Lifetime Probes Research Articles

Advancements in phasor-based FLIM: multi-component analysis and lifetime probes in biological imaging.

Fluorescence lifetime imaging microscopy (FLIM) is a reliable method that achieves imaging by detecting fluorescence lifetimes within samples. Owing to its unique temporal characteristic, it can complement fluorescence intensity measurement. Technological and methodological advancements in FLIM have broadened its applications across various domains. The processing of fluorescence lifetime data is crucial for enhancing the speed and accuracy of imaging. Thus, various lifetime fitting algorithms have been developed to improve the imaging speed. The phasor analysis (PA) method is an approach for processing fluorescence lifetime data, capable of directly converting lifetime signals into visual graphics without fitting, which outperforms traditional approaches in speed. Furthermore, lifetime probes with distinct lifetimes are readily implemented for visualization and cluster analysis combined with PA, facilitating the prediction of specific biological states or functions. This review examines various lifetime probes employed in phasor-based FLIM and discusses their roles in the PA method. The methods for multi-component PA within complex biological environments were also described. Additionally, we focused on the advantages of the phasor vector rule and the unmixing of multi-component analysis based on PA. The integration of lifetime probes with phasor-based FLIM facilitates rapid and intuitive detection methods for analyzing complex biological environments.

Mechanosensitive fluorescence lifetime probes for investigating the dynamic mechanism of ferroptosis

Deciphering the dynamic mechanism of ferroptosis can provide insights into pathogenesis, which is valuable for disease diagnosis and treatment. However, due to the lack of suitable time-resolved mechanosensitive tools, researchers have been unable to determine the membrane tension and morphology of the plasma membrane and the nuclear envelope during ferroptosis. With this research, we propose a rational strategy to develop robust mechanosensitive fluorescence lifetime probes which can facilitate simultaneous fluorescence lifetime imaging of the plasma membrane and nuclear envelope. Fluorescence lifetime imaging microscopy using the unique mechanosensitive probes reveal a dynamic mechanism for ferroptosis: The membrane tension of both the plasma membrane and the nuclear envelope decreases during ferroptosis, and the nuclear envelope exhibits budding during the advanced stage of ferroptosis. Significantly, the membrane tension of the plasma membrane is always larger than that of the nuclear envelope, and the membrane tension of the nuclear envelope is slightly larger than that of the nuclear membrane bubble. Meanwhile, the membrane lesions are repaired in the low-tension regions through exocytosis.

Achieving Cross Time-Domain Multiplexed Signal Cascade and Cancer Exosomes Identification by Bridging Long Lifetime Phosphor to NIR-II Lanthanide Energy Transfer.

Designing lanthanide luminescence lifetime sensors in the second near-infrared (NIR-II) window holds great potentials for physiological studies. However, the single lifetime signal is confined to one or two orders of magnitude of signal variation, which limits the sensitivity of lifetime probes. In this study, a lifetime cascade system, i.e., ZGO:Mn, Eu-DNA-1/TCPP-PEI70K@Yb-AptEpCAM, with a variety of signals (τm, τn, τµ, τm/τn and τm/τµ) is constructed for exosome identification using time-domain multiplexing. The sensitized ligand TCPP acts as both target-modulated switch and a bridge for connecting long lifetime ZGO:Mn, Eu-DNA-1 emitter to lanthanide Yb3+. This drives successive dual-path energy transfer and forms two D(donor)-A(acceptor) pairs. The lifetime variation is dominantly modulated by arranging TCPP as energy intermediate relay to covert milliseconds to nanoseconds to microseconds. It enables a broad lifetime range of six orders of magnitude. The presence of exosome specifically recognizes aptamers on TCPP-PEI70K@Yb-AptEpCAM to impede D-A pairs and reverse multiplexed response signals of the lifetime cascade system. The ratio lifetime signals τm/τn and τm/τµ achieve prominent exosome quantification and exosome type differentiation attributed to signal amplification. The cascade system relying on lifetime criteria can realize precise quantization and provide an effective strategy for subsequent physiological study.

Visualizing pH and Mass Transport at Hydrogen Evolving Electrodes with Fluorescence Lifetime Microscopy

