Fluorescence Lifetime Studies Research Articles

Ratiometric Fluorescence Sensing of Cu(II): Elucidation of FRET Mechanism and Bio‐Imaging Application

AbstractRhodamine‐formylaniline conjugate RFA has been developed as a Cu2+‐selective ratiometric fluorescent probe. The probe shows high selectivity towards Cu2+ ion through both its absorption and emission properties; and has been characterized by 1H NMR, FTIR, ESI‐MS spectroscopy. Sensing of Cu2+ proceed through FRET process which is nicely depicted from steady‐state and fluorescence lifetime studies. From the measurement of fluorescence lifetime, the FRET mechanism has been established undoubtedly. The important parameters regarding FRET, namely the energy transfer efficiency (ΦEt) and the Förster distance (R0) have also been calculated by fluorescence measurement. The origin of different emission bands has been distinguished by recording excitation spectra. The lowest detection limit (DL) for Cu2+ ion was 4.2 ×10−7 M in Tris‐HCl buffered MeCN: H2O (10 mM, 1:1 v/v).

A Selective Turn off Fluorescence Sensor Based on Propranolol-SDS Assemblies for Fe3+ Detection.

A fluorophore modulation with sodium dodecyl sulphate (SDS) assemblies for the selective and sensitive sensing of Fe3+ ions in aqueous solution is illustrated in this work. Emission spectral characteristics of fluorescent molecule, propranolol (PPH) was intact in presence of metal ions. While on modulation with SDS assemblies, PPH was transformed into a tuneable sensor for Fe3+ ions. This sensor ensemble was not only highly sensitive towards Fe3+ ions in aqueous solution with detection limits lower than 3μM but also possess high discriminating efficiency in presence of other metal ions like Cu2+, Pb2+, Zn2+, Ni2+, Fe2+, Cd2+, Co2+, Al3+, Mg2+, Hg2+ and Mn2+. The electrostatic interaction of the anionic group of surfactants with the metal cations significantly increases the communication between metal ions and PPH moiety which results in the quenching of PPH fluorescence. We have employed fluorescence steady state and lifetime studies to understand the metal sensing behaviour of the PPH-SDS sensor system. Principal component analysis (PCA) was used to evaluate the discriminative ability of the developed sensor system towards Fe3+ ions.

Energy dissipation mechanisms in the FCPb light-harvesting complex of the diatom Cyclotella meneghiniana

Transient absorption spectroscopy has been applied to investigate the energy dissipation mechanisms in the nonameric fucoxanthin-chlorophyll-a,c-binding protein FCPb of the centric diatom Cyclotella meneghiniana. FCPb complexes in their unquenched state were compared with those in two types of quenching environments, namely aggregation-induced quenching by detergent removal, and clustering via incorporation into liposomes. Applying global and target analysis, in combination with a fluorescence lifetime study and annihilation calculations, we were able to resolve two quenching channels in FCPb that involve chlorophyll-a pigments for FCPb exposed to both quenching environments. The fast quenching channel operates on a timescale of tens of picoseconds and exhibits similar spectral signatures as the unquenched state. The slower quenching channel operates on a timescale of tens to hundreds of picoseconds, depending on the degree of quenching, and is characterized by enhanced population of low-energy states between 680 and 710 nm. The results indicate that FCPb is, in principle, able to function as a dissipater of excess energy and can do this in vitro even more efficiently than the homologous FCPa complex, the sole complex involved in fast photoprotection in these organisms. This indicates that when a complex displays photoprotection-related spectral signatures in vitro it does not imply that the complex participates in photoprotection in vivo. We suggest that FCPa is favored over FCPb as the sole energy-regulating complex in diatoms because its composition can more easily establish the balance between light-harvesting and quenching required for efficient photoprotection.

