Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases.
Ontology highlight
ABSTRACT:
SUBMITTER: Jena S
PROVIDER: S-EPMC7679178 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
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
Project description:The removal of varnish from the surface is a key step in painting conservation. Varnish removal is traditionally monitored by examining the painting surface under ultraviolet illumination. We show here that by imaging the fluorescence lifetime instead, much better contrast, sensitivity, and specificity can be achieved. For this purpose, we developed a lightweight (4.8 kg) portable instrument for macroscopic fluorescence lifetime imaging (FLIM). It is based on a time-correlated single-photon avalanche diode (SPAD) camera to acquire the FLIM images and a pulsed 440 nm diode laser to excite the varnish fluorescence. A historical model painting was examined to demonstrate the capabilities of the system. We found that the FLIM images provided information on the distribution of the varnish on the painting surface with greater sensitivity, specificity, and contrast compared to the traditional ultraviolet illumination photography. The distribution of the varnish and other painting materials was assessed using FLIM during and after varnish removal with different solvent application methods. Monitoring of the varnish removal process between successive solvent applications by a swab revealed an evolving image contrast as a function of the cleaning progress. FLIM of dammar and mastic resin varnishes identified characteristic changes to their fluorescence lifetimes depending on their ageing conditions. Thus, FLIM has a potential to become a powerful and versatile tool to visualise varnish removal from paintings.Graphical abstract
| S-EPMC10276100 | biostudies-literature
Project description:Changes in chromatin structure are key determinants of genomic responses. Thus, methods that enable such measurements are instrumental for investigating genome regulation and function. Here, we report further developments and validation of a streamlined method of histone-based fluorescence lifetime imaging microscopy (FLIM) that robustly detects chromatin compaction states in fixed and live cells, in 2D and 3D. We present a quality-controlled and detailed method that is simpler and faster than previous methods, and uses FLIMfit open-source software. We demonstrate the versatility of this chromatin FLIM through its combination with immunofluorescence and implementation in immortalised and primary cells. We applied this method to investigate the regulation of chromatin organisation after genotoxic stress and provide new insights into the role of ATM in controlling chromatin structure independently of DNA damage. Collectively, we present an adaptable chromatin FLIM method for examining chromatin structure and establish its utility in mammalian cells.
| S-EPMC5898265 | biostudies-literature
Project description:Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that are added to live cells and have fluorescent properties. Engineering efforts aim to improve performance and add versatile features. Current efforts focus on adding multiplexing capabilities to tag and visualize multiple RNAs simultaneously in the same cell. Here, we present the fluorescence lifetime-based platform Riboglow-FLIM. Our system requires a smaller tag and has superior cell contrast when compared with intensity-based detection. Because our RNA tags are derived from a large bacterial riboswitch sequence family, the riboswitch variants add versatility for using multiple tags simultaneously. Indeed, we demonstrate visualization of two RNAs simultaneously with orthogonal lifetime-based tags.
| S-EPMC9935525 | biostudies-literature
Project description:For monitoring the intracellular pathway of small interfering RNA (siRNA), both strands were labelled at internal positions by two ATTO dyes as an interstrand Förster resonance energy transfer pair. siRNA double strands show red emission and a short donor lifetime as readout, whereas siRNA antisense single strands show green emission and a long donor lifetime. This readout signals if GFP silencing can be expected (green) or not (red). We attached both dyes to three structurally different alkyne anchors by postsynthetic modifications. There is only a slight preference for the ribofuranoside anchors with the dyes at their 2'-positions. For the first time, the delivery and fate of siRNA in live HeLa cells was tracked by fluorescence lifetime imaging microscopy (FLIM), which revealed a clear relationship between intracellular transport using different transfection methods and knockdown of GFP expression, which demonstrates the potential of our siRNA architectures as a tool for future development of effective siRNA.
| S-EPMC8453559 | biostudies-literature
Project description:Incorporation of thymidine analogues in replicating DNA, coupled with antibody and fluorophore staining, allows analysis of cell proliferation, but is currently limited to monolayer cultures, fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation, S phase progression over several division cycles, effects of anti-proliferative drugs and other applications. It is based on the prominent (~ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain, Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content, multi-parametric dynamic analyses, far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures, complex 3D tissue models of tumor cell spheroids and intestinal organoids, and in physiological study with metformin treatment.
| S-EPMC5156356 | biostudies-literature
Project description:Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.
| S-EPMC6744410 | biostudies-literature
Project description:We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100's to 1000's of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. KD values broadly agree with published biochemical measurements.
| S-EPMC4919659 | biostudies-literature
Project description:The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe-biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.
| S-EPMC10681155 | biostudies-literature
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