Fluorescence Time-correlated Single Photon Counting Research Articles

Compaction of Calf Thymus DNA by a Potential One-Head-Two-Tail Surfactant: Properties of Nanomaterials and Biological Testing for Gene Delivery.

A one-head-two-tail cationic surfactant, Dilauryldimethylammonium bromide (DDAB) has shown a great extent of calf thymus DNA (ct-DNA) compaction being adsorbed on the surfaces of negatively charged SiO2 nanoparticles (NPs). DDAB molecules show high adsorption efficiency and induce many positive surface charges per-unit surface area of the SiO2 NPs compared to cationic Gemini (12-6-12) and conventional (DTAB) surfactants in an aqueous medium at pH 7.4, as evident from zeta potential and EDAX data. Transmission electron microscopy and field emission scanning electron microscopy images, along with ethidium bromide exclusion assay and DLS data support the compaction of ct-DNA. Fluorescence microscopic images show that in the presence of SiO2 NPs, DDAB can perform 50% compaction of ct-DNA at a concentration ∼58% and ∼99% lower than that of 12-6-12 and DTAB, respectively. Better ct-DNA compaction by DDAB is evident compared to other Gemini surfactants (12-4-12 and 12-8-12) as well reported before. Time-correlated single photon counting fluorescence intensity decay measurements of a probe DAPI in ct-DNA have revealed the average lifetime value that is decreased by ∼61% at 2.5 μM of DDAB in the presence of SiO2 NPs as compared to a decrease by only ∼29% in its absence, supporting NPs-induced stronger surfactant binding with ct-DNA. Fluorescence lifetime data have also demonstrated the crowding effect of NPs. At 2.5 μM of DDAB, both fast and slow rotational relaxation components of DAPI contribute almost equally to depolarization with the absence of NPs; however, with the presence of NPs, ∼96% weightage of the anisotropy decay is for the fast component. The present DDAB-SiO2 NPs combination has proved to be an excellent gene delivery system based on the cell viability in the mouse mammary gland adenocarcinoma cells (4T1) and human embryonic kidney (HEK) 293 cell lines, and in vitro and in vivo studies.

Membrane binding and pore forming insertion of PEX5 into horizontal lipid bilayer.

The assembly of the peroxisomal translocon involves the transition of a soluble form of the peroxisomal targeting receptor PEX5 into a membrane-bound form, which becomes an integral membrane component of the import pore for peroxisomal matrix proteins. How this transition occurs is still a mystery. We addressed this question using a artificial horizontal bilayer in combination with fluorescence time-correlated single photon counting (TCSPC) and electrophysiological channel recording. Purified human isoform PEX5L and truncated PEX5L(1-335) lacking the cargo binding domain were selectively labeled with thiol-reactive Atto-dyes. Diffusion coefficients of labeled protein in solution show that PEX5L is monomeric with a rather compact spherical conformation, while the truncated protein appeared in a more extended conformation. Labeled PEX5L and the truncated PEX5L(1-335) bind stably to horizontal bilayer thereby accumulating around 100-fold. The diffusion coefficients of the membrane-bound PEX5L forms are 3-4 times lower than in solution, indicating the formation of larger complexes. Electrophysiological single channel recording shows that membrane-bound labeled and non-labeled PEX5L, but not the truncated PEX5L(1-335), can form ion conducting membrane channels. The data suggest that PEX5L is the pore-forming component of the oligomeric peroxisomal translocon and that spontaneous PEX5L membrane surface binding might be an important step in its assembly.