Synthesis, Photophysical Properties and Application of New Porphyrin Derivatives for Use in Photodynamic Therapy and Cell Imaging

New derivatives of tetrakis(4-carboxyphenyl) porphyrin were designed, synthesized and characterized by IR, proton NMR and mass spectroscopy. The ground and excited state nature of new derivatives were examined using UV-Vis. absorption and fluorescence spectroscopy, fluorescence quantum yield and fluorescence lifetime studies. The singlet oxygen quantum yield of each synthesized derivative of porphyrin was estimated for their further efficacy as potential photosensitizer in biological studies. The significant photophysical data of all synthesized derivatives was supplementary accessed to examine the cell imaging and cytotoxicity against two cancer cell lines viz. MBA-MD-231 and A375. The fluorescence lifetime, fluorescence quantum yield and efficiency of singlet oxygen generation suggests alkyl amine and alkyl hydrazide linked new porphyrin photosensitizers can be useful for PDT agent in cancer treatment.

A pyrene based fluorescent turn on chemosensor for detection of Cu2+ ions with antioxidant nature

The Copper ion recognition ability of a pyrene based sensor (1) was explored. A selective Cu2+ ion induced colorimetric naked eye changes and a turn on emission was observed in acetonitrile medium. The results were monitored through UV–vis spectrophotometry, steady state emission and fluorescence life time studies. The outcome revealed a selective and sensitive Cu2+ recognition with a detection limit of 2.5 × 10−6 M. The compound exhibited aggregation induced emission (AIE) in water-acetonitrile mixture where its critical aggregate concentration was measured by tensiometry at the solvent fraction of maximum AIE. Moreover the compound exhibited an antioxidant nature in DPPH (2,2-diphenyl-1-picrylhydrazyl) assay.

Functional Molecular System of Bis(pyrazolyl)pyridine Derivatives: Photophysics, Spectroscopy, Computation, and Ion Sensing.

A new series of conjugated donor−π–acceptor type of 2,6-bis(pyrazolyl)pyridine derivatives (compounds IK-(3–9)) have been synthesized via Horner–Wadsworth–Emmons (HWE) reaction, starting from a common phosphonate precursor and diverse donor aromatic aldehydes and characterized by routine spectral analysis including elemental analysis. Compound IK-2, one of the starting precursors, and molecule IK-3, the first member of the donor−π–acceptor series, are additionally characterized by single-crystal X-ray structure determination. Compounds IK-2 and IK-3 are crystallized in P1̅ (triclinic) and P21/c (monoclinic) space groups, respectively. The absorption maxima in the electronic spectra of the title compounds shift mainly due to intramolecular charge transfer (ICT) between different donor (dibutyl and cyclic pyrrolidine) groups and the acceptor moiety [2,6-bis(pyrazolyl) pyridine]. Solution-state emission spectral studies of all these compounds show large solvent sensitive behavior with significant amounts of Stokes shifts. The large solvent dependence of the emission indicates that the excited state is stabilized in more polar solvents due to the ICT. All chromophores exhibit solid-state fluorescence behavior except compound IK-7. The role of the position and nature of the donor functionalities in the conjugated backbone of overall donor moiety of compounds IK-(3–9), on the electronic absorption properties of the title chromophores has been demonstrated, which has further been corroborated by density functional theory (DFT) and time-dependent DFT (TDDFT) computational studies. The emission spectral results of compounds IK-3, IK-5, and IK-7 have also been supported by the DFT and TDDFT calculations. A fluorescence lifetime study on this series also shows that the excited states are stabilized in more polar solvents. Finally, one of the chromophores (chromophore IK-4) in the title series has been shown to act as a selective molecular sensor (turn-off switch) for the Cu(II) ion.

Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recognition by Damage Recognition Protein Rad4

How DNA-binding proteins search for and identify their target sites buried amid a vast excess of non-target sites remains a central question in biology. This study is aimed at elucidating the mechanism by which DNA damage recognition protein XPC recognizes diverse DNA lesions, and initiates DNA repair. Structural studies of Rad4 (yeast ortholog of XPC) bound to model lesions showed that DNA is unwound and bent at the lesion site and the damaged nucleotides are flipped out. Previous studies using laser T-jump perturbation approaches indicated that Rad4 interrogates potential sites by unwinding DNA on ~100-500 μs and eventually flips out damaged nucleotides on ~5-10 ms. However, these ‘ensemble’ measurements could not capture intrinsic DNA deformability that is potentially critical in the ability of Rad4/XPC to distinguish damaged from undamaged sites. Previous studies were also limited to measurements on short, linear DNA oligomers, while DNA in our cells is typically bent and supercoiled, which is expected to have a profound impact on DNA damage recognition. In this study, I employed an innovative fluorescence lifetime approach with fluorescent probes exquisitely sensitive to DNA twisting/bending fluctuations and mapped the range of conformations accessible to mismatched DNA substrates of varying Rad4-binding specificities. My results reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity mismatched DNA, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound. Conversely, nonspecific mismatched DNA retained a largely homogeneous, B-DNA-like conformation. Surprisingly, even mismatched-DNA specifically bound to Rad4 remained highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions. Using T-jump, I unveiled the rates of intrinsic DNA conformational dynamics in mismatched DNA. Remarkably, these rates remained unaltered in the presence of Rad4. Finally, I examined the effect of DNA bending strain on the intrinsic deformability at the mismatched sites by extending the fluorescence lifetime studies in the context of DNA minicircles. These studies revealed amplified distortions for both matched and mismatched sites in DNA minicircles, with further distortions detected upon Rad4 binding to mismatched sites. Taken together, my results strongly suggest that intrinsic DNA deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating ‘conformational capture’ of pre-distorted damaged sites.

Probing the temperature-dependent changes of the interfacial hydration and viscosity of Tween20 : cholesterol (1 : 1) niosome membrane using fisetin as a fluorescent molecular probe.

A detailed photophysical study of fisetin in a Tween20 : cholesterol (1 : 1) niosome membrane has been carried out. Fisetin is found to partition well into the Tween20 : cholesterol (1 : 1) niosome membrane at low temperature (Kp = 2.7 × 104 M-1 at 10 °C). Cetylpyridinium chloride quenching study confirms the location of fisetin molecules in the interfacial domain of Tween20 : cholesterol (1 : 1) niosome membrane. The emission from the prototropic forms of fisetin (neutral form, excited state anion, ground state anion and phototautomer form) is found to sensitively reflect the local heterogeneities in Tween20 : cholesterol (1 : 1) niosome membrane. The shift in anionic emission maximum with variation in temperature shows the sensitivity of fisetin towards water accessibility at the interfacial domain of Tween20 : cholesterol (1 : 1) niosome membrane. Zeta potential value confirms that there is no role of surface charge in the multiple prototropism of fisetin in Tween20 : cholesterol (1 : 1) niosome membrane. The microviscosity changes with temperature, as reflected in fluorescence anisotropy values of fisetin phototautomeric species FT*, give information about the temperature-induced changes in the motional resistance offered by the interfacial domain of the niosomal membrane to small molecules. A temperature-dependent fluorescence lifetime study confirms the distribution of FT* in the two different sites of niosomal interfacial domain, i.e. water-deficient inner site and water-accessible outer site. This heterogeneity in distribution of FT* is further confirmed through time-resolved fluorescence anisotropy decay resulting in two different rotational time constants (faster component of ∼1.04 ns originates from water-accessible outer site and slower component of ∼16.50 ns originates from water-deficient inner site). The interfacial location of fisetin in Tween20 : cholesterol (1 : 1) niosome membrane has an important implication with regards to antioxidant activity as confirmed from a DPPH radical scavenging study.

FRET Between Riboflavin and 9-Anthraldehyde Based Fluorescent Organic Nanoparticles Possessing Antibacterial Activity.

