Fluorescent Core Research Articles

The Combination of a Donor-Acceptor TADF and a MR-TADF Emitting Core Results in Outstanding Electroluminescence Performance.

Here the utility and potential of an emitter design are demonstrated, consisting of a narrowband-emitting multiresonant thermally activated delayed fluorescent (MR-TADF) core that is decorated with a suitably higher energy donor-acceptor TADF moiety. Not only does this D-A TADF group offer additional channels for triplet exciton harvesting and confers faster reverse intersystem crossing (RISC) kinetics but it also acts as a steric shield, insulating the emissive MR-TADF core from aggregation-caused quenching. Two emitters, DtCzBN-CNBT1 and DtCzBN-CNBT2, demonstrate enhanced photophysical properties leading to outstanding performance of the organic light-emitting diodes (OLEDs). DtCzBN-CNBT2, containing a D-A TADF moiety, has a faster kRISC (1.1×105 s-1) and higher photoluminescence quantum yield (ΦPL: 97%) compared to DtCzBN-CNBT1 (0.2×105 s-1, ΦPL: 90%), which contains a D-A moiety that itself is not TADF. The sensitizer-free OLEDs with DtCzBN-CNBT2 achieve a record-high maximum external quantum efficiency (EQEmax) of 40.2% and showed milder efficiency roll-off (EQE1000 of 20.7%) compared to the DtCzBN-CNBT1-based devices (EQEmax of 37.1% and EQE1000 of 11.9%).

Facile, sustainable, and innovative inclusion of a highly fluorescent isoindole-sulfonate-based probe for the quantification of fluvoxamine, a CNS antidepressant drug; comprehensive method sustainability evaluation

The suggested study employs a specific technique to ensure the sustainability and ecological friendliness of an analyte evaluation procedure. The utilization of various recently developed methodologies as potential indicators of ecological sustainability and feasibility necessitates attention and consideration. The established analytical methodology employs a novel reagent mixture composed of phthalic dialdehyde and sulfite ion. This reagent is used to create a new fluorescent core that can be monitored using fluorimetric techniques at a wavelength of 385 nm after being excited at 320 nm. Condensation of the amino group of fluvoxamine can occur in a pH-controlled solution containing phthalaldehyde/sulfite. The present investigation presents a new, environmentally sustainable, and accurate fluorometric technique for identifying the medication. A linear relationship was detected for fluvoxamine over the 0.06–1.50 μg mL−1 concentration range. The method's validation and quantum yield determination were assessed following the International Council for Harmonization's (I.C.H.) established guidelines. The results exhibit remarkable precision and accuracy, consistent with the different methodologies published in the literature. Subsequently, an assessment was conducted to evaluate the environmental sustainability of the suggested technique, employing a range of contemporary indicators for determining environmental safety. The WAC concept, which combines ecological and operational elements and makes use of the Green/Red/Blue (RGB 12) scheme, interestingly identifies the current analytical method as white. Finally, BAGI, a novel tool for evaluating the method's applicability, was used in this study and showed that the planned method achieved a high applicability score.

Validation of ZIP4 as a tumour-associated antigen for nanotargeting

Pancreatic ductal adenocarcinoma remains a highly aggressive and untreatable cancer. There is a need to develop a new PDAC-associated antigen-targeting drug delivery system to tackle this disease. We validated choosing ZIP4 as a putative target in PDAC theranostics. We developed a nanosystem composed of a fluorescent polystyrene core coated with gold nanoparticles onto which a ZIP4-specific polyclonal antibody is attached. The polystyrene core’s fluorescence properties allow the nanosystem tracking by intravital imaging. We also developed two ZIP4-expressing cell lines by stably transfecting HEK293 and RWP1 cells with a ZIP4-coding plasmid that simultaneously provides cells with puromycin resistance. We studied the cell internalisation of the as-synthesised nanoparticles and demonstrated that ZIP4-expressing HEK293 and ZIP4-expressing RWP1 cells tended to take up more ZIP4-targeting nanoparticles. Moreover, we observed that ZIP4-targeting nanoparticles accumulated more in ZIP4-expressing HEK293 and RWP1 tumours when injected intravenously in a subcutaneous xenograft and an orthotopic in vivo model, respectively. Furthermore, the administration of these nanoparticles did not induce any significant systemic toxicity as determined by histological analysis of all organs. Altogether, these results provide the first evidence of the feasibility of using a ZIP4-targeting nanosystem further to design efficient therapeutic and diagnostic tools for PDAC.