Scrutinizing the stability of haemoglobin in 1,2,4-triazolium based ionic liquid

In this work, N-1 substituted 1,2,4-triazolium-based cation and trifluoromethanesulfonate anion based IL has been synthesized and used as a solvent system to stabilize haemoglobin (Hb). The conformational stability of Hb at different concentrations of developed IL was inspected through spectroscopic techniques like UV–Vis, steady-state fluorescence, fluorescence time-correlated single photon counting (TCSPC), temperature dependent UV–Vis, fluorescence, and circular dichroism (CD). Hb structure is stabilized in the IL concentration range 0.001 to 0.01 M; Hb structure starts opening up in the IL concentration range 0.05 to 0.1 M; at 0.2 M IL concentration, denaturation of Hb takes place. Further, the nature of the interaction between IL and Hb was verified through a competitive hydrophobic fluorescence probe, 1-Anilino-8-Naphthalene Sulfonate (ANS), and it was observed that ANS does not interact with IL till 0.01 M IL concentration. Finally, the experiments illustrate that the IL concentration plays a significant role in stabilizing/destabilizing a particular protein.

Investigations of the Energy Transfer in the Phycobilisome Antenna of Arthrospira platensis Using Femtosecond Spectroscopy

Understanding the energy transfer in phycobilisomes extracted from cyanobacteria can be used for building biomimetic hybrid systems for optimized solar energy collection and photocurrent amplification. In this paper, we applied time-resolved absorption and fluorescence spectroscopy to investigate the ultrafast dynamics in a hemidiscoidal phycobilisome obtained from Arthrospira platensis. We obtained the steady-state and time-resolved optical properties and identified the possible pathways of the excitation energy transfer in the phycobilisome and its components, phycocyanin and allophycocyanin. The transient absorption data were studied using global analysis and revealed the existence of ultrafast kinetics down to 850 fs in the phycobilisome. The fluorescence lifetimes in the nanosecond time-scale assigned to the final emitters in each sample were obtained from the time-correlated single photon counting fluorescence experiments.

Photophysical Properties of Fluorescent Self-Assembled Peptide Nanostructures for Singlet Oxygen Generation.

In this work, a drug delivery system for perillyl alcohol based on the peptide self-assembly containing 3-(2-benzothiazolyl)-7-(diethylamino)coumarin (C6) as a fluorescent additive is obtained, and its photophysical characteristics as well as its release dynamics were studied by steady-state and time-resolved fluorescence spectroscopy. Results proved the dynamics of drug release from the peptide nanostructures and showed that the system formed by the self-assembled peptide and C6, along with perillyl alcohol, presents unique photophysical properties that can be exploited to generate singlet oxygen (1O2) upon irradiation, which is not achieved by the sole components. Through epifluorescence microscopy combined with time-correlated single photon counting fluorescence spectroscopy, the release mechanism was proven to occur upon peptide structure interconversion, which is controlled by environmental changes.

Fluorescence Resonance Energy Transfer, Small-Angle Neutron Scattering, and Dynamic Light Scattering Study on Interactions of Gemini Surfactants Having Different Spacer Groups with Protein at Various Regions of Binding Isotherms.

The binding interactions of three gemini surfactants having different spacer groups (12-4-12, 12-8-12, and 12-4(OH)-12) with a high concentration (150 μM) of bovine serum albumin (BSA) at various regions of binding isotherms have been studied by means of steady-state fluorescence and fluorescence anisotropy, time-correlated single-photon counting fluorescence of trans-2-[4-(dimethylamino)styryl]benzothiazole, small-angle neutron scattering (SANS), and dynamic light scattering (DLS) measurements. The fluorescence resonance energy transfer phenomenon between the twisted intramolecular charge transfer fluorescent molecule, trans-2-[4-(dimethylamino)styryl]benzothiazole as an acceptor, and tryptophan 213 (Trp-213) of BSA as a donor has been successfully used to probe the binding interactions of gemini surfactants with protein at all regions of binding isotherms. The increasing order of energy transfer efficiency at a higher concentration range of surfactants is 12-8-12 > 12-4-12 > 12-4(OH)-12. Stronger binding of micelles of gemini surfactant molecules having a comparatively more hydrophobic spacer group with the hydrophobic segments of the protein results in closer approach of trans-2-[4-(dimethylamino)styryl]benzothiazole molecules solubilized in micelles to Trp-213. The average excited-state lifetimes become shorter with a trend of increase in contribution from the fast component and decrease in contribution from the slow component to the decay with increasing concentration of a surfactant. The nonradiative rate constant of trans-2-[4-(dimethylamino)styryl]benzothiazole increases with increasing concentration of a surfactant because the average microenvironment around it in protein–surfactant aggregates is more polar as compared to that in native protein. SANS and DLS measurements were carried out for the study of the structural deformations in the protein, on enhancement of the concentration of the gemini surfactants. The necklace and bead model has been used for the analysis of SANS data for the protein–surfactant complexes. At a higher concentration range, 12-8-12 and 12-4-12 have a slightly smaller fractal dimension and a larger correlation length as compared to 12-4(OH)-12. DLS data show that the increasing order of hydrodynamic diameter for the complexes of protein with three gemini surfactants in their high concentration range is 12-4(OH)-12 < 12-4-12 < 12-8-12.