The aqueous suspension of fluorescent nanoparticles were prepared by using 9-anthradehdye derivative (AH). The nanoparticles (AHNPs) were characterized using DLS-zeta sizer and SEM techniques. The photo physical properties of nanoparticles and precursor were measured and compared using UV-absorption spectroscopy, fluorescence spectroscopy and fluorescence lifetime studies. The significant overlap between fluorescence spectrum of AHNPs and excitation spectrum of Riboflavin (RF) led us to explore Fluorescence Resonance Energy Transfer (FRET) studies between AHNPs and RF in aqueous medium. The mechanism of FRET from AHNPs to RF discussed on spectral observations, thermodynamic parameters and changes produces in fluorescence lifetime in absence and presence of different concentrations of RF to AHNPs. The limit of detection for RF (0.071µM) is considerably low compared with reported methods. Thus, we explore AHNPs as novel nano probe for quantitative determination of RF in pharmaceutical samples based on FRET study. In addition with this, AHNPs has excellent antibacterial activity than the bulk material for two different bacteria culture viz. E. coli and Bacillus sps. Graphical Abstract 9-anthradehdye based fluorescent nanoparticles (AHNPs) explores as nano probe to detect Riboflavin (RF) in aqueous medium based on Fluorescence Resonance Energy Transfer (FRET) studies. The proposed analytical method successfully applied for quantitative determination of RF in pharmaceutical samples. In addition, with this, AHNPs has excellent antibacterial activity than the bulk material for two different bacteria culture suspension viz. E. coli and Bacillus sps.

Photochemical Degradation of Curcumin: a Mechanism for Aqueous Based Sensing of Fluoride.

The present work describes the enhanced photochemical degradation of natural dye Curcumin in acetonitrile-water mixture in the presence of fluoride upon irradiation with light. The strong basicity of fluoride modifies the solvent environment around Curcumin molecule leading to alkaline mediated degradation of Curcumin which is further accelerated by irradiation with light. The photochemical degradation of Curcumin is studied using absorption and fluorescence spectroscopy and verified using infrared spectroscopy and fluorescence lifetime studies. The results of the work indicate that the method of Curcumin irradiation can be used as a sensing technique for fluoride detection in a wide range.

Photoinduced interaction of arylamine dye with carbon quantum dots ensued from Centella asiatica

Developing new carbon nano-materials and interaction studies with organic dye molecules are raising interest in the current scenario for photovoltaic applications. Herein, a simple, economical and green method is adopted for the synthesis of carbon quantum dots via hydrothermal process using Centella asiatica as a carbon source (CQDs). The synthesized CQDs were characterized by vital techniques such as atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), UV−vis absorption, steady-state and time-resolved fluorescence spectroscopy. The results reveal that the synthesized CQDs were spherical shape with an average diameter of ~ 2nm. It shows the emission maximum at 440nm with a quantum yield of 3% and it exhibit broad and excitation-dependent emission spectra, which is a unique property of CDs. Further, fluorescence quenching of an arylamine dye, 2-Cyano-3-(4-diphenylamino-phenyl)-acrylic acid (TCC) with CQDs has also been studied. The quenching mechanism of TCC with CQDs was evaluated by Stern–Volmer equation and the time-resolved fluorescence lifetime studies. The results explored that the dominant process of fluorescence quenching is attributed to electron transfer from excited TCC to CQDs. As a consequence of the work, CQDs can be used as a acceptor material for various emerging fields such as in photovoltaics, photocatalytic water treatment and etc.