Crescent-shaped Janus nanoassemblies of gold nanoparticles and AIEgens: Enhancing plasmonic and fluorescent activities for colorimetric and ratiometric fluorescence lateral flow immunoassay

Herein, crescent-shaped Janus nanoassemblies (Au-AIENPs) with high plasmonic and fluorescent activities are prepared by co-assembling oleylamine-coated gold nanoparticles (OA-AuNPs) and red-emitting aggregation-induced emission luminogens (AIEgens) in separate compartments of polymer nanoparticles. The obtained Au-AIENPs show typical Janus heterostructures, where AIEgens preferentially aggregate to form fluorescent core and OA-AuNPs are distributed in the polymer matrix to form a crescent-shaped plasmonic shell, thus resulting in effective spatial separation of OA-AuNPs and AIEgens to achieve the balance between plasmonic and fluorescent signals and reduce the mutual interference between signals. Taking advantage of the excellent plasmonic and fluorescent activities of Au-AIENPs, we successfully established a colorimetric and ratiometric fluorescence dual-mode lateral flow immunoassay (Au-AIENPs-RLFIA) for the visual and quantitative detection of aflatoxin B1 (AFB1) in corn sample. Under the developed conditions, the visual detection limit (vLOD) of the Au-AIENPs-RLFIA for colorimetric and ratiomentic fluorescence detection was 0.62 ng/mL and 0.02 ng/mL, respectively, while the quantitative LOD (qLOD) for ratiomentic fluorescence mode was as low as 0.0076 ng/mL. The above results indicate that the designed Janus Au-AIENPs are promising as dual-signal output probes and hold great potential for improving flexible dual-mode detection of various targets on the LFIA platform.

Water‐Dispersible BODIPY Multifunctionalized Silicon Oxide Nanoparticles for Glutathione Sensing

AbstractGlutathione (GSH), a thiol containing small peptide, plays pivotal roles in maintaining cellular redox balance, metabolism, detoxification, and scavenging of free radicals. Aberrant GSH levels in cells and tissues are associated with various disorders, underscoring the importance of accurate GSH detection for clinical diagnosis and therapy monitoring. Several molecular probes have been designed as fluorescent‐based GSH sensors. However, their water insolubility and the need of using organic cosolvents hinder their applicability on biological samples. Alternatively, nanomaterials have proven to be highly promising for boosting the precision of treatments and enhancing the accuracy of diagnosing diseases, thanks to their compatibility with biological environments and improved cell uptake. Here, the synthesis and characterization of a boron‐dipyrromethene (BODIPY)‐based probe (PB) are reported, incorporating a fluorescent BODIPY core, chlorine substituents for reaction with GSH, and a linking moiety for conjugation to the surface of silicon oxide nanoparticles (SONPs). Functionalized SONPs with PB are also characterized at the nanoscale using high‐resolution transmission electron microscopy (HR‐TEM), dynamic light scattering (DLS), Zeta potential, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), UV–Vis absorption, and fluorescence spectroscopies, confirming the surface functionalization and water‐dispersibility of functionalized SONPs with PB. GSH sensing is evaluated in aqueous solution, conjugated to SONPs, and in living cells, showing promising potential for ratiometric GSH detection.