Lipid and DNA interaction with the triorganotin dimethylaminophenylazobenzoates studied by DSC and spectroscopy methods

The aim of the study was to investigate and compare the interaction of tributyltin (TBTA) and triphenyltin (TPhTA) 2-[4 (dimethylamino)phenylazo]benzoates complexes with the lipid membrane and plasmid DNA. Both TBTA and TPhTA belong to the group of promising anticancer agents. Differential scanning calorimetry method was employed to examine the heat signal associated with phase transitions of liposomes formed from dipalmitoylphosphatidylcholine (DPPC) or DPPC/cholesterol and modified with TBTA and TPhTA. The investigated compounds influence the thermotropic parameters of the DPPC and DPPC/cholesterol lipid bilayer in a slightly different way: Although the temperature of the main phase transition of the DPPC remains almost unaffected, the addition of TPhTA can abolish sub- and pre-transition when added in sufficiently high concentrations. At the same time, the addition of the TBTA practically does not change the sub-transition and only slightly influences the pre-transition. The effect of organotin compounds on plasmid DNA was investigated using single-molecule florescence technique and time-correlated single-photon counting fluorescence correlation spectroscopy. Influence of TBTA and TPhTA on DNA resulted only in fractional conformation change which did not lead to the tightly packed conformation however, analysis of the diffusion times indicates that TPhTA interacts with DNA in a different mode than TBTA.

Rotor-Based Organelle Viscosity Imaging

Microscopic viscosity is a critical parameter in cellular processes such as diffusion-controlled reactions and the correct folding of nascent proteins. However, a lack of a suitable technology has hampered the analysis of this fundamental parameter within the organelles of living cells. We have established a new technique called Rotor-based organelle viscosity imaging (ROVI). In our technique, environment-sensitive fluorescent dyes termed ‘molecular rotors’ are directed via active targeting to specific intracellular compartments of live cells for the real time dynamic measurement of microscopic viscosity. Molecular rotors are small synthetic fluorophores whose photophysical properties strongly depend upon the viscosity and molecular crowding of their immediate environment. By measuring the fluorescence lifetime of molecular rotors via time-correlated single photon counting fluorescence lifetime imaging (TCSPC FLIM) it is possible to quantitatively determine the microviscosity of the local environment. Using ROVI, we were able to measure and compare the microviscosity in multiple cellular compartments, namely the cytosol, nucleus, ER and mitochondria. Microviscosity was measured both basally and during manipulations that alter organelle function. We report significant variations of microviscosity between the studied organelles. In addition to providing measurements of microviscosity in multiple compartments of the cell with a single ‘molecular rotor’ probe, we also report striking variation of microviscosity between and within individual matrices of mitochondria, the cell's energy repository. Furthermore, we were able to detect changes of ER microviscosity upon drug administration. The ability to measure the viscoelastic response of the ER upon stress is extremely important and highly pertinent to health and disease, as well as being novel fundamental science. We believe that ROVI offers novel avenues to study cell biology and can aid to monitoring the progression and treatment of diseases.

Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector

We perform wide-field time-correlated single photon counting-based fluorescence lifetime imaging (FLIM) with a crossed delay line anode image intensifier, where the pulse propagation time yields the photon position. This microchannel plate-based detector was read out with conventional fast timing electronics and mounted on a fluorescence microscope with total internal reflection (TIR) illumination. The picosecond time resolution of this detection system combines low illumination intensity of microwatts with wide-field data collection. This is ideal for fluorescence lifetime imaging of cell membranes using TIR. We show that fluorescence lifetime images of living HeLa cells stained with membrane dye di-4-ANEPPDHQ exhibit a reduced lifetime near the coverslip in TIR compared to epifluorescence FLIM.

Effect of Polymethylene Spacer of Cationic Gemini Surfactants on Solvation Dynamics and Rotational Relaxation of Coumarin 153 in Aqueous Micelles.

The present work demonstrates the solvation dynamics and rotational relaxation of Coumarin 153 (C-153) in the micelles of a series of cationic gemini surfactants, 12-s-12, 2Br(-) containing a hydrophobic polymethylene spacer with s = 3, 4, 6, 8, 12. Steady-state and time-correlated single-photon counting (TCSPC) fluorescence spectroscopic techniques have been used to carry out this study. Steady-state and TCSPC fluorescence data suggest that C-153 molecules are located at the Stern layer of micelles. While probe molecules feel more or less the same micropolarity in the micellar phase, the microviscosity of micelles decreases with spacer chain length. Solvation dynamics at the Stern layer is bimodal in nature with fast solvation as a major component. Counter ions and water molecules bonded with the polar headgroups of surfactant molecules are responsible for the slow component. Average solvation time increases with spacer chain length because of the increased degree of counter ion dissociation. Some water molecules are involved in the solvation of counter ions themselves, resulting in the decrease in "free" water molecules to be available for the solvation of C-153. The hydrophobic spacer chain also has an effect on increasing the solvation time with increasing chain length. The average rotational relaxation time for C-153 decreases with spacer chain length with a rapid decrease at s > 4. The anisotropy decay of C-153 in micelles is biexponential in nature. The slow rotational relaxation is due to the lateral diffusion of C-153 in micelles. Lateral diffusion is much faster than the rotational motion of a micelle as a whole. The rotational motion of the micelle as a whole becomes faster with the decreasing size of micelles.

Fluorescence Lifetime Analysis of Graphene Quantum Dots

Fitting of the fluorescence lifetimes for fluorophores can be achieved using various techniques. Problems arise when the fluorophore sample itself is heterogeneous and the fluorescence decay is far from exponential, as a single sample may have a distribution of lifetimes. We study the fitting of fluorescence lifetimes for two systems, a homogeneous and near-exponential fluorescein dye and a highly heterogeneous system of graphene quantum dots. We fit a variety of different models to experimental time-correlated single photon counting fluorescence data from each system, evaluating the validity of each model for the two systems. We find that, for the near-exponential system, there is little difference between the fit of each model. However, for strongly nonexponential behavior, the models give quite different estimates of mean and standard deviation, indicating that appropriate model choice and assessment is crucial in obtaining meaningful lifetimes from fluorescence lifetime data. Moreover, we perform simu...

Interaction of new butyltin citrate complex with lipid model membrane and DNA

Organotin(IV) complexes show a wide variety of biological activity: bactericidal, fungicidal, caricidal, and pesticidal. Many of them are more effective than traditional heavy metal anticancer drugs. The aim of the present study was to investigate the interaction of a new dibutyltin complex with citric acid (DBTC) with lipid membrane and plasmid DNA. The effect of this compound on the multilamellar liposomes formed with dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dimyristoylphosphatidylethanolamine was studied mainly by the means of differential scanning calorimetry and additionally by the steady-state fluorimetry. Calorimetrical results together with fluorescence spectroscopy suggest that investigated complex interacts with lipids and probably locates itself in the hydrophilic part of the membrane. The effect of DBCT on plasmid DNA was investigated using single-molecule florescence technique and time-correlated single photon counting fluorescence correlation spectroscopy. We could conclude then that DBTC–DNA interaction occurs due to the interaction with the DNA phosphate group and charge neutralization which then leads to DNA compaction.

