Increase In Fluorescence Lifetime Research Articles

Spectroscopic investigation of the quadruplex DNA‐binding properties of 9‐aryl‐substituted isoquinolinium derivatives

The spectroscopic investigation of the binding properties of berberine‐type 9‐aryl‐substituted isoquinolinium derivatives with G‐quadruplex DNA (G4‐DNA) are presented. Photometric titrations show that these ligands bind with high affinity to the telomeric G4‐DNA form 22AG (K = 1.0–44 ´ 105 M−2). Furthermore, fluorimetric analysis of thermal DNA denaturation (FRET melting) reveals a significant thermal stabilization of G4‐DNA 22AG upon association with the methoxy‐substituted derivatives. As an analytically useful property, the derivatives with a phenyl substituent or with additional electron‐donating groups show a very weak fluorescence intensity, which increased significantly upon G4‐DNA complexation (fluorescence light‐up effect). Additional time‐resolved fluorescence spectroscopy indicated increased fluorescence lifetimes of the DNA‐bound 9‐(4‐methoxy­phenyl)­substituted derivative, when interacting with the quadruplex forming strand 22AG. Notably, the changes of the steady‐state and time‐resolved emission properties of the ligand are more pronounced with G4‐DNA than with duplex DNA so that the combination of these complementary methods may be used for the selective G4‐DNA detection.

Fluorescence lifetime of plant leaves with sub-nanosecond resolution

The study aimed to develop a measurement apparatus for in vivo chlorophyll-a (Chl-a) fluorescence decay measurements of plants by means of time correlated single photon counting. In this approach, sub-nanosecond laser pulses with a repetition rate of 10 MHz are applied to excite the sample, followed by the analysis of arrival times of the emitted fluorescence photons. Photon statistics are generated by iteratively fitting the sum of two exponential functions. The tool was tested on both plastid and in vivo leaf samples of Savoy cabbage (Brassica oleracea var. sabauda) with 3–4 subsequent leaves giving a complete sample coverage starting from the outermost. The Chl-a fluorescence lifetime exhibited a gradual increase in both the isolated plastid suspensions and the in vivo leaf samples towards the innermost leaf layers explained by an increase of natural absence of light (etiolation syndrome). Furthermore, cadmium stress and iron deficiency were investigated on treated sugar beet (Beta vulgaris) samples in vivo using TCSPS measurements. The reduced fluorescence quenching resulted in an increased fluorescence lifetime. Finally, a long-term (10 week) testing of the setup was carried out on Chl-retaining resurrection Haberlea rhodopensis plants protecting themselves by an elevated non-photochemical quenching yielding a decrease of fluorescence lifetime during their desiccation.

Intercavity polariton slows down dynamics in strongly coupled cavities

Band engineering stands as an efficient route to induce strongly correlated quantum many-body phenomena. Besides inspiring analogies among diverse physical fields, tuning on demand the group velocity is highly attractive in photonics because it allows unconventional flows of light. Λ-schemes offer a route to control the propagation of light in a lattice-free configurations, enabling exotic phases such as slow-light and allowing for highly optical non-linear systems. Here, we realize room-temperature intercavity Frenkel polaritons excited across two strongly coupled cavities. We demonstrate the formation of a tuneable heavy-polariton, akin to slow light, appearing in the absence of a periodic in-plane potential. Our photonic architecture based on a simple three-level scheme enables the unique spatial segregation of photons and excitons in different cavities and maintains a balanced degree of mixing between them. This unveils a dynamical competition between many-body scattering processes and the underlying polariton nature which leads to an increased fluorescence lifetime. The intercavity polariton features are further revealed under appropriate resonant pumping, where we observe suppression of the polariton fluorescence intensity.