Anti-OTC antibody-conjugated fluorescent magnetic/silica and fluorescent hybrid silica nanoparticles for oxytetracycline detection

Abstract This study presents two alternative fluorescent nanoparticle-based oxytetracycline (OTC) detection methods in milk samples. Rhodamine 6G-coated fluorescent hybrid silica nanoparticles and fluorescent magnetic/silica nanoparticles functionalized with anti-OTC antibodies were used in this test. The sandwich test format was utilized to compare anti-OTC antibody-conjugated fluorescent magnetic/silica nanoparticles with OTC/OTC antibody-conjugated fluorescent hybrid silica nanoparticles in an Eppendorf tube with magnetic separators. The magnetic separator helps to quickly retain all of the OTC captured by fluorescent magnetic core–shell nanoparticles in the milk sample. As a result, the assay time was dramatically shortened. The obtained linear range was 1.34 × 10−6 to 2.10 × 10−8 (M) (R 2 = 0.9954), the detection limit was 4.76 ng/mL, and the total assay time was 90 min. This approach was used to determine the OTC concentration in milk samples, and the maximum percentage (%) of interference was less than 3.0%, with a recovery rate of greater than 97.0%. This approach offers a high potential for residue detection in milk samples. With a total analysis period of less than 90 min, this approach provided the best way to determine the capture and detector nanoparticles’ response.

Click generated photochromic naphthalenediimide-dithienylethene diad: Application in deciphering secret codes

Photochromic molecules with efficient fluorescence switching capability remain a core research interest among the scientific community for the application in security technologies and anti-counterfeiting measures. A dithienylethene-naphthalene diimide diad was designed and synthesized using Cu(I) catalysed click reaction between a fluorescent core substituted naphthalene diimide (cNDI) and photochromic dithienylethene (DTE) molecule, which demonstrated excellent photocyclization (ksolution = 0.0359 s−1, ksolid = 0.0122 s−1) and photocycloreversion (ksolution = 0.01737 s−1, ksolid = 0.00456 s−1) dynamics with high fluorescence quantum yield (5o ΦF = 0.39, PSS ΦF = 0.05). The fluorescence property of the molecule was switched ‘on-off’ by UV and visible light irradiation respectively via Förster resonance energy transfer (FRET) mechanism in solution state as well as in solid state, which was further corroborated with DFT studies. In view of efficient fluorescence switching behaviour and high fatigue resistance properties (25 cycles), the molecule was successfully applied in secret code encryption-decryption purposes using barcode demonstrating real-life applicability of the molecule in security technologies.

Cyanine-induced apoptosis for cancer therapy

Apoptosis is a programmed cell death characterized by cell rounding, chromatin condensation and clumping, cytoplasmic collapse, and the formation of apoptotic bodies. Recently, increasing scientific research has disclosed the close relationship between apoptosis and cyanine. Cyanine, due to its excellent fluorescent characteristics, is used in constructing vital fluorescent cores for fluorescent probes. It has been extensively utilized in targeted labeling for growing cells and tumor phototherapy. Cyanine-derived compounds have provided new approaches for treating different cancers by inducing tumor cell apoptosis. This review introduces the molecular mechanism of apoptosis, essential apoptosis-related proteins, and the pathways related to tumor cell apoptosis. Furthermore, it systematically discusses the function of cyanine in cancer therapy by inducing apoptosis. A deeper understanding of cyanine-induced apoptosis may offer new options for cancer treatment and new strategies for developing and utilizing cyanine.

Rotational diffusion of colloidal microspheres near flat walls.