Effect of Hydroxyl Group Substituted Spacer Group of Cationic Gemini Surfactants on Solvation Dynamics and Rotational Relaxation of Coumarin-480 in Aqueous Micelles

The solvation dynamics and rotational relaxation of Coumarin 480 (C-480) have been investigated in the micelles of a series of gemini surfactants, 12-4(OH)n-12 (n = 0, 1, and 2), with increasing hydroxyl group substitution within the spacer group. Steady-state and time-correlated single photon counting (TCSPC) fluorescence spectroscopic techniques have been used to carry out such study. Steady-state and TCSPC fluorescence data support the location of probe molecule at the Stern layer. The solvation dynamics is found to be slower on hydroxyl substitution of spacer group due to the formation of hydrogen bonds between water molecules and hydroxyl group(s) of spacer group. Such kind of hydrogen bonding protects the probe molecule from its contact with water molecules and also results in restricted mobility of water molecules. The average rotational relaxation time increases on increasing number of substituted hydroxyl group on a spacer group. It is because of formations of more and more close packed micelles and larger extent of intermolecular hydrogen bonding interactions between C-480 and hydroxyl group(s). For micelles of each of 12-4-12 and 12-4(OH)-12, the slow rotational relaxation is dominated by the lateral diffusion of the fluorophore along the spherical surface of the micelle. However, for 12-4(OH)2-12, the slow rotational relaxation is mainly due to the rotational motion of the micelle as a whole. Because of high microviscosity of micelles of 12-4(OH)2-12 and greater extent of hydrogen bonding interactions with C-480, the relaxation time corresponding to the lateral diffusion of the fluorophore is very high in this case.

Fluorescence Lifetime Imaging of Biosensor Peptide Phosphorylation in Single Live Cells

Better living through biochemistry: Phosphorylation of a Cy5-labeled Abl kinase peptide biosensor resulted in an extension of fluorescence lifetime in live cells. Time-correlated single photon counting fluorescence lifetime imaging (FLIM) enabled excellent signal-to-noise to visualize the subcellular patterns of this sensor (see picture). This strategy should be generalizable to other peptide-based kinase substrates for imaging of kinase activity in single cells.

Monitoring the distribution of warfarin in blood plasma.

Warfarin is an anticoagulant drug extensively used in the treatment and prevention of thrombotic disorders. Previous studies have shown that warfarin binds extensively to blood plasma proteins and that only a small fraction of the drug is unbound and thus available for therapeutic function. Both warfarin's narrow therapeutic window and the susceptibility of anticoagulant function to patient-dependent factors necessitate regular monitoring. In this study, we have shown that the lifetimes for each of the various bound and free forms of the drug in blood plasma can be quantified in situ by time-correlated single-photon counting fluorescence spectroscopy over the clinically significant concentration range. A relationship between the blood coagulation and the distribution of fluorescence lifetimes was observed. The in situ detection of clinically relevant concentrations of warfarin in its respective bound and unbound forms could provide a prognostic tool for use in patient treatment.

Tryptophan side chain conformers monitored by NMR and time‐resolved fluorescence spectroscopies

We have inserted a tryptophan (F77W) in the core of the regulatory domain of cardiac troponin C (cNTnC), and previously determined the structure of this mutant with and without the cosolvent trifluoroethanol (TFE). Interestingly, the orientations of the indole side chain of the Trp are in opposite directions in the two structures (Julien et al., Protein Sci 2009; 18:1165-1174). Fluorescence decay experiments for single Trp-containing proteins often show several lifetimes, which have been interpreted as reflecting conformational heterogeneity of the Trp side chain resulting from different rotamers. To test this interpretation, we monitored the effect of TFE on wild type, F77W and F77W-V82A calcium-saturated cNTnC using 2D (13)C-HSQC NMR and time-correlated single photon counting fluorescence spectroscopies. The time dependence of the Trp fluorescence decay was fit with three lifetimes. Addition of TFE caused a gradual, but limited decrease of the lifetimes due to dynamic quenching. For F77W cNTnC, the amplitude fractions of the lifetimes also changed upon addition of TFE-the long lifetime increased from 13 to 29%, while the middle lifetime decreased from 63 to 50% and the short lifetime remained relatively unchanged. For F77W-V82A cNTnC, comparable NMR changes are observed, confirming the switch in rotamer conformation, but only much smaller changes in fluorescence decay parameters were detected. These data indicate that the balance between the rotamer states can be changed without changing the lifetime amplitude fractions appreciably, and suggest that the environment(s) of the indole ring, responsible for the different lifetimes, can result from factors other than the intrinsic rotamer state of the tryptophan.