Photophysical, rotational and translational properties of Radachlorin photosensitizer upon binding to serum albumins

IntroductionAlthough photophysical properties of Radachlorin photosensitizer (PS) were extensively studied in solutions and cells, no data is available on variations of its characteristics upon binding to serum albumins, which are major transporters in blood and nutrients in cell culture media. ObjectivesThe primary objective of this study was to analyze changes in photophysical properties of Radachlorin molecules upon their binding to human and bovine serum albumins at different microenvironment properties. MethodsExperiments were performed using time-resolved fluorescence spectroscopy and fluorescence recovery after photobleaching. Variations in fluorescence spectra and lifetime, fluorescence anisotropy, rotational and translational diffusion of PS molecules upon binding to albumins were studied in normal, basic and acidic conditions and at different concentrations of albumin and PS molecules. ResultsRadachlorin molecules effectively bind to both types of serum albumins, which causes changes in photophysical properties of the PS. A minor red shift of the fluorescence spectrum, an increase in fluorescence lifetime and anisotropy and substantial decrease of translational and rotational mobility of PS molecules were observed upon their binding to albumins. The analysis of rotational diffusion time provided robust evaluation of the bound fraction of PS molecules. Both the highly acidic microenvironment and increase in alcohol concentration above 40% resulted in detachment of PS molecules from albumins. Photophysical properties of Radachlorin in complexes with BSA and HSA were found to be slightly different. ConclusionsBinding of Radachlorin photosensitizer to either BSA or HSA affects significantly its photophysical properties, which may also vary with microenvironment acidity and alcohol concentration.

Photo-Controlled Nano-Supramolecular Size and Reversible Luminescent Behaviors Based on Cucurbit[7]uril Cascaded Assembly.

Supramolecular luminescent material with switchable behavior and photo-induced aggregation with emission enhancement is a current research hot spot. Herein, a size-tunable nano-supramolecular assembly with reversible photoluminescent behavior was constructed by noncovalent polymerization of diarylethene-bridged bis(coumarin) derivative (DAE-CO), cucurbit[7]uril (CB[7]), and β-cyclodextrin-grafted hyaluronic acid (HACD). Benefiting from the macrocyclic confinement effect, the guest molecule DAE-CO was included into the cavity of CB[7] to give enhanced fluorescence emission of the resulting DAE-CO⊂CB[7]2 with longer lifetime at 432 nm to 1.43 ns, thereby further enhancing fluorescence output and lifetime (1.46 ns) when further assembled with HACD, compared with the free DAE-CO (0.95 ns). In addition, DAE-CO, DAE-CO⊂CB[7]2, and DAE-CO⊂CB[7]2&HACD all possessed characteristics of aggregation-induced emission and reversible photo-switched structural interconversion, exhibiting an obvious photophysical activation phenomenon of self-aggregation into larger nanoparticles with increase in fluorescence emission intensity, lifetime, and size after irradiation, which could be increased step by step with the alternating irradiation of 254 nm (5 min) or >600 nm (30 s) repeated 7 times. These supramolecular assemblies were successfully used in the tumor cells' targeted imaging and anti-counterfeiting because of the capability of HACD for recognizing specific receptors overexpressed on the surface of tumor cells and the excellent photo-regulated switch ability of DAE-CO, providing an approach of constructing photo-induced emission-enhanced luminescent materials.

Fluorescence Lifetime of Chlorophyll α in Oxidized Edible Vegetable Oil.

To promote the application of time-resolved fluorescence in oxidation determination of edible vegetable oil, fluorescence lifetime of chlorophyll α in oxidized edible vegetable oils was recorded and analyzed by simulated microenvironment experiments and spectral methods. It was showed that fluorescence lifetime of chlorophyll α decreased with the increase of polarity in the early stage of oxidation, and increased with the increase of viscosity in the later stage of oxidation. Conjugation effect and hydrogen bonding existed in the microenvironment of oxidized edible vegetable oil were considered to be the factors leading to the increase of fluorescence lifetime. The change mechanism of fluorescence lifetime in oxidized edible vegetable oil was supplied, which was considered to be strong support for the application of time-resolved fluorescence.

Two-photon fluorescence lifetime imaging microscopy of NADH metabolism in HIV-1 infected cells and tissues.

