Search Results (26)

This study presents a novel ratiometric fluorescent sensor based on Förster resonance energy transfer (FRET) between glutathione (GSH)-coated CdTe quantum dots (CdTe/GSH QDs) and bovine serum albumin (BSA)-coated Au nanoclusters (AuNCs/BSA) for dopamine (DA) detection. The nanoparticles were characterized using transmission electron microscopy (TEM), zeta potential measurements, Fourier transform infrared (FTIR) spectroscopy, UV-Vis absorption and fluorescence spectroscopy. Key FRET parameters, including energy transfer efficiency (E), donor–acceptor distance (r), Förster distance (R₀), and the overlap integral (J), were determined. The interactions between the CdTe/GSH-AuNCs/BSA conjugate and DA were investigated, revealing a dual mechanism of QDs fluorescence quenching that involves both energy and electron transfer. The average lifetime values and spectral profiles of CdTe/GSH QDs, both in the absence and presence of DA, suggest a dynamic fluorescence quenching process. The variation in the ratiometric signal with increasing DA concentration demonstrated a linear response within the range of 0–250 µM, with a correlation coefficient of 0.9963 and a detection limit of 6.9 nM. This proposed nanosensor exhibited selectivity against potential interfering substances, including urea, glucose, BSA, GSH, citric acid, and metal ions such as Na⁺ and Ca²⁺. The conjugate also demonstrates excellent cytocompatibility and enhances cell proliferation in HeLa epithelial cells, making it suitable for biological applications. It was successfully employed for DA detection in urine samples, achieving recoveries ranging from 99.1% to 104.2%. The sensor is highly sensitive, selective, rapid, and cost-effective, representing a promising alternative for DA detection across various sample types. Full article

Protein–protein interactions (PPIs) play a crucial role in various biological processes, including signal transduction, transcriptional regulation, and metabolic pathways. Over the years, many methods have been developed to study PPIs, such as yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), pull-down assays, and surface plasmon resonance (SPR). However, each of these techniques has its own limitations, including false positives, a lack of specific binding partners, and restricted interaction zones. Fluorescence resonance energy transfer (FRET) has emerged as a powerful technique for investigating PPIs, offering several advantages over traditional methods. Recent advancements in fluorescence microscopy have further enhanced its application in PPI studies. In this review, we summarize recent developments in FRET-based approaches and their applications in PPIs research over the past five years, including conventional FRET, time-resolved FRET (TR-FRET), fluorescence lifetime imaging microscopy-FRET (FLIM-FRET), single-molecule FRET (smFRET), fluorescence cross-correlation spectroscopy FRET (FCCS-FRET), and provide guidance on selecting the most appropriate method for PPIs studies. Full article

Time-resolved fluorescence techniques, such as fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS), and time-resolved fluorescence spectroscopy, are ideally suited for investigating molecular dynamics and interactions in biological and chemical systems. However, the analysis and interpretation of these datasets require advanced computational tools capable of handling diverse models and datasets. This paper presents a comprehensive software solution designed for model generation and analysis of time-resolved fluorescence data with a strong focus on fluorescence for quantitative structural analysis and biophysics. The software supports the integration of multiple fluorescence techniques and provides users with robust tools for performing complex model analysis across diverse experimental data. By enabling global analysis, model generation, data visualization, and sampling over model parameters, the software enhances the interpretability of intricate fluorescence phenomena. By providing flexible modeling capabilities, this solution offers a versatile platform for researchers to extract meaningful insights from time-resolved fluorescence data, aiding in the understanding of dynamic biomolecular processes. Full article