Fast time-correlated single-photon counting fluorescence lifetime acquisition using a 100 MHz semiconductor excitation source

The fast and efficient collection of time-resolved fluorescence decay data is shown using a high repetition rate semiconductor laser excitation source optimally matched to low dead-time counting electronics. The 100 MHz repetition rate allows a measurement range of 10 ns without re-excitation of the sample. Rapid acquisition of time-resolved fluorescence data is essential for microscopy applications, such as fluorescence lifetime imaging. With this in mind we measured a representative dye (lifetime 376 ps) to determine the fastest collection time for a single exponential decay. Other dyes were also used with lifetimes ranging from ∼90 ps to 4 ns.

Characterization of guest molecule concentration dependent nanotubes of β-cyclodextrin and their secondary assembly: Study with trans-2-[4(dimethylamino)styryl]benzothiazole, a TICT-fluorescence probe

The formation of nanotubes of β-cyclodextrins (β-CD) and their secondary assembly induced by trans-2-[4-(dimethylamino)styryl]benzothiazole (DMASBT) as a guest molecule has been explained using twisted intramolecular charge transfer (TICT) fluorescence, steady state fluorescence anisotropy, time-correlated single-photon counting fluorescence decay of DMASBT, atomic force microscopy and also transmission electron microscopy. It has been demonstrated that although at lower concentration of β-CD, DMASBT molecules form simple adduct of 1:1 stoichiometry but they form extended nanotubes at relatively high concentration of β-CD which further lead to the secondary assembly through intertubular hydrogen bondings forming rod-like structures. The changes in fluorescence properties of DMASBT yield critical aggregation concentration (CAC) of β-CD very close to 2.5 mM. The extent of formation of these supramolecular structures is dependent on the concentration of guest molecule. It has been observed that perchlorate ion (ClO 4 −) reduces the stability of supramolecular structures at higher concentration of it but at low concentration (<7 mM) it enhances the stability by providing the anchor sites for intermolecular hydrogen bonding between neighboring β-CD molecules. The polarity and viscosity dependent TICT fluorescence characteristics of DMASBT are exploited to determine the micropolarity and microviscosity of environment around it inside the nanotubes.

MAPK-activated Protein Kinase-2 (MK2)-mediated Formation and Phosphorylation-regulated Dissociation of the Signal Complex Consisting of p38, MK2, Akt, and Hsp27

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H(2)O(2) or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H(2)O(2) and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knock-out mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex.

Broadband optical limiting performance of polymer-wrapped carbon nanotubes in the orange-NIR region

Soluble multi-walled carbon nanotubes (MWNTs) have been obtained by noncovalent modification with poly [2-methoxy,5-(2′-ethyl-hexyloxy)-p-phenylene vinylene] (MEH-PPV). For the composite MWNT/MEH-PPV, there is π–π interaction between the MEH-PPV and MWNTs in addition to the wrapping of the polymer. The nonlinear optical transmittance was measured using a nanosecond optical parametric oscillator pumped with a Nd:YAG system. Excellent optical limiting performance of the composite MWNT/MEH-PPV was observed both in the visible region of 590–680 nm and at the wavelength of 1064 nm. By means of time-correlated single-photon counting fluorescence measurement, an explanation based on the nonlinear absorption of MWNT dominated by the intermolecular energy transfer was proposed.