Influences of urea on the interaction of 32π-Norcorrole with bovine serum albumin

ABSTRACTThe aim of the present study is to investigate the urea-induced denaturation of bovine serum albumin. The native and denatured bovine serum albumin interactions with 32π-Norcorrole were investigated, respectively. The circular dichroism spectra indicated that the α-helix content of bovine serum albumin reduces in the presence of urea and 32π-Norcorrole. The Stern–Volmer quenching constant indicated that both dynamic and static quenching exist in the interaction; moreover, the denaturation of bovine serum albumin leads to the decrease of Stern–Volmer quenching constant. Binding constant illustrated that the native bovine serum albumin has stronger combination capacity than the denatured bovine serum albumin. The fluorescence lifetime studies demonstrated that denaturation lead to the fluorescence decay of bovine serum albumin. The binding sites experiment and molecular docking studies demonstrated that the binding site of 32π-Norcorrole on bovine serum albumin is mainly located in site I.

Unravel the interaction of protoporphyrin IX with reduced graphene oxide by vital spectroscopic techniques

Probing interaction between dyes and reduced graphene oxide (rGO) is of contemporary research interest. Since, rGO is widely used as electron acceptor in photovoltaic and optoelectronic devices. Hence, we have investigated the interaction between protoporphyrin IX (PPIX) and rGO by vital spectroscopic techniques. The adsorption of PPIX on rGO is studied by Attenuated total reflection-Fourier transform infrared (ATR-FTIR) and X-ray photoelectron spectroscopic (XPS) measurements. The fluorescence quenching measurements are also performed and the fluorescence intensity of PPIX is quenched by rGO. The quenching of PPIX with rGO is evaluated by the Stern–Volmer equation and time-resolved fluorescence lifetime studies. The results revealed that the fluorescence quenching of PPIX with rGO is due to the static quenching mechanism. The dominant process for this quenching has been attributed to the process of electron transfer from excited state PPIX to rGO. Fluorescence lifetime measurements were used to calculate the rate of electron transfer process between excited state of PPIX and rGO. Transient absorption studies demonstrated the formation of PPIX cation radical for the evidence of electron transfer between PPIX and rGO.

Stabilization of whey protein isolate (WPI) through interactions with sugar beet pectin (SBP) induced by controlled dry-heating

It has been shown previously that reacting whey protein isolate (WPI) with sugar beet pectin (SBP) through a Maillard type reaction in a dry-state led to noticeable improvement in the physical, chemical and functional properties of the end products. Studies on the changes in the molecular structure of whey protein as a result of the reaction were carried out in this work to provide a molecular understanding of these observed improvements. The effect of interacting and conjugating with SBP on the apparent secondary structure of WPI was examined by far-UV circular dichroism (CD) spectroscopy and followed by spectral deconvolution. Results showed that the structure of WPI, especially the antiparallel β-sheet element, was preserved by reacting with SBP at the weight ratios of WPI:SBP = 3:1, 2:1, and 1:1. The analysis of difference UV-VIS spectra indicated significant changes occurred in the molecular electronic states of both WPI and the feruloyl moieties of SBP. Initial Maillard reaction products were detected in the form of the FRET complexes with WPI as the donor, and SBP, the acceptor by the studies of the steady-state fluorescence and the lifetime of the excited states. The tertiary structural contacts in WPI, probed by the intrinsic Trp fluorescence, were disrupted significantly upon reacting with SBP at an equal ratio (1:1). The thermal stability of WPI, however, at both secondary and tertiary structural levels, was greatly improved by the dry-heating induced reaction with SBP at all levels. The thermal stability of the feruloyl groups of SBP was also enhanced by reacting with WPI. Furthermore, the fluorescence lifetime studies definitively identified the distinctive advanced Maillard reaction end products (AMREPs) formed between WPI and SBP at varying ratios.

Experimental and Quantum Chemical Calculations of Imidazolium Appended Naphthalene Hybrid in Different Biomimicking Aqueous Interfaces.