The chemical industry needs electrochemical solutions to avoid large CO₂ emissions. However, many of these electrochemical processes suffer from poor mass transport around the catalyst. To optimize these technologies, we need to gain a better understanding of the local processes at the electrode. This could give us ideas of new catalyst and reactor designs to enhance mass transport.The pH is a parameter that helps us to understand these processes. This is because the catalyst stability and selectivity of many reactions is strongly dependent on the local pH. You need a non-invasive technique to measure in operando spatial maps of pH. Optical techniques are a logical route for this.Fluorescence Lifetime Imaging Microscopy (FLIM) is a technique that measures the fluorescence lifetime of a probe molecule. It is friendly to use, as it is self-referencing (i.e. you do not need to calibrate your system for every new measurement). Many electrochemical applications, like seawater electrolysis, CO2 reduction and alkaline water electrolysis, tend to work in alkaline conditions (pH >8). Unfortunately commercial FLIM probes are only available in neutral and mildly acidic pH (4 to 8), owing to their use in biological systems.We have developed new fluorescence lifetime probes based on the 1-methyl-7-amino-quinolinium fluorophore molecules, that allow pH measurements in the range of pH 5.5-13. We have used the probes to visualise the local pH around a hydrogen evolving electrode and can show the boundary layer that forms close to the electrode and investigate how it is disturbed by gas bubbles at a rate of 3 fps and resolution of 4 μm. Figure 1

Harnessing Dual-Fluorescence Lifetime Probes to Validate Regulatory Mechanisms of Organelle Interactions.

Organelles are dynamic yet highly organized to preserve cellular homeostasis. However, the absence of time-resolved molecular tools for simultaneous dual-signal imaging of two organelles has prevented scientists from elucidating organelle interaction regulatory mechanisms on a nanosecond timescale. To date, the regulatory mechanisms governing the interaction between endoplasmic reticulum (ER) and autophagosomes are unknown. In this study, we propose a strategy for developing dual-fluorescence lifetime probes localized to the endoplasmic reticulum and autophagosomes to investigate their interaction regulatory mechanisms. Using the robust probe CF2, we investigated the regulatory mechanisms between ER and autophagosomes and discovered the following: (i) motile autophagosome in ER tips drives the ER tubule to grow and slide; (ii) the ER reticulate tubule forms a three-way junction centered on the autophagosome; (iii) ER autophagy is a type of cell damage index during drug-induced apoptosis. Thus, this study advances our knowledge of organelle interaction regulatory mechanisms, shedding light on the identification of therapeutic targets for neurodegenerative diseases.

A Comparative Study of High-Contrast Fluorescence Lifetime Probes for Imaging Amyloid in Tissue.

Optical imaging of protein aggregates in living and post-mortem tissue can often be impeded by unwanted fluorescence, prompting the need for novel methods to extract meaningful signal in complex biological environments. Historically, benzothiazolium derivatives, prominently Thioflavin T, have been the state-of-the-art fluorescent probes for amyloid aggregates, but their optical, structural, and binding properties typically limit them to in vitro applications. This study compares the use of novel uncharged derivative, PAP_1, with parent Thioflavin T as a fluorescence lifetime imaging probe. This is applied specifically to imaging recombinant α-synuclein aggregates doped into brain tissue. Despite the 100-fold lower brightness of PAP_1 compared to that of Thioflavin T, PAP_1 binds to α-synuclein aggregates with an affinity several orders of magnitude greater than Thioflavin T; thus, we observe a specific decrease in the fluorescence lifetime of PAP_1 bound to α-synuclein aggregates, resulting in a separation of >1.4 standard deviations between PAP_1-stained brain tissue background and α-synuclein aggregates that is not observed with Thioflavin T. This enables contrast between highly fluorescent background tissue and amyloid fibrils that is attributed to the greater affinity of PAP_1 for α-synuclein aggregates, avoiding the substantial off-target staining observed with Thioflavin T.

Fluorescence lifetime probes for detection of RNA degradation

To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

Protein Supercomplex Recording in Living Cells Via Position-Specific Fluorescence Lifetime Sensors.

Our group has previously established a strategy utilizing fluorescence lifetime probesto image membrane protein supercomplex (SC) formation in situ. We showed that a probe at the interface between individualmitochondrial respiratory complexes exhibits a decreased fluorescence lifetime when a supercomplex is formed. This is caused by electrostatic interactions with the adjacent proteins. Fluorescence lifetime imaging microscopy (FLIM) records the resulting decrease of the lifetime of the SC-probe. Here we present the details of our method for performingSC-FLIM, including theevaluation of fluorescence lifetimes from the FLIM images. Tovalidatethe feasibility of the technique for monitoring adaptive SC formation, we compare data obtained under different metabolic conditions. The results confirm that SC formation is dynamic.

Evaluation of Ebselen-azadioxatriangulenium as redox-sensitive fluorescent intracellular probe and as indicator within a planar redox optode

Redox homeostasis is essential for cellular survival and is therefore tightly regulated. Visualizing changes in the local redox state is essential for detecting oxidative stress, especially with the help of red, long emission lifetime probes. We synthesized a new reversible, redox-sensitive fluorescent probe, ebselen-azadioxatriangulenium (Ebselen-ADOTA), and characterized its potential use for intra- and extracellular redox sensing. The probe consists of a photostable triangulenium-based fluorophore (emission maximum at 564 nm) modulated by a redox-active ebselen motif. The reduced form only shows around 1/10 of the emission intensity of the oxidized form. The Ebselen-ADOTA probe exhibited reversible oxidation and reduction in solution under extracellular conditions. In addition, Ebselen-ADOTA can visualize an increase in oxidative stress within anaerobic bacteria when exposing them to air, the probe did not readily respond to changes in the redox environment inside mammalian cells, probably due to unfavorable localization, aggregation or binding to cysteine containing proteins. When incorporated inside a polymer matrix (hydrogel D4 mixed with poly(acrylic acid)), the probe showed a rapid and fully reversible response to redox changes, thus enabling the preparation of a redox-sensitive optode.