Photosystem I in most organisms contains long-wavelength or “Red” chlorophylls (Chls) absorbing light beyond 700 nm. At cryogenic temperatures, the Red Chls become quasi-traps for excitations as uphill energy transfer is blocked. One pathway for de-excitation of the Red Chls is via transfer to the oxidized RC (P700⁺), which has broad absorption in the near-infrared region. This study investigates the excitation dynamics of Red Chls in Photosystem I from the cyanobacterium Thermostichus vulcanus at cryogenic temperatures (77 K) and examines the role of the oxidized RC in modulating their fluorescence kinetics. Using time-resolved fluorescence spectroscopy, the kinetics of Red Chls were recorded for samples with open (neutral P700) and closed (P700⁺) RCs. We found that emission lifetimes in the range of 710–720 nm remained unaffected by the RC state, while more red-shifted emissions (>730 nm) decayed significantly faster when the RC was closed. A kinetic model describing the quenching by the oxidized RC was constructed based on simultaneous fitting to the recorded fluorescence emission in Photosystem I with open and closed RCs. The analysis resolved multiple Red Chl forms and variable quenching efficiencies correlated with their spectral properties. Only the most red-shifted Chls, with emission beyond 730 nm, are efficiently quenched by P700⁺, with rate constants of up to 6 ns⁻¹. The modeling results support the notion that structural and energetic disorder in Photosystem I can have a comparable or larger effect on the excitation dynamics than the geometric arrangement of Chls. Full article

Fluorescence lifetime imaging microscopy is sensitive to molecular interactions and environments. In homo-dyne frequency-domain fluorescence lifetime imaging microscopy, images of fluorescence objects are acquired at different phase settings of the detector. The detected intensity as a function of detector phase is a sinusoidal function that is sensitive to the lifetime of the fluorescent species. In this paper, the theory of phase-sensitive fluorescence image correlation spectroscopy is described. In this version of lifetime imaging, image correlation spectroscopy analysis (i.e., spatial autocorrelation) is applied to successive fluorescence images acquired at different phase settings of the detector. Simulations of different types of lifetime distributions reveal that the phase-dependent density of fluorescent objects is dependent on the heterogeneity of lifetimes present in the objects. We provide an example of this analysis workflow to a cervical cancer cell stained with a fluorescent membrane probe. Full article

Background/Objectives: Despite the role of metabolism in breast cancer metastasis, we still cannot predict which breast tumors will progress to distal metastatic lesions or remain dormant. This work uses metabolic imaging to study breast cancer cell lines (4T1, 4T07, and 67NR) with differing metastatic potential in a 3D collagen gel bioreactor system. Methods: Within the bioreactor, hyperpolarized magnetic resonance spectroscopy (HP-MRS) is used to image lactate/pyruvate ratios, while fluorescence lifetime imaging microscopy (FLIM) of endogenous metabolites measures metabolism at the cellular scale. Results: HP-MRS results showed no lactate peak for 67NR and a comparatively large lactate/pyruvate ratio for both 4T1 and 4T07 cell lines, suggestive of greater pyruvate utilization with greater metastatic potential. Similar patterns were observed using FLIM with significant increases in FAD intensity, redox ratio, and NAD(P)H lifetime. The lactate/pyruvate ratio was strongly correlated to NAD(P)H lifetime, consistent with the role of NADH as an electron donor for the glycolytic pathway, suggestive of an overall upregulation of metabolism (both glycolytic and oxidative), for the 4T07 and 4T1 cell lines compared to the non-metastatic 67NR cell line. Conclusions: These findings support a complementary role for HP-MRS and FLIM enabled by a novel collagen gel bioreactor system to investigate metastatic potential and cancer metabolism. Full article

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute a fundamental part of these systems. Moreover, the drug–liposome model serves as a robust platform for investigating interaction processes at both cellular and molecular levels. To advance our understanding of drug carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), leveraging the unique benefits of two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) as a simplified model system for cell membranes, labelled with the amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC₁₈(3)). Additionally, large unilamellar vesicles (LUVs) functioned as a drug carrier system, incorporating the spectrally distinct fluorescent sulforhodamine 101 (SRh101) as a surrogate drug. The investigation emphasized the diverse interactions between GUVs and LUVs based on the charged lipids employed. We examined the exchange kinetics and structural alterations of liposome carriers during the uptake process. Our study underscores the significance of employing 2P excitation in conjunction with FLIM and FCS. This powerful combination offers a valuable methodological approach for studying liposome interactions, positioning them as an exceptionally versatile model system with a distinct technical advantage. Full article