Rapid detection of microbial-induced cellular changes during the course of an infection is critical to understanding pathogenesis and immunological homeostasis. In the last two decades, fluorescence imaging has received significant attention for its ability to help characterize microbial induced cellular and tissue changes in in vitro and in vivo settings. However, most of these methods rely on the covalent conjugation of large exogenous probes and detection methods based on intensity-based imaging. Here, we report a quantitative, intrinsic, label-free, and minimally invasive method based on two-photon fluorescence lifetime (FLT) imaging microscopy (2p-FLIM) for imaging 1,4-dihydro-nicotinamide adenine dinucleotide (NADH) metabolism of virally infected cells and tissue sections. To better understand virally induced cellular and tissue changes in metabolism we have used 2p-FLIM to study differences in NADH intensity and fluorescence lifetimes in HIV-1 infected cells and tissues. Differences in NADH fluorescence lifetimes are associated with cellular changes in metabolism and changes in cellular metabolism are associated with HIV-1 infection. NADH is a critical co-enzyme and redox regulator and an essential biomarker in the metabolic processes. Label-free 2p-FLIM application and detection of NADH fluorescence using viral infection systems are in their infancy. In this study, the application of the 2p-FLIM assay and quantitative analyses of HIV-1 infected cells and tissue sections reveal increased fluorescence lifetime and higher enzyme-bound NADH fraction suggesting oxidative phosphorylation (OxPhos) compared to uninfected cells and tissues. 2p-FLIM measurements improve signal to background, fluorescence specificity, provide spatial and temporal resolution of intracellular structures, and thus, are suitable for quantitative studies of cellular functions and tissue morphology. Furthermore, 2p-FLIM allows distinguishing free and bound populations of NADH by their different fluorescence lifetimes within single infected cells. Accordingly, NADH fluorescence measurements of individual single cells should provide necessary insight into the heterogeneity of metabolic activity of infected cells. Implementing 2p-FLIM to viral infection systems measuring NADH fluorescence at the single or subcellular level within a tissue can provide visual evidence, localization, and information in a real-time diagnostic or therapeutic metabolic workflow.

Two-Photon Excited Fluorescence Lifetime Imaging of Tetracycline-Labeled Retinal Calcification.

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE). We found that these mineral deposits could be imaged by fluorescence using tetracycline antibiotics as specific stains. Binding of tetracyclines to the minerals was accompanied by increases in fluorescence intensity and fluorescence lifetime. The lifetimes for tetracyclines differed substantially from the known background lifetime of the existing natural retinal fluorophores, suggesting that calcification could be visualized by lifetime imaging. However, the excitation wavelengths used to excite these lifetime changes were generally shorter than those approved for retinal imaging. Here, we show that tetracycline-stained drusen in post mortem human retinas may be imaged by fluorescence lifetime contrast using multiphoton (infrared) excitation. For this pilot study, ten eyes from six anonymous deceased donors (3 female, 3 male, mean age 83.7 years, range 79-97 years) were obtained with informed consent from the Maryland State Anatomy Board with ethical oversight and approval by the Institutional Review Board.

1H- and 2H-1,2,3-triazoles hybrids: Comparative study of photophysical properties

The photophysical properties of three types of hybrid molecules, containing two 1,2,3-triazole isomers with different spatial arrangements and set of surrounding substituents, were investigated. The effect of the heterocyclic scaffold construction and the nature and position of the substituents were revealed by spectral and computational investigations. UV–Vis absorption spectra showed maxima with high molar extinction coefficients. Moderate blue emission (QYs up to 42.2%) and average fluorescence life-time of 1.05–4.53 ns were determined. Positive solvatochromism and intramolecular charge transfer were detected and confirmed by the quantum mechanical calculations. A peculiar effect of a hydrogen bond formation was registered in MeOH, EtOH, i-PrOH, ethylene glycol (EG) solutions and water mixtures. The compounds exhibited an Aggregation Induced Emission Enhancement (AIEE) effect in DMSO-H2O mixtures with a 1.3–3.1 fold increase of QYs and a 1.1–2.8 fold increase of fluorescence life-time. Molecular geometries and electronic characteristics were studied at (Time Dependent) Density Functional Theory ((TD-)DFT) level. The biological behavior of 2H-bis[1,2,3]triazolo)[5,1-a:4′,5′-c]isoquinolines was studied by means of confocal microscopy experiments, which showed that these compounds successfully penetrate the cell membranes, accompanied by bright emission in the cytoplasmic region of the confocal micrographs.