The effect of solvent polarity and micellar headgroup on a newly designed imidazolium based ionic liquid (IL) conjugated with naphthalene, 1,2-dimethyl-3-((6-(octyloxy)naphthalen-2-yl)methyl)-1H-imidazol-3-ium chloride (IN-O8-Cl), was studied using steady state and time-resolved fluorescence techniques. We observed that the dipole moment in the excited state is remarkably higher than the ground state. The effect of micellar surface charge on the photophysics of IN-O8-Cl in aqueous phase at room temperature was investigated. Formation of premicellar aggregates in sodium dodecylsulfate (SDS) was perceived; further the microenvironment of IN-O8-Cl was examined using steady-state fluorescence spectroscopy. Micropolarity of the micellar environment of SDS was found to be lower than that of cetyltrimethylammonium bromide (CTAB) and triton X-100 (TX100) following the order SDS < TX-100 < CTAB. The binding constant (Kb) and edge excitation red shift (EERS) from the emission maximum suggest that the probe binds strongly to the micelles. Multiexponential behavior was observed in time-resolved fluorescence lifetime studies in all micellar environments. We have observed an increase in rotational correlation time as we move from pure aqueous phase to solution containing surfactants of different head charge. Varieties of spectral parameters were used to justify the region in which the probe is present. The experimentally obtained dipole moment data were justified and explained by the DFT calculations of the electronic properties of IN-O8-Cl molecules in gas phase and in selected solvents.

Fundamental Study of Electrospun Pyrene-Polyethersulfone Nanofibers Using Mixed Solvents for Sensitive and Selective Explosives Detection in Aqueous Solution.

Fluorescent pyrene-polyethersulfone (Py-PES) nanofibers were prepared through electrospinning technique using mixed solvents. The effects of mixed solvent ratio and polymer/fluorophore concentrations on electrospun nanofiber's morphology and its sensing performance were systematically investigated and optimized. The Py-PES nanofibers prepared under optimized conditions were further applied for highly sensitive detection of explosives, such as picric acid (PA), 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) in aqueous phase with limits of detection (S/N = 3) of 23, 160, 400, and 980 nM, respectively. The Stern-Volmer (S-V) plot for Py excimer fluorescence quenching by PA shows two linear regions at low (0-1 μM) and high concentration range (>1 μM) with a quenching constant of 1.263 × 10(6) M(-1) and 5.08 × 10(4) M(-1), respectively. On the contrary, S-V plots for Py excimer fluorescence quenching by TNT, DNT, and RDX display an overall linearity in the entire tested concentration range. The fluorescence quenching by PA can be attributed to the fact that both photoinduced electron transfer and energy transfer are involved in the quenching process. In addition, pyrene monomer fluorescence is also quenched and exhibits different trends for different explosives. Fluorescence lifetime studies have revealed a dominant static quenching mechanism of the current fluorescent sensors for explosives in aqueous solution. Selectivity study demonstrates that common interferents have an insignificant effect on the emission intensity of the fluorescent nanofibers in aqueous phase, while reusability study indicates that the fluorescent nanofibers can be regenerated. Spiked real river water sample was also tested, and negligible matrix effect on explosives detection was observed. This research provides new insights into the development of fluorescent explosive sensor with high performance.

A stable, efficient and reusable CdS–SnO2 heterostructured photocatalyst for the mineralization of Acid Violet 7 dye

We report the photocatalytic activity of a stable and efficient CdS–SnO2 composite for the complete degradation and mineralization of Acid Violet 7 (AV 7, 50ppm) dye. The composite catalyst exhibits higher photocatalytic activity compared to pure CdS and SnO2. Fluorescence lifetime study shows that the photogenerated charge carriers have higher lifetime in the composite than that in single phase CdS. The enhanced photocatalytic activity is attributed to the increased lifetime of the photogenerated charge carriers in the heterostructured CdS–SnO2 catalyst. Total organic carbon (TOC) analysis indicates that there is hardly any organic carbon in the solution after irradiation for 150min. Characterization of the catalyst before and after photocatalysis does not show the presence of dye in it in the adsorbed form, suggesting complete mineralization of the dye by the photocatalytic reaction. Experiments with different quenchers indicate that photogenerated holes (h+), superoxide radical anions (O2−) and hydroxyl radicals (OH) play significant roles in the degradation reaction. The composite catalyst exhibits good photo stability and there is no significant deterioration of the catalyst up to 6 cycles of photocatalytic reaction. Present study provides insights for improving the photocatalytic activity and stability of CdS photocatalyst.