Fluorescence lifetime measurements of blood or plasma offer valuable insights into the microenvironment and molecular interactions of fluorophores, particularly concerning albumin. Neutrophil- and hypoxia-induced oxidative stress in COVID-19 pneumonia patients leads to hyperinflammation, various oxidative modifications of blood proteins, and potential alterations in the fluorescence lifetime of tryptophan-containing proteins, especially albumin. The objective of this study was to investigate the efficacy of time-resolved fluorescence spectroscopy of blood and plasma as a prompt diagnostic tool for the early diagnosis and severity assessment of COVID-19-associated pneumonia. This study examined a cohort of sixty COVID-19 patients with respiratory symptoms. To investigate whether oxidative stress is the underlying cause of the change in fluorescence lifetime, human serum albumin was treated with chloramine T. The time-resolved spectrometer Life Spec II (Edinburgh Instruments Ltd., Livingston, UK), equipped with a sub-nanosecond pulsed 280 nm diode, was used to measure the fluorescence lifetime of blood and plasma. The findings revealed a significant reduction in the fluorescence lifetime of blood (diluted 200 times) and plasma (diluted 20 times) at 360 nm in COVID-19 pneumonia patients compared with their respective values recorded six months post-infection and those of healthy individuals. Significant negative correlations were observed between the mean fluorescence lifetime of blood and plasma at 360 nm and several severity biomarkers and advanced oxidation protein products, while a positive correlation was found with albumin and the albumin–globulin ratio. The time-resolved fluorescence spectroscopy method demonstrates the potential to be used as a preliminary screening technique for identifying patients who are at risk of developing severe complications. Furthermore, the small amount of blood required for the measurements has the potential to enable a rapid fingerstick blood test. Full article

Designing phosphors that are excited by blue light is extraordinarily important for white light-emitting diodes (w-LEDs). In the present study, a new Ruddlesden–Popper type of SZO:xEu³⁺ (x = 0.01~0.10) phosphors was developed using solid-state reactions. Interestingly, a Eu³⁺ doping-induced phase transformation from the Sr₃Zr₂O₇ (cubic) to the SrZrO₃ (orthorhombic) phase was observed, and the impact of the occupied sites of Eu³⁺ ions on the lifetime of Sr₃Zr₂O₇:xEu³⁺ phosphors is discussed in detail. Diffuse reflectance spectroscopy results showed that the band gap of SZO:xEu³⁺ phosphors gradually increased from 3.48 eV for undoped Sr₃Zr₂O₇ hosts to 3.67 eV for SZO:0.10Eu³⁺ samples. The fluorescence excitation spectrum showed that ultraviolet (300 nm), near-ultraviolet (396 nm) and blue light (464 nm) were all effective excitation pump sources of Sr₃Zr₂O₇:xEu³⁺ phosphors, and the strongest emission at 615 nm originated from an electric dipole transition (⁵D₀→⁷F₂). CIE coordinates moved from orange (0.5969, 0.4267) to the reddish-orange region (0.6155, 0.3827), and the color purity also increased. The fabricated w-LED was placed on a 460 nm chip with a mixture of YAG:Ce³⁺ and SZO:0.1Eu³⁺ samples and showed “warm” white light with a color rendering index (CRI) of 81.8 and a correlation color temperature (CCT) of 5386 K, indicating great potential for application in blue chip white LEDs. Full article

Molecular probes based on the excited-state intramolecular proton-transfer (ESIPT) mechanism have emerged to be attractive candidates for various applications. Although the steady-state fluorescence mechanisms of these ESIPT-based probes have been reported extensively, less information is available about the fluorescence lifetime characteristics of newly developed NIR-emitting dyes. In this study, four NIR-emitting ESIPT dyes with different cyanine terminal groups were investigated to evaluate their fluorescence lifetime characteristics in a polar aprotic solvent such as CH₂Cl₂. By using the time-correlated single-photon counting (TCSPC) method, these ESIPT-based dyes revealed a two-component exponential decay (τ₁ and τ₂) in about 2–4 nanoseconds (ns). These two components could be related to the excited keto tautomers. With the aid of model compounds (5 and 6) and low-temperature fluorescence spectroscopy (at −189 ℃), this study identified the intramolecular charge transfer (ICT) as one of the major factors that influenced the τ values. The results of this study also revealed that both fluorescence lifetimes and fractional contributions of each component were significantly affected by the probe structures. Full article