Bifunctional Single-Molecular Fluorescent Probe: Visual Detection of Mitochondrial SO2 and Membrane Potential.

Mitochondrial membrane potential (MMP) and sulfur dioxide (SO2) significantly affect the mitochondrial state. In this work, TC-2 and TC-8 were constructed through side-chain engineering, in which TC-2 bearing the poorer hydrophobicity could localize on mitochondria better. Interestingly, short-wave emission was captured due to the sensitive response of TC-2 to SO2 (LOD = 13.8 nM). Meanwhile, the probe could bind with DNA, presenting enhanced long-wave emission. Encouragingly, TC-2 could migrate from mitochondria to the nucleus when MMP was decreased, accompanied by the increase of fluorescence lifetime (9-fold). Hence, TC-2 could be used for dual-channel monitoring of mitochondrial SO2 and MMP, which showed a completely different pathway from the commercial MMP detectors JC-1/JC-10. The cellular experiments showed that MMP was gradually decreased due to reactive oxygen species-triggered oxidative stress, and the SO2 level was up-regulated simultaneously. Overall, this work proposed a new method to investigate and diagnose the mitochondrial-related diseases.

Enhanced Solid-State Fluorescence of Flavin Derivatives by Incorporation in the Metal-Organic Frameworks MIL-53(Al) and MOF-5.

The flavin derivatives 10-methyl-isoalloxazine (MIA) and 6-fluoro-10-methyl-isoalloxazine (6F-MIA) were incorporated in two alternative metal-organic frameworks, (MOFs) MIL-53(Al) and MOF-5. We used a post-synthetic, diffusion-based incorporation into microcrystalline MIL-53 powders with one-dimensional (1D) pores and an in-situ approach during the synthesis of MOF-5 with its 3D channel network. The maximum amount of flavin dye incorporation is 3.9 wt% for MIA@MIL-53(Al) and 1.5 wt% for 6F-MIA@MIL-53(Al), 0.85 wt% for MIA@MOF-5 and 5.2 wt% for 6F-MIA@MOF-5. For the high incorporation yields the probability to have more than one dye molecule in a pore volume is significant. As compared to the flavins in solution, the fluorescence spectrum of these flavin@MOF composites is broadened at the bathocromic side especially for MIA. Time-resolved spectroscopy showed that multi-exponential fluorescence lifetimes were needed to describe the decays. The fluorescence-weighted lifetime of flavin@MOF of 4 ± 1 ns also corresponds to those in solution but is significantly prolonged compared to the solid flavin dyes with less than 1 ns, thereby confirming the concept of "solid solutions" for dye@MOF composites. The fluorescence quantum yield (ΦF) of the flavin@MOF composites is about half of the solution but is significantly higher compared to the solid flavin dyes. Both the fluorescence lifetime and quantum yield of flavin@MOF decrease with the flavin loading in MIL-53 due to the formation of various J-aggregates. Theoretical calculations using plane-wave and QM/MM methods are in good correspondence with the experimental results and explain the electronic structures as well as the photophysical properties of crystalline MIA and the flavin@MOF composites. In the solid flavins, π-stacking interactions of the molecules lead to a charge transfer state with low oscillator strength resulting in aggregation-caused quenching (ACQ) with low lifetimes and quantum yields. In the MOF pores, single flavin molecules represent a major population and the computed MIA@MOF structures do not find π-stacking interactions with the pore walls but only weak van-der-Waals contacts which reasons the enhanced fluorescence lifetime and quantum yield of the flavins in the composites compared to their neat solid state. To analyze the orientation of flavins in MOFs, we measured fluorescence anisotropy images of single flavin@MOF-5 crystals and a static ensemble flavin@MIL53 microcrystals, respectively. Based on image information, anisotropy distributions and overall curve of the time-resolved anisotropy curves combined with theoretical calculations, we can prove that all fluorescent flavins species have a defined and rather homogeneous orientation in the MOF framework. In MIL-53, the transition dipole moments of flavins are orientated along the 1D channel axis, whereas in MOF-5 we resolved an average orientation that is tilted with respect to the cubic crystal lattice. Notably, the more hydrophobic 6F-MIA exhibits a higher degree order than MIA. The flexible MOF MIL-53(Al) was optimized essentially to the experimental large-pore form in the guest-free state with QuantumEspresso (QE) and with MIA molecules in the pores the structure contracted to close to the experimental narrow-pore form which was also confirmed by PXRD. In summary, the incorporation of flavins in MOFs yields solid-state materials with enhanced rigidity, stabilized conformation, defined orientation and reduced aggregations of the flavins, leading to increased fluorescence lifetime and quantum yield as controllable photo-luminescent and photo-physical properties.

