emission signals of three metal nanoshells could be distinguished from one another on the cell images. This feature may offer an opportunity to detect multiple target molecules in a single cell imaging scan when the metal nanoshells are bound with various targets in the cells.

| S-EPMC3130523 | biostudies-literature

Fluorescent vinblastine probes for live cell imaging.

Project description:Herein we describe the synthesis of several fluorescent analogues of the clinically approved microtubule destabilizing agent vinblastine. The evaluated probes are the most potent described and provides the first example of uptake, distribution and live cell imaging using this well known antimitotic agent.

| S-EPMC4970878 | biostudies-literature

Quinolinium-Based Fluorescent Probes for Dynamic pH Monitoring in Aqueous Media at High pH Using Fluorescence Lifetime Imaging.

Project description:Spatiotemporal pH imaging using fluorescence lifetime imaging microscopy (FLIM) is an excellent technique for investigating dynamic (electro)chemical processes. However, probes that are responsive at high pH values are not available. Here, we describe the development and application of dedicated pH probes based on the 1-methyl-7-amino-quinolinium fluorophore. The high fluorescence lifetime and quantum yield, the high (photo)stability, and the inherent water solubility make the quinolinium fluorophore well suited for the development of FLIM probes. Due to the flexible fluorophore-spacer-receptor architecture, probe lifetimes are tunable in the pH range between 5.5 and 11. An additional fluorescence lifetime response, at tunable pH values between 11 and 13, is achieved by deprotonation of the aromatic amine at the quinolinium core. Probe lifetimes are hardly affected by temperature and the presence of most inorganic ions, thus making FLIM imaging highly reliable and convenient. At 0.1 mM probe concentrations, imaging at rates of 3 images per second, at a resolution of 4 μm, while measuring pH values up to 12 is achieved. This enables the pH imaging of dynamic electrochemical processes involving chemical reactions and mass transport.

| S-EPMC10226165 | biostudies-literature

Tomographic fluorescence lifetime multiplexing in the spatial frequency domain.

Project description:The ability to simultaneously recover multiple fluorophores within biological tissue (multiplexing) can have important applications for tracking parallel disease processes in vivo. Here we present a novel method for rapid and quantitative multiplexing within a scattering medium, such as biological tissue, based on fluorescence lifetime contrast. This method employs a tomographic inversion of the asymptotic (late) portion of time-resolved spatial frequency (SF) domain measurements. Using Monte Carlo simulations and phantom experiments, we show that the SF-asymptotic time domain (SF-ATD) approach provides a several-fold improvement in relative quantitation and localization accuracy over conventional SF-time domain inversion. We also show that the SF-ATD approach can exploit selective filtering of high spatial frequencies to dramatically improve reconstruction accuracy for fluorophores with subnanosecond lifetimes, which is typical of most near-infrared fluorophores. These results suggest that the SF-ATD approach will serve as a powerful new tool for whole-body lifetime multiplexing.

| S-EPMC6457659 | biostudies-literature

Fluorescence lifetime multiplexing with fluorogen activating protein FAST variants.

Project description:In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.

| S-EPMC11219735 | biostudies-literature

Molecular probes for fluorescence lifetime imaging.

Project description:Visualization of biological processes and pathologic conditions at the cellular and tissue levels largely relies on the use of fluorescence intensity signals from fluorophores or their bioconjugates. To overcome the concentration dependency of intensity measurements, evaluate subtle molecular interactions, and determine biochemical status of intracellular or extracellular microenvironments, fluorescence lifetime (FLT) imaging has emerged as a reliable imaging method complementary to intensity measurements. Driven by a wide variety of dyes exhibiting stable or environment-responsive FLTs, information multiplexing can be readily accomplished without the need for ratiometric spectral imaging. With knowledge of the fluorescent states of the molecules, it is entirely possible to predict the functional status of biomolecules or microevironment of cells. Whereas the use of FLT spectroscopy and microscopy in biological studies is now well-established, in vivo imaging of biological processes based on FLT imaging techniques is still evolving. This review summarizes recent advances in the application of the FLT of molecular probes for imaging cells and small animal models of human diseases. It also highlights some challenges that continue to limit the full realization of the potential of using FLT molecular probes to address diverse biological problems and outlines areas of potential high impact in the future.

| S-EPMC4482119 | biostudies-literature

Near-infrared fluorescence lifetime pH-sensitive probes.

Project description:We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pK(a) of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pK(a) ? 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pK(a) to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (?1.16 ns in acidic DMSO) and deprotonated (?1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescence-lifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes.

| S-EPMC3077686 | biostudies-literature

Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes.

Project description:Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.

| S-EPMC4873741 | 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