Indolizine‐Based Donors as Organic Sensitizer Components for Dye‐Sensitized Solar Cells

Strong electron‐donating functionality is desirable for many organic donor‐π‐bridge‐acceptor (D‐π‐A) dyes. Strategies for increasing the electron‐donating strength of common nitrogen‐based donors include planarization of nitrogen substituents and the use of low resonance‐stabilized energy aromatic ring‐substituted nitrogen atoms. Organic donor motifs based on the planar nitrogen containing heterocycle indolizine are synthesized and incorporated into dye‐sensitized solar cell (DSC) sensitizers. Resonance active substitutions at several positions on indolizine in conjugation with the D‐π‐A π‐system are examined computationally and experimentally. The indolizine‐based donors are observed to contribute electron density with strengths greater than triarylamines and diarylamines, as evidenced by UV/Vis, IR absorptions, and oxidation potential measurements. Fluorescence lifetime studies in solution and on TiO2 yield insights in understanding the performance of indolizine‐based dyes in DSC devices.

Versatile Pulsed 560 nm Laser Source for Time-Resolved Microscopy and Spectroscopy

Fluorophores have become a standard tool in life science and biophysics as well as material sciences. Nowadays, fluorophore position and concentration are often not sufficient to answer questions in flurophore dynamics, such as diffusion behavior or molecular interactions. These aspects become accessible with the application of time-resolved fluorescence microscopy and spectroscopy when the fluorophores are excited through pulsed lasers. The large variety of fluorophores requires full spectral coverage through available pulsed lasers.Here, we present a new pulsed laser, filling the existing gap at 560 nm pulsed excitation. This freely triggerable laser source can operate in a wide range of repetition frequencies from 1 MHz up to 80 MHz which makes it easy to adapt the pulse period to different fluorescence lifetimes. Synchronization to other lasers or scanning devices is possible as well as burst operation.The wavelength around 560 nm is appropriate for a wide range of applications, especially in the life sciences where state of the art red fluorescent proteins excited at about 560 nm are essential. A pulsed 559 nm laser enables the long sought Fluorescence Lifetime studies like FLIM (Fluorescence Lifetime Imaging Microscopy) or Fluorescence Lifetime (Cross-)Correlation Spectroscopy (FL(C)CS) involving Red Fluorescent Proteins like mCherry and with that provided access to molecular interaction studies and background reduction. Implemented in a confocal microscope like the MicroTime 200 (PicoQuant) this laser becomes a versatile tool.

Single-Molecule Fret Detects Intermediates and Fast Dynamics of DNA Holliday Junctions

Holliday junctions are four-way DNA junctions that occur during genetic recombination. In previous studies it was demonstrated that in the presence of Mg2+ ions these junctions continually switch between two stacking conformations with rates strongly dependent on the ion concentration. In the present study we performed FRET studies using confocal single molecule detection with diffusing molecules or evanescent excitation of immobilized single molecules as well as ensemble fluorescence lifetime studies. By this means the full time range of fluorescence from ns to s could be accessed in order to capture all dynamic ranges. A complex Mg-dependent kinetics was found. The simplest model that describes the observed kinetics consists of four states, with only two distinct FRET levels. All methods and analysis techniques consistently reveal continuous interconversion also in absence of Mg2+ whereas binding of Mg2+ locks the junctions in the respective states. Our results shape a new view on structural properties of four-way DNA junctions, identifying a dynamic equilibrium instead of an accumulation of a single open structure at low Mg2+.