Fluorescent Correlation Spectroscopy (FCS) allows us to determine interactions of labeled proteins or changes in the oligomeric state. The FCS method needs a low amount of fluorescent dye, near nanomolar concentrations. To control the amount of fluorescent dye, we used new photoconvertible FP SAASoti. This work is devoted to the proof of principle of using photoconvertible proteins to measure caspase enzymatic activity in a single live cell. The advantage of this approach is that partial photoconversion of the FP makes FCS measurements possible when studying enzymatic reactions. To investigate the process, in vivo we used HeLa cell line expressing the engineered FRET sensor, SAASoti-23-KFP. This FRET sensor has a cleavable (DEVD) sequence in the linker between two FPs for the detection of one of the key enzymes of apoptosis, caspase-3. Caspase-3 activity was detected by registering the increase in the fluorescent lifetimes of the sensor, whereas the diffusion coefficient of SAASoti decreased. This can be explained by an increase in the total cell viscosity during apoptosis. We can suppose that in the moment of detectible caspase-3 activity, cell structure already has crucial changes in viscosity. Full article

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs. Full article

Nanoformulations offer multiple advantages over conventional drug delivery, enhancing solubility, biocompatibility, and bioavailability of drugs. Nanocarriers can be engineered with targeting ligands for reaching specific tissue or cells, thus reducing the side effects of payloads. Following systemic delivery, nanocarriers must deliver encapsulated drugs, usually through nanocarrier degradation. A premature degradation, or the loss of the nanocarrier coating, may prevent the drug’s delivery to the targeted tissue. Despite their importance, stability and degradation of nanocarriers in biological environments are largely not studied in the literature. Here we review techniques for tracing the fate of nanocarriers, focusing on nanocarrier degradation and drug release both intracellularly and in vivo. Intracellularly, we will discuss different fluorescence techniques: confocal laser scanning microscopy, fluorescence correlation spectroscopy, lifetime imaging, flow cytometry, etc. We also consider confocal Raman microscopy as a label-free technique to trace colocalization of nanocarriers and drugs. In vivo we will consider fluorescence and nuclear imaging for tracing nanocarriers. Positron emission tomography and single-photon emission computed tomography are used for a quantitative assessment of nanocarrier and payload biodistribution. Strategies for dual radiolabelling of the nanocarriers and the payload for tracing carrier degradation, as well as the efficacy of the payload delivery in vivo, are also discussed. Full article

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (μM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos. Full article

Luminescent lanthanide-containing biocompatible nanosystems represent promising candidates as nanoplatforms for bioimaging applications. Herein, citrate-functionalized calcium-doped terbium phosphate hydrate nanophosphors of the rhabdophane type were prepared at different synthesis times and different Ca²⁺/Tb³⁺ ratios by a bioinspired crystallization method consisting of thermal decomplexing of Ca²⁺/Tb³⁺/citrate/phosphate/carbonate solutions. Nanoparticles were characterized by XRD, TEM, SEM, HR-TEM, FTIR, Raman, Thermogravimetry, inductively coupled plasma spectroscopy, thermoanalysis, dynamic light scattering, electrophoretic mobility, and fluorescence spectroscopy. They displayed ill-defined isometric morphologies with sizes ≤50 nm, hydration number n ~ 0.9, tailored Ca²⁺ content (0.42–8.11 wt%), and long luminescent lifetimes (800–2600 µs). Their relative luminescence intensities in solid state are neither affected by Ca²⁺, citrate content, nor by maturation time for Ca²⁺ doping concentration in solution below 0.07 M Ca²⁺. Only at this doping concentration does the maturation time strongly affect this property, decreasing it. In aqueous suspensions, neither pH nor ionic strength nor temperature affect their luminescence properties. All the nanoparticles displayed high cytocompatibility on two human carcinoma cell lines and cell viability correlated positively with the amount of doping Ca²⁺. Thus, these nanocrystals represent promising new luminescent nanoprobes for potential biomedical applications and, if coupled with targeting and therapeutic moieties, they could be effective tools for theranostics. Full article