Multiphoton FLIM Analyses of Native and UVA-Modified Synthetic Melanins.

To better understand the impact of solar light exposure on human skin, the chemical characterization of native melanins and their structural photo-modifications is of central interest. As the methods used today are invasive, we investigated the possibility of using multiphoton fluorescence lifetime (FLIM) imaging, along with phasor and bi-exponential fitting analyses, as a non-invasive alternative method for the chemical analysis of native and UVA-exposed melanins. We demonstrated that multiphoton FLIM allows the discrimination between native DHI, DHICA, Dopa eumelanins, pheomelanin, and mixed eu-/pheo-melanin polymers. We exposed melanin samples to high UVA doses to maximize their structural modifications. The UVA-induced oxidative, photo-degradation, and crosslinking changes were evidenced via an increase in fluorescence lifetimes along with a decrease in their relative contributions. Moreover, we introduced a new phasor parameter of a relative fraction of a UVA-modified species and provided evidence for its sensitivity in assessing the UVA effects. Globally, the fluorescence lifetime properties were modulated in a melanin-dependent and UVA dose-dependent manner, with the strongest modifications being observed for DHICA eumelanin and the weakest for pheomelanin. Multiphoton FLIM phasor and bi-exponential analyses hold promising perspectives for in vivo human skin mixed melanins characterization under UVA or other sunlight exposure conditions.

Cholesterol as a tool to probe the role of membrane in cochlear hair cell mechanotransduction.

Most mechanically gated channels are sensitive to force translated through the membrane. The lipid bilayer can modulate channel function directly through lipid/protein interactions or indirectly based on membrane mechanical properties. Cholesterol is an important component of the membrane and a major modulator of membrane mechanical properties. While other ion channels and mechanically gated channels can be activated or modulated by changes to the membrane cholesterol, the functional role of membrane cholesterol in regulation of mammalian cochlear mechanotransduction (MET) channels has not been closely investigated. Using whole-cell patch clamping and live-cell fluorescence lifetime imaging (FLIM) of a viscosity-sensitive molecular rotor BODIPY 1c for the first time in the inner ear, we examined the role of membrane cholesterol in modulating the MET response properties of rat cochlear hair cells. Molecular rotors are fluorophores for which the fluorescence lifetime (the average time a fluorophore remains in the excited state) increase with increasing viscosity of their immediate environment. We confirmed extraction of cholesterol, using methyl β cyclodextrin (MβCD), with reduced filipin staining in both inner and outer hair bundles. MβCD results in reversible reduction in fluorescence lifetime in hair bundles, suggesting initial reduction followed by gradual recovery in the stereocilia membrane viscosity. MβCD reversibly increases the channel resting open probability, suggesting that cholesterol depletion increases force transfer to the MET channel. Together this data suggests that the cell membrane is part of the force relay machinery to the MET channel and could possibly interact directly with components of the MET machinery. Further studies are needed to generate causal link between MET channel gating and membrane mechanics.