Recently, colloids with an off-center fluorescent core and homogeneous composition have been developed to measure the rotational diffusivity of microparticles using 3D confocal microscopy in refractive index-matched suspensions. Here, we show that the same particles may be imaged using a standard fluorescence microscope to yield their rotational diffusion coefficients. Trajectories of the off-center core may be combined with known expressions for the correlation decay of particle orientations to determine an effective rotational diffusivity. For sedimented particles, we also find the rotational diffusivity about axes perpendicular and parallel to the interface by adding some bright field illumination and simultaneously tracking both the core and the particle. Trajectories for particles of different sizes yield excellent agreement with hydrodynamic models of rotational diffusion near flat walls, taking the sedimentation-diffusion equilibrium into account. Finally, we explore the rotational diffusivity of particles in crowded two-dimensional monolayers, finding a different reduction of the rotational motion about the two axes depending on the colloidal microstructure.

Preparation and characterization of dually fluorescent core–shell particles and dual-sensing membranes for simultaneous detection of pH and dissolved oxygen

In this study, two dually fluorescent core–shell particles based on the response of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) and platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) were prepared for simultaneous detection of dissolved oxygen (pO2) and pH. The first involved forming a core–shell structure by capturing PtP in polystyrene (PS) particles as the core (PS@PtP) and then coating HPTS on the surface of PS@PtP via a supporting layer of melamine–formaldehyde resin as the shell. The second comprised covalently binding HPTS to a shell layer of sol–gel coated onto the surface of PS@PtP. The fluorescent particles from either method were immobilized in a supporting polymer matrix of ethyl cellulose (EC) and polyurethane hydrogel HydroMed D4 (D4) to produce the dual-sensing membranes. The resulting two dual-sensing membranes showed high sensitivity to pO2 in the range of 0–100% pO2 and to pH in the range of pH 5–9. They also showed high reproducibility, less interference, and long-term stability. A ratiometric fluorescence method was also applied to the dual emissions from HPTS and PtP to correct for a variety of analyte-independent factors and to provide precise quantitative analysis.

Roth's switch-on fluoremetric probe for green tracking and quantifying of 1.4-dihydropyridine medication: Evaluation of greenness, whiteness, and blueness

Facile incorporation of a fluorescence-modulating moiety to decorate a molecular derivatized system for an ultrasensitive quantifying of an individual of 1,4-dihydropyridine antihypertensive medication, amlodipine (AML). The strategy is based on a derivatized condensation reaction in an alkaline buffered solution between AML and Roth's reagent system. The produced fluorescent core was tracked by the fluorimetric means at 443 nm, subsequent to excitation at 338 nm. A linear response was observed within the 0.05–0.75 μg mL−1 concentration range of AML with LOD and LOQ of 0.0132 and 0.0424 μg mL−1, respectively. The validation of the method and the determination of its quantum yield were evaluated in accordance with the guidelines established by the ICH. The proposed investigation was utilized to precisely assess the specified pharmaceutical product in both bulk powder and dosage forms, with no impact from excipients. The planned system was finally assessed from an ecological standpoint using various modern ecological metrics. Additionally, the research strived to achieve two primary objectives centered around environmental and sustainability-related aspects. Two green metrics, the Green Certificate Classification (GCC) and the Analytical Greenness software (AGREE), were implemented to accomplish this evaluation. Furthermore, the developed method's sustainability levels were assessed by considering its whiteness scores. These scores were derived in accordance with the principles of White Analytical Chemistry (WAC). The evaluation was carried out using an assessment tool known as (RGB12), which takes into consideration the red, green, and blue color components. As well as a recently introduced tool known as the Blue Applicability Grade Index (BAGI) was used for assessing the practicality (blueness) of the fluorometric methods.