Targeted fluorescence lifetime probes reveal responsive organelle viscosity and membrane fluidity.

The only way to visually observe cellular viscosity, which can greatly influence biological reactions and has been linked to several human diseases, is through viscosity imaging. Imaging cellular viscosity has allowed the mapping of viscosity in cells, and the next frontier is targeted viscosity imaging of organelles and their microenvironments. Here we present a fluorescent molecular rotor/FLIM framework to image both organellar viscosity and membrane fluidity, using a combination of chemical targeting and organelle extraction. For demonstration, we image matrix viscosity and membrane fluidity of mitochondria, which have been linked to human diseases, including Alzheimer’s Disease and Leigh’s syndrome. We find that both are highly dynamic and responsive to small environmental and physiological changes, even under non-pathological conditions. This shows that neither viscosity nor fluidity can be assumed to be fixed and underlines the need for single-cell, and now even single-organelle, imaging.

Ab Initio Prediction of Fluorescence Lifetimes Involving Solvent Environments by Means of COSMO and Vibrational Broadening.

The fluorescence lifetime is a key property of fluorophores that can be utilized for microenvironment probing, analyte sensing, and multiplexing as well as barcoding applications. For the rational design of lifetime probes and barcodes, theoretical methods have been developed to enable the ab initio prediction of this parameter, which depends strongly on interactions with solvent molecules and other chemical species in the emitteŕs immediate environment. In this work, we investigate how a conductor-like screening model (COSMO) can account for variations in fluorescence lifetimes that are caused by such fluorophore-solvent interactions. Therefore, we calculate vibrationally broadened fluorescence spectra using the nuclear ensemble method to obtain distorted molecular geometries to sample the electronic transitions with time-dependent density functional theory (TDDFT). The influence of the solvent on fluorescence lifetimes is accounted for with COSMO. For example, for 4-hydroxythiazole fluorophore containing different heteroatoms and acidic and basic moieties in aprotic and protic solvents of varying polarity, this approach was compared to experimentally determined lifetimes in the same solvents. Our results demonstrate a good correlation between theoretically predicted and experimentally measured fluorescence lifetimes except for the polar solvents ethanol and acetonitrile that can specifically interact with the heteroatoms and the carboxylic acid of the thiazole derivative.

Diazaoxatriangulenium: synthesis of reactive derivatives and conjugation to bovine serum albumin.

The azaoxa-triangulenium dyes are characterised by emission in the red and a long fluorescence lifetime (up to 25 ns). These properties have been widely explored for the azadioxatrianguelnium (ADOTA) dye. Here, the syntheses of reactive maleimide and NHS-ester forms of the diazaoxatriangulenium (DAOTA) system are reported. The DAOTA fluorophore was conjugated to bovine serum albumin (BSA) and investigated in comparison to the corresponding ADOTA-BSA conjugate. It was found that the fluorescence of DAOTA experienced a significantly higher degree of solvent quenching if compared to ADOTA as non-conjugated dyes in aqueous solution, while the fluorescence quenching observed upon conjugation to BSA was significantly reduced for DAOTA when compared to ADOTA. The differences in observed quenching for the conjugates can be explained by the different electronic structures of the dyes, which renders DAOTA significantly less prone to reductive photoinduced electron transfer (PET) quenching from e.g. tryptophan. We conclude that DAOTA, with emission in the red and inherent resistance to PET quenching, is an ideal platform for the development of long fluorescence lifetime probes for time-resolved imaging and fluorescence polarisation assay.

Angular distributions as lifetime probes

If new TeV scale particles are discovered, it will be important to determine their width. There is, however, a problematic region, where the width is too small to be determined directly, and too large to generate a secondary vertex. For a collection of colored, spin polarized particles, hadronization depolarizes the particles prior to their decay. The amount of depolarization can be used to probe the lifetime in the problematic region. In this paper we apply this method to a realistic scenario of a top-like particle that can be produced at the LHC. We study how depolarization affects the angular distributions of the decay products and derive an equation for the distributions that is sensitive to the lifetime.