Ultrafast laser filament-induced fluorescence for detecting uranium stress in Chlamydomonas reinhardtii

Plants and other photosynthetic organisms have been suggested as potential pervasive biosensors for nuclear nonproliferation monitoring. We demonstrate that ultrafast laser filament-induced fluorescence of chlorophyll in the green alga Chlamydomonas reinhardtii is a promising method for remote, in-field detection of stress from exposure to nuclear materials. This method holds an advantage over broad-area surveillance, such as solar-induced fluorescence monitoring, when targeting excitation of a specific plant would improve the detectability, for example when local biota density is low. After exposing C. reinhardtii to uranium, we find that the concentration of chlorophyll a, chlorophyll fluorescence lifetime, and carotenoid content increase. The increased fluorescence lifetime signifies a decrease in non-photochemical quenching. The simultaneous increase in carotenoid content implies oxidative stress, further confirmed by the production of radical oxygen species evidence in the steady-state absorption spectrum. This is potentially a unique signature of uranium, as previous work finds that heavy metal stress generally increases non-photochemical quenching. We identify the temporal profile of the chlorophyll fluorescence to be a distinguishing feature between uranium-exposed and unexposed algae. Discrimination of uranium-exposed samples is possible at a distance of sim 35 m with a single laser shot and a modest collection system, as determined through a combination of experiment and simulation of distance-scaled uncertainty in discriminating the temporal profiles. Illustrating the potential for remote detection, detection over 125 m would require 100 laser shots, commensurate with the detection time on the order of 1 s.

Isomeric probes for monitoring mitochondrial membrane potential under fluorescence lifetime imaging microscopy

The mitochondrial membrane potential (ΔΨm) plays indispensable roles in important life processes such as cell metabolism and signal transduction. Fluorescent probes that can observe ΔΨm by fluorescence lifetime imaging are important molecular tools for accurate visualization and in-depth study of ΔΨm. However, such probes have been rarely reported, mainly because the deficiency of valid design strategies. In this work, we proposed a new strategy to alter the fluorescence lifetime of probes via changing their subcellular immigration, and developed two viscosity-responsive isomeric probes (PQO and PQP) to monitor ΔΨm under fluorescence lifetime imaging microscopy. These two probes were designed to target mitochondria in living cells while exhibit certain affinity to RNA. As a result, they stained mitochondria in healthy cells, while left mitochondria and bound to RNA when the ΔΨm decreased. Both PQO and PQP could respond to the viscosity changes via fluorescence lifetime, and showed significantly increased fluorescence lifetime upon binding to RNA. Consequently, the two probes displayed evidently different fluorescence lifetime in cells with high and low levels of ΔΨm, and thus enabled visualization of ΔΨm under fluorescence lifetime imaging microscopy.

3-aminoquinoline: a turn-on fluorescent probe for preferential solvation in binary solvent mixtures

3-Aminoquinoline (3AQ) has been used as a fluorescent probe for preferential solvation in hexane-ethanol solvent mixtures. Results of the present experiment have been put into context by comparison with prior observations with 5-aminoquinoline (5AQ) as the probe. 3AQ exhibits a relatively small change of dipole moment (Δμ = 2.2 D) upon photoexcitation, compared to 5AQ (Δμ = 6.1D), which might appear to be a hindrance in the way of its use as a solvation probe. Indeed, the values of parameters like spectral shifts are smaller for the present experiment with 3AQ. At the smallest concentration of alcohol used, its local mole fraction around the probe is significantly lower than in the previous experiments with 5AQ. However, these apparent disadvantages are outweighed by the significant increase in fluorescence intensity and lifetime observed with increasing concentration of ethanol in the solvent mixture, as opposed to the drastic fluorescence quenching that occurs for 5AQ. This is a marked advantage in the use of 3AQ in studies like the present one. The local mole fraction of ethanol and preferential solvation index experienced by 3AQ are in line with those reported for 5AQ. The disadvantage of the smaller magnitude of Δμ persists in the time resolved fluorescence experiments, for solvent mixtures with very low ethanol content. Negligible wavelength dependence of fluorescence transients of 3AQ is observed for x p = 0.002,. However, this effect is outweighed at higher alcohol concentrations, for which nanosecond dynamics of preferential solvation is observed.