Fluorescent protein chromophore-based photosensitizer modified with anthracenyl for lysosome-targeted photodynamic therapy and two-photon fluorescence imaging

In this work, two cis-anthracenyl fluorescent protein chromophore analogs, AnFP-1 and AnFP-2 were synthesized by [2 + 3] cycloaddition for photodynamic therapy and two-photon fluorescence imaging. We combine the anthracenyl group and fluorescent core imidazolinone to form the dye AnFP-1, and then the morpholine indole structure was introduced via Knoevenagel reaction to obtain the dye AnFP-2. The heavy-atom-free photosensitizing dyes AnFP-1 and AnFP-2 have satisfied yields of singlet oxygen, measured to be 30% and 21% in methanol solution, respectively. The tetrazolium blue (MTT) experiment showed that the survival rate of A549 cells was over 92% without light exposure and under 460 nm light exposure for 10 min, the half-inhibitory concentrations (IC50) of the photosensitizing dyes AnFP-1 and AnFP-2 were 3.7 μmol•L−1 and 4.1 μmol•L−1, respectively. Acridine orange (AO)/Ethidium bromide (EB) double staining experiments showed that the two photosensitizing dyes could generate singlet oxygen and induce apoptosis in cancerous cells under irradiation. In addition, the photosensitizing dye AnFP-2 has high specificity to lysosomes which improve the photodynamic effect. Its Pearson co-localization correlation coefficient is 93%. In biological imaging, AnFP-1 and AnFP-2 showed satisfactory two-photon fluorescence imaging in zebrafish with 800 nm femtosecond laser excitation. According to these results, AnFP-1 and AnFP-2 can be used in photodynamic therapy and two-photon fluorescence imaging, which lays the foundation for developing new photosensitizing dyes.

Polysiloxane-based hyperbranched fluorescent probe for dynamic visualization of HClO in lysosomes and vivo.

As a type of reactive oxygen species, hypochlorous acid (HClO) is associated with inducing oxidative stress in lysosomes. Once its concentration is abnormal, it may lead to lysosomal rupture and subsequent apoptosis. Meanwhile, this may provide new inspiration for cancer treatment. Therefore, it is crucial to visualize HClO in lysosomes at the biological level. So far, numerous fluorescent probes have emerged to identify HClO. However, fluorescent probes that combine low biotoxicity with lysosome-targetable properties are scarce. In this paper, hyperbranched polysiloxanes were modified by embedding perylenetetracarboxylic anhydride red fluorescent cores with naphthalimide derivative green fluorophores to synthesize novel fluorescent probe (PMEA-1). PMEA-1 was a lysosome-targetable fluorescent probe with unique dual emission, high biosafety, and good response speed. PMEA-1 exhibited excellent sensitivity and responsiveness to HClO in PBS solution and could dynamically visualize HClO fluctuations in cells and zebrafish. Simultaneously, PMEA-1 also had monitoring ability for HClO produced in the process of cellular ferroptosis. In addition, the bioimaging results indicated that PMEA-1 was capable of accumulating within the lysosomes. We anticipate that PMEA-1 will broaden the application of silicon-based fluorescent probes in the field of fluorescence imaging.

A fluorescent magnetic core–shell nanosensor for detection of copper ions in natural waters

A nanosensor based on magnetic core–shell nanoparticles functionalized with rhodamine derivative, N-(3-carboxy)acryloyl rhodamine B hydrazide (RhBCARB), using (3-aminopropyl)triethoxysilane (APTES) as a linker, has been synthesized for detection of Cu(II) ions in water. The magnetic nanoparticle and the modified rhodamine were fully characterized, showing a strong orange emission sensitive to Cu(II) ions. The sensor shows a linear response from 10 to 90 µg L−1, detection limit of 3 µg L−1 and no interference of Ni(II), Co(II), Cd(II), Zn(II), Pb(II), Hg(II) and Fe(II) ions. The nanosensor performance is similar to those described in the literature, being a viable option for the determination of Cu(II) ions in natural waters. In addition, the magnetic sensor can be easily removed from the reaction medium with the aid of a magnet and its signal recovered in acidic solution, allowing its reuse in subsequent analysis.

Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling.

Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive β-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.

