Fluorescence Decay Parameters Research Articles

Detection of HOCl-driven degradation of the pericardium scaffolds by label-free multiphoton fluorescence lifetime imaging

Artificial biomaterials can significantly increase the rate of tissue regeneration. However, implantation of scaffolds leads not only to accelerated tissue healing but also to an immune response of the organism, which results in the degradation of the biomaterial. The synergy of the immune response and scaffold degradation processes largely determines the efficiency of tissue regeneration. Still, methods suitable for fast, accurate and non-invasive characterization of the degradation degree of biomaterial are highly demandable. Here we show the possibility of monitoring the degradation of decellularized bovine pericardium scaffolds under conditions mimicking the immune response and oxidation processes using multiphoton tomography combined with fluorescence lifetime imaging (MPT-FLIM). We found that the fluorescence lifetimes of genipin-induced cross-links in collagen and oxidation products of collagen are prominent markers of oxidative degradation of scaffolds. This was verified in model experiments, where the oxidation was induced with hypochlorous acid or by exposure to activated neutrophils. The fluorescence decay parameters also correlated with the changes of micromechanical properties of the scaffolds as assessed using atomic force microscopy (AFM). Our results suggest that FLIM can be used for quantitative assessments of the properties and degradation of the scaffolds essential for the wound healing processes in vivo.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Interrogation of tumor metabolism in tissue samples ex vivo using fluorescence lifetime imaging of NAD(P)H

Exploring metabolism in human tumors at the cellular level remains a challenge. The reduced form of metabolic cofactor NAD(P)H is one of the major intrinsic fluorescent components in tissues and a valuable indicator of cellular metabolic activity. Fluorescence lifetime imaging (FLIM) enables resolution of both the free and protein-bound fractions of this cofactor, and thus, high sensitivity detection of relative changes in the NAD(P)H-dependent metabolic pathways in real time. However, the clinical use of this technique is still very limited. The applications of metabolic FLIM could be usefully expanded by probing cellular metabolism in tissues ex vivo. For this, however, the development of appropriate tissue preservation protocols is required in order to maintain the optical metabolic characteristics in the ex vivo sample in a state similar to those of the tumor in vivo. Using mouse tumor models of different histological types—colorectal cancer, lung carcinoma and melanoma—we tested eight different methods of tissue handling by comparing NAD(P)H fluorescence decay parameters ex vivo and in vivo as measured with two-photon excited FLIM microscopy. It was found that the samples placed in 10% BSA on ice immediately after excision maintained the same fluorescence lifetimes and free/bound ratios as measured in vivo for at least 3 hours. This protocol was subsequently used for metabolic assessments in fresh postoperative samples from colorectal cancer patients. A high degree of inter- and intra-tumor heterogeneity with a trend to a more oxidative metabolism was detected in T3 colorectal tumors in comparison with normal tumor-distant colon samples. These results suggest that the methodology developed on the basis of FLIM of NAD(P)H in tissues ex vivo show promise for interrogating the metabolic state of patients’ tumors.

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

Photophysical and photoconductive aspects of donor-acceptor low band gap conjugated copolymer

This article presents the photophysical and photoconductive properties of a newly synthesized low band gap Donor-Acceptor conjugated poly (benzothiadiazole-triphenylamine) co-polymer labelled as P(BTZ-TPA) in which 2,1,3 benzothiadiazole (BTZ) act as the electron deficient moiety and triphenylamine (TPA) is used as donor moiety. Photophysical properties of this synthesized polymer were analyzed by recording steady state absorption and emission spectra of dilute solution and thin films respectively. In addition to this, solvatochromic experiments were carried out using a binary mixture consisting of toluene and acetonitrile to understand the solvent dependence on the ground state and excited states of this polymer. The optical characterization revealed high degree of intramolecular charge transfer through the polymer backbone. The P(BTZ-TPA) thin films exhibit good fluorescence emission with emission peak around 615 nm. Quantum yield of the fluorescence emission is measured in relative method. Also, fluorescence decay parameters were examined using time correlated single-photon counting (TCSPC) technique. Carrier generation and transport of the generated carriers upon photo excitation is studied in pristine polymer films as well as in P(BTZ-TPA):PCBM blend films, by performing photoconductivity studies using broad spectral source and lasers of wavelengths 488 nm and 632 nm. P(BTZ-TPA) thin films exhibited good photoresponse over the entire visible region, with promising internal photocurrent efficiency. Quenching of fluorescence is observed in PCBM blend films. This drastic quenching of fluorescence emission is assigned to the charge transfer interaction between co-polymer and PCBM, and is confirmed by substantial increment in photoconductivity of the blend films. An internal photocurrent efficiency of 17.4% was obtained in the blend films for a biasing field of 10V/μm.

Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein.

The present work reports a new strategy for triblock-copolymer-assisted refolding of sodium dodecyl sulfate (SDS)-induced unfolded serum protein human serum albumin (HSA) by mixed-micelle formation of SDS with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer EO20PO68EO20 (P123) under physiological conditions. The steady-state and time-resolve fluorescence results show that the unfolding of HSA induced by SDS occurs in a stepwise manner through three different phases of binding of SDS, which is followed by a saturation of interaction. Interestingly, the addition of polymeric surfactant P123 to the unfolded protein results in the recovery of ∼87% of its α-helical structure, which was lost during SDS-induced unfolding. This is further corroborated by the return of the steady-state and time-resolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial nativelike condition. The isothermal titration calorimetry (ITC) data also substantiates that there is almost no interaction between P123 and the native state of the protein. However, the mixed-micelle formation, accompanied by substantial binding affinities, removes the bound SDS molecules from the scaffolds of the unfolded state of the protein. On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refolding the protein back to its nativelike state.

Two-photon autofluorescence lifetime imaging of human skin papillary dermis in vivo: assessment of blood capillaries and structural proteins localization

The papillary dermis of human skin is responsible for its biomechanical properties and for supply of epidermis with chemicals. Dermis is mainly composed of structural protein molecules, including collagen and elastin, and contains blood capillaries. Connective tissue diseases, as well as cardiovascular complications have manifestations on the molecular level in the papillary dermis (e.g. alteration of collagen I and III content) and in the capillary structure. In this paper we assessed the molecular structure of internal and external regions of skin capillaries using two-photon fluorescence lifetime imaging (FLIM) of endogenous compounds. It was shown that the capillaries are characterized by a fast fluorescence decay, which is originated from red blood cells and blood plasma. Using the second harmonic generation signal, FLIM segmentation was performed, which provided for spatial localization and fluorescence decay parameters distribution of collagen I and elastin in the dermal papillae. It was demonstrated that the lifetime distribution was different for the inner area of dermal papillae around the capillary loop that was suggested to be due to collagen III. Hence, we propose a generalized approach to two-photon imaging of the papillary dermis components, which extends the capabilities of this technique in skin diagnosis.

Photon Counting Data Analysis: Application of the Maximum Likelihood and Related Methods for the Determination of Lifetimes in Mixtures of Rose Bengal and Rhodamine B.

It is often convenient to know the minimum amount of data needed to obtain a result of desired accuracy and precision. It is a necessity in the case of subdiffraction-limited microscopies, such as stimulated emission depletion (STED) microscopy, owing to the limited sample volumes and the extreme sensitivity of the samples to photobleaching and photodamage. We present a detailed comparison of probability-based techniques (the maximum likelihood method and methods based on the binomial and the Poisson distributions) with residual minimization-based techniques for retrieving the fluorescence decay parameters for various two-fluorophore mixtures, as a function of the total number of photon counts, in time-correlated, single-photon counting experiments. The probability-based techniques proved to be the most robust (insensitive to initial values) in retrieving the target parameters and, in fact, performed equivalently to 2-3 significant figures. This is to be expected, as we demonstrate that the three methods are fundamentally related. Furthermore, methods based on the Poisson and binomial distributions have the desirable feature of providing a bin-by-bin analysis of a single fluorescence decay trace, which thus permits statistics to be acquired using only the one trace not only for the mean and median values of the fluorescence decay parameters but also for the associated standard deviations. These probability-based methods lend themselves well to the analysis of the sparse data sets that are encountered in subdiffraction-limited microscopies.

Room temperature fluorescence and phosphorescence study on the interactions of iodide ions with single tryptophan containing serum albumins

In this study, the influence of heavy-atom perturbation, induced by the addition of iodide ions, on the fluorescence and phosphorescence decay parameters of some single tryptophan containing serum albumins isolated from: human (HSA), equine (ESA) and leporine (LSA) has been studied. The obtained results indicated that, there exist two distinct conformations of the proteins with different exposure to the quencher. In addition, the Stern–Volmer plots indicated saturation of iodide ions in the binding region. Therefore, to determine quenching parameter, we proposed alternative quenching model and we have performed a global analysis of each conformer to define the effect of iodide ions in the cavity by determining the value of the association constant. The possible quenching mechanism may be based on long-range through-space interactions between the buried chromophore and quencher in the aqueous phase. The discrepancies of the decay parameters between the albumins studied may be related with the accumulation of positive charge at the main and the back entrance to the Drug Site 1 where tryptophan residue is located.