Increase of Fluorescence of Humic-Like Substances in Interaction with Cd(II): a Photoinduced Charge Transfer Approach.

Described is the enhancement of fluorescence intensity due to the interaction of a humic-like substance (HLS 1%) extracted from process water (PW) and Cd(II) ions in aqueous solution. Using Canonical Polyadic/Parallel Factor Analysis (CP/PARAFAC), two main components were seen that contributed to fluorescence, the first one increased it and the second one kept it constant in both static and dynamic fluorescence studies. Two-dimensional FTIR analysis indicated that the interaction of HLS 1% and Cd(II) ions occurred in the following order of affinity with the groups: C-O bonds in polysaccharides > C-O bonds in carboxylic acid. The results obtained suggest that the increase in fluorescence intensity and lifetime suggest a photoinduced charge transfer (PCT) between Cd(II) ions and carboxylic acid groups present in HLS 1%.

Accumulation and penetration behavior of hypericin in glioma tumor spheroids studied by fluorescence microscopy and confocal fluorescence lifetime imaging microscopy

Glioblastoma WHO IV belongs to a group of brain tumors that are still incurable. A promising treatment approach applies photodynamic therapy (PDT) with hypericin as a photosensitizer. To generate a comprehensive understanding of the photosensitizer-tumor interactions, the first part of our study is focused on investigating the distribution and penetration behavior of hypericin in glioma cell spheroids by fluorescence microscopy. In the second part, fluorescence lifetime imaging microscopy (FLIM) was used to correlate fluorescence lifetime (FLT) changes of hypericin to environmental effects inside the spheroids. In this context, 3D tumor spheroids are an excellent model system since they consider 3D cell–cell interactions and the extracellular matrix is similar to tumors in vivo. Our analytical approach considers hypericin as probe molecule for FLIM and as photosensitizer for PDT at the same time, making it possible to directly draw conclusions of the state and location of the drug in a biological system. The knowledge of both state and location of hypericin makes a fundamental understanding of the impact of hypericin PDT in brain tumors possible. Following different incubation conditions, the hypericin distribution in peripheral and central cryosections of the spheroids were analyzed. Both fluorescence microscopy and FLIM revealed a hypericin gradient towards the spheroid core for short incubation periods or small concentrations. On the other hand, a homogeneous hypericin distribution is observed for long incubation times and high concentrations. Especially, the observed FLT change is crucial for the PDT efficiency, since the triplet yield, and hence the O2 activation, is directly proportional to the FLT. Based on the FLT increase inside spheroids, an incubation time > 30 min is required to achieve most suitable conditions for an effective PDT.Graphical abstract

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Visualization of Macrophase Separation and Transformation in Immiscible Polymer Blends

Label-Free Characterization of Amyloids and Alpha-Synuclein Polymorphs by Exploiting Their Intrinsic Fluorescence Property.

Conventional in vitro aggregation assays often involve tagging with extrinsic fluorophores, which can interfere with aggregation. We propose the use of intrinsic amyloid fluorescence lifetime probed using two-photon excitation and represented by model-free phasor plots as a label-free assay to characterize the amyloid structure. Intrinsic amyloid fluorescence arises from the structured packing of β-sheets in amyloids and is independent of aromatic-based fluorescence. We show that different amyloids [i.e., α-Synuclein (αS), β-Lactoglobulin (βLG), and TasA] and different polymorphic populations of αS (induced by aggregation in salt-free and salt buffers mimicking the intra-/extracellular environments) can be differentiated by their unique fluorescence lifetimes. Moreover, we observe that disaggregation of the preformed fibrils of αS and βLG leads to increased fluorescence lifetimes, distinct from those of their fibrillar counterparts. Our assay presents a medium-throughput method for rapid classification of amyloids and their polymorphs (the latter of which recent studies have shown lead to different disease pathologies) and for testing small-molecule inhibitory compounds.