Antibody Functionalization of Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticle Probes for Advanced Intracellular Labeling and Optical Super Resolution Microscopy

Fluorescent labeling of cellular substructures is commonly performed using antibody–organic dye conjugates. Organic dyes do not exhibit ideal optical properties in terms of brightness and photostability, however, in particular when it comes to advanced optical super-resolution microscopy (SRM) applications. Here, we demonstrate the efficient conjugation of widely available secondary antibodies and cationic species to ultrasmall (sub-10 nm) fluorescent silica core─poly(ethylene glycol) shell (core–shell) aluminosilicate nanoparticles (aC′ dots) encapsulating different color dyes for specific targeting and high-quality fluorescence imaging of structures of the cytoskeleton (tubulin and actin) and nucleus, respectively. We show that the different color aC′ dots provide enhanced brightness and photostability relative to their parent dyes. As recently discovered, we further demonstrate that they exhibit photo-induced blinking with low ON–OFF duty cycles enabling optical SRM, for example, in the form of stochastic optical reconstruction microscopy (STORM), without the need for complex imaging setups or cocktails. After carefully optimizing Ab–aC′ dot conjugation as well as cell structure labeling protocols in fixed and permeabilized HeLa and MDA-MB-231 cells, we demonstrate three-color STORM and exemplify improved resolution compared to standard antibody–dye conjugates. This work paves the way to next-generation multifunctional optical probes based on ultrasmall silica nanoparticle platforms for advanced applications in bioimaging, nanomedicine, and beyond.

Xanthene, cyanine, oxazine and BODIPY: the four pillars of the fluorophore empire for super-resolution bioimaging.

In the realm of biological research, the invention of super-resolution microscopy (SRM) has enabled the visualization of ultrafine sub-cellular structures and their functions in live cells at the nano-scale level, beyond the diffraction limit, which has opened up a new window for advanced biomedical studies to unravel the complex unknown details of physiological disorders at the sub-cellular level with unprecedented resolution and clarity. However, most of the SRM techniques are highly reliant on the personalized special photophysical features of the fluorophores. In recent times, there has been an unprecedented surge in the development of robust new fluorophore systems with personalized features for various super-resolution imaging techniques. To date, xanthene, cyanine, oxazine and BODIPY cores have been authoritatively utilized as the basic fluorophore units in most of the small-molecule-based organic fluorescent probe designing strategies for SRM owing to their excellent photophysical characteristics and easy synthetic acquiescence. Since the future of next-generation SRM studies will be decided by the availability of advanced fluorescent probes and these four fluorescent building blocks will play an important role in progressive new fluorophore design, there is an urgent need to review the recent advancements in designing fluorophores for different SRM methods based on these fluorescent dye cores. This review article not only includes a comprehensive discussion about the recent developments in designing fluorescent probes for various SRM techniques based on these four important fluorophore building blocks with special emphasis on their effective integration into live cell super-resolution bio-imaging applications but also critically evaluates the background of each of the fluorescent dye cores to highlight their merits and demerits towards developing newer fluorescent probes for SRM.

Fluorescent naphthalimide boronates as theranostics: structural investigations, confocal fluorescence and multiphoton fluorescence lifetime imaging microscopy in living cells.

New design and synthetic strategies were developed to generate functional phenyl boronic acid (BA)-based fluorescent probes incorporating the 1,8-naphthalimide (NI) tag. This fluorescent core was anchored onto the BA unit through small organic linkers consisting of nitrogen groups which can arrest, and internally stabilise the phenyl-boronate units. The newly synthesised fluorophores were characterised spectroscopically by NMR spectroscopy and mass spectrometry and evaluated for their ability to bind to a naturally occurring polysaccharide, β-d-glucan in DMSO and simultaneously as act as in vitro cell imaging reagents. The uptake of these new NI-boronic acid derivatives was studied living cancer cells (HeLa, PC-3) in the presence, and absence, of β-d-glucan. Time-correlated single-photon counting (TCSPC) of DMSO solutions and two-photon fluorescence-lifetime imaging microscopy (FLIM) techniques allowed an insight into the probes' interaction with their environment. Their cellular uptake and distributions were imaged using laser scanning confocal fluorescence microscopy under single- and two-photon excitation regimes (λmax 910 nm). FLIM facilitated the estimation of the impact of the probe's cellular surroundings using the fluorophore lifetime. The extent to which this was mediated by the β-d-glucan was visualised by 2-photon FLIM in living cells. The fluorescence lifetime observed under a range of temperatures varied appreciably, indicating that changes in the environment can be sensed by these probes. In all cases, the cellular membrane penetration of these new probes was remarkable even under variable temperature conditions and localisation was widely concentrated in the cellular cytoplasm, without specific organelle trapping: we conclude that these new probes show promise for cellular imaging in living cancer cells.