BSA Au Clusters as a Probe for Enhanced Fluorescence Detection Using Multipulse Excitation Scheme

Although BSA Au clusters fluoresce in red region (λmax: 650 nm), they are of limited use due to low fluorescence quantum yield (~6%). Here we report an enhanced fluorescence imaging application of fluorescent bio-nano probe BSA Au clusters using multipulse excitation scheme. Multipulse excitation takes advantage of long fluorescence lifetime (> 1 µs) of BSA Au clusters and enhances its fluorescence intensity 15 times over short lived cellular auto-fluorescence. Moreover we have also shown that by using time gated detection strategy signal (fluorescence of BSA Au clusters) to noise (auto-fluorescence) ratio can be increased by 30 fold. Thereby with multipulse excitation long lifetime probes can be used to develop biochemical assays and perform optical imaging with zero background.

Dinuclear Ruthenium(II) Complexes as Two‐Photon, Time‐Resolved Emission Microscopy Probes for Cellular DNA

The first transition-metal complex-based two-photon absorbing luminescence lifetime probes for cellular DNA are presented. This allows cell imaging of DNA free from endogenous fluorophores and potentially facilitates deep tissue imaging. In this initial study, ruthenium(II) luminophores are used as phosphorescent lifetime imaging microscopy (PLIM) probes for nuclear DNA in both live and fixed cells. The DNA-bound probes display characteristic emission lifetimes of more than 160 ns, while shorter-lived cytoplasmic emission is also observed. These timescales are orders of magnitude longer than conventional FLIM, leading to previously unattainable levels of sensitivity, and autofluorescence-free imaging.

DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins

Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

The development of responsive, luminescent lifetime probes based upon axially functionalised fac-[Re(CO)3(di-imine)(L)]+ complexes

The syntheses of four new ditopic ligands (L1-4), based on N,N-functionalised 4-aminomethylpyridine, was achieved via a reductive amination methodology. The ligands react with fac-[Re(CO)(3)(di-imine)(MeCN)](OTf) to give the mixed-ligand species fac-[Re(CO)(3)(di-imine)(L1-4)](OTf). X-Ray crystallography has been used to structurally characterise one of the complexes confirming that the axial ligand (L4) coordinates via the pyridine unit: by adopting this binding mode the axial ligand therefore provides an additional binding site for metal cations. Each of the complexes displayed room temperature phosphorescence from (3)MLCT excited states. In addition the complexes based upon L1-3 also possessed ligand-centred fluorescence, originating from either the quinoline or 8-hydroxyquinoline units of the axial ligands. The luminescent properties of the complexes were monitored in the presence of metal dications of physiological and toxicological importance (Cu(II), Zn(II) and Hg(II)). As well as emission intensity changes the results show that these probes firstly, display highly modulated (3)MLCT lifetimes thus allowing differentiation between the different dications and secondly, can demonstrate selectivity in an ionic mixture, as judged by (3)MLCT lifetime measurements.

High‐aperture cryogenic light microscopy

We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.

Detection of single nucleotide polymorphisms through the application of lifetime fluorescent probes

Fluorescence-based approaches for genotyping single nucleotide polymorphisms (SNPs) commonly involve enzymatic cleavage, stringent control of hybridization temperature, or extensive post amplification clean up. Our research to SNP genotyping employs fluorescence lifetime probes, which consist of a single fluorophore that is covalently bound to a oligonucleotide complementary to a SNP allele. We require that the matched and mismatched forms correspond to different lifetimes of the fluorophore. In other words, fluorophore lifetime reports whether the base has entered into the double strand or is mismatched. The constraints on hybridization conditions are greatly relaxed and enzymatic cleavage is not required. This fluorescent lifetime-based technology allows resolving SNP alleles in both the homozygous and heterozygous states using a single lifetime probe. Initial studies have been conducted on the human β-globin gene, known to cause sickle cell anemia. Our results show the fluorophore lifetime is measurably different for the mutant and wild forms. We are currently characterizing the effects of probe/target base pair mismatch beyond the initial SNP. Preliminary studies suggest fluorescence polarization data likely provides additional specificity. This research was supported by the North Dakota INBRE grant.

Covalently-attached 1-pyrenylmethyl groups as a probe of temperature-dependent relaxation processes in polyethylene films

1-Pyrenylmethyl lumophores have been attached covalently to chains of several types of polyethylene (crystallinities 37–74%). The temperature dependence of their fluorescence between 40 and 400 K, as well as that of ultra-high-molecular-weight polyethylene (85% crystallinity) containing covalently-attached 1-pyrenyl groups, has been combined with data from DSC measurements and information concerning the properties of films to determine the utility and limitations of the fluorescence changes to detect the onset temperatures of α -, β - and γ -relaxation processes in the polyethylenes. The relative sensitivities of fluorescence from covalently-attached pyrenyl and anthryl (with a much shorter excited singlet-state lifetime) probes to each of the relaxation processes are compared in the same films. The probe whose excited singlet-state lifetime better matches the rate associated with a relaxation process is better able to detect it.