Magnetoresponsive fluorescent core-shell nanoclusters for biomedical applications.

Nowadays, superparamagnetic iron oxide nanoparticles (SPIONs) have a dominant role in many subfields of biomedicine. Owing to their peculiar properties, they can be employed for magnetic separation, drug delivery, diagnostics, and hyperthermia treatments. However, these magnetic nanoparticles (NPs) suffer from low unit magnetization due to size constraints (up to 20-30 nm) to exhibit superparamagnetic character. In this work, we have designed and synthesized superparamagnetic nanoclusters (SP-NCs) with diameters of up to 400 nm with high unit magnetization for enhanced loading capacity. These were synthesized with conventional or microwave-assisted solvothermal methods, in the presence of either of the two biomolecules (citrate or l-lysine) as the capping agent. Primary particle size, SP-NC size, surface chemistry, and the resultant magnetic properties were observed to be significantly influenced by the choice of synthesis route and capping agent. Selected SP-NCs were then coated with a fluorophore-doped silica shell to provide fluorescence properties, in the near-infrared spectrum region, while silica provided high chemical and colloidal stability. Heating efficiency studies were performed under alternating magnetic field on the synthesized SP-NCs, highlighting their potential in hyperthermia treatment. We envision that their enhanced magnetically-active content, fluorescence, magnetic property, and heating efficiency will pave the way to more effective uses in biomedical applications.

Prospects of Genetically Encoded Flim Indicators for the Quantitative Assessment of Intracellular Parameters

A significant number of genetically encoded indicators based on fluorescent proteins that allow detecting changes in various parameters: membrane potential shift, pH, concentrations of hydrogen peroxide, lactate, pyruvate, NAD+/NADH, ATP, calcium cations, etc. have been created. Some of them (for example, indicators of calcium cations and hydrogen peroxide) are successfully used by numerous groups of researchers in experiments in vivo. The fundamental limitation of most fluorescent indicators is their ability to only qualitatively assess the change in the parameter of interest in the cell. The nature of this limitation is that it is difficult to compare the absolute value of the measured parameter and the detected fluorescent signal for various reasons. Quantitative measurements impose an additional requirement on the indicator signal: it should not depend on the technical features of the experiment (such as the level of protein expression, the type and power of the excitation source, a set of fluorescent filters, etc.). The fulfillment of this requirement may be associated with the detection of the indicator signal in the time domain, where the main characteristic of the signal is the fluorescence lifetime. The fluorescence lifetime is an absolute value, insensitive to changes in the intensity of the excitation source, detection settings, losses in the optical path or sample, changes in the concentration of the fluorophore in the sample, photobleaching. Thus, when calibrating an indicator in the time domain, a specific value of the measured parameter is associated with a specific value of the fluorescence lifetime. The time-resolved imaging mode imposes its own limitations on the properties of the indicator's fluorescent core. Thus, the lifetime of its fluorescence should change in proportion to the value of the measured parameter, which is not characteristic of any fluorophore. In this work, we make a brief review of the principles of detection of the fluorescent signal of genetically encoded indicators, focusing on current approaches related to the analysis of time-resolved data.