Real-Time Visualization of Tissue Surface Biochemical Features Derived From Fluorescence Lifetime Measurements

Fiber based fluorescence lifetime imaging has shown great potential for intraoperative diagnosis and guidance of surgical procedures. Here we describe a novel method addressing a significant challenge for the practical implementation of this technique, i.e., the real-time display of the quantified biochemical or functional tissue properties superimposed on the interrogated area. Specifically, an aiming beam (450 nm) generated by a continuous-wave laser beam was merged with the pulsed fluorescence excitation light in a single delivery/collection fiber and then imaged and segmented using a color-based algorithm. We demonstrate that this approach enables continuous delineation of the interrogated location and dynamic augmentation of the acquired frames with the corresponding fluorescence decay parameters. The method was evaluated on a fluorescence phantom and fresh tissue samples. Current results demonstrate that 34 frames per second can be achieved for augmenting videos of 640 × 512 pixels resolution. Also we show that the spatial resolution of the fluorescence lifetime map depends on the tissue optical properties, the scanning speed, and the frame rate. The dice similarity coefficient between the fluorescence phantom and the reconstructed maps was estimated to be as high as 93%. The reported method could become a valuable tool for augmenting the surgeon's field of view with diagnostic information derived from the analysis of fluorescence lifetime data in real-time using handheld, automated, or endoscopic scanning systems. Current method provides also a means for maintaining the tissue light exposure within safety limits. This study provides a framework for using an aiming beam with other point spectroscopy applications.

Neuroprotection by caloric restriction is associated with a distinct state of hippocampal energy metabolism linked to PGC-1alpha and GSK3beta

The hippocampus is critical for cognition and memory formation and is vulnerable to age-related atrophy and loss of function. These phenotypes are attenuated by caloric restriction (CR), a dietary intervention that delays aging. Here we show significant effects of aging and CR on hippocampal energy metabolism that implicate metabolic pathways in neuronal protection. Using high-resolution quantitative imaging, we detected significant regional differences in activity of the mitochondrial electron transport system (ETS) in mouse hippocampi. Mitochondrial activity was lower in aged mice, and the impact of age was region specific. Multi-photon laser scanning microscopy revealed age- and region-specific differences within the dentate gyrus (DG), where levels of autofluroescence of NAD derived metabolic cofactors declined and chemical properties were altered. There was a significant regional impact of age to decrease levels of PGC-1alpha, a master regulator of ETS gene expression. Rather than reversing the impact of age, we found CR induced a distinct metabolic state. Fluorescence intensity of NAD(P) H was higher in the DG of CR animals, but CR had no impact on fluorescence decay parameters. Levels of PGC-1alpha were lower with CR, as were levels of GSK3beta, a key regulator of PGC-1alpha turnover and activity, and these differences were conserved in rhesus monkeys. These data reveal significant changes in hippocampal energy metabolism with age and suggest CR’s prevention of age-related functional decline involves extensive metabolic reprogramming.

2-Aminopurine as a fluorescent probe of DNA conformation and the DNA-enzyme interface.

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

Time-resolved fluorescence spectroscopy and ultrasound backscatter microscopy for nondestructive evaluation of vascular grafts.

Quantitative and qualitative evaluations of structure and composition are important in monitoring development of engineered vascular tissue both in vitro and in vivo. Destructive techniques are an obstacle for performing time-lapse analyses from a single sample or animal. This study demonstrates the ability of time-resolved fluorescence spectroscopy (TRFS) and ultrasound backscatter microscopy (UBM), as nondestructive and synergistic techniques, for compositional and morphological analyses of tissue grafts, respectively. UBM images and integrated backscatter coefficients demonstrate the ability to visualize and quantify postimplantation changes in vascular graft biomaterials such as loss of the external elastic lamina and intimal/medial thickening over the grafted region as well as graft integration with the surrounding tissue. TRFS results show significant changes in spectra, average lifetime, and fluorescence decay parameters owing to changes in collagen, elastin, and cellular content between normal and grafted tissue regions. These results lay the foundation for the application of a catheter-based technique for in vivo evaluation of vascular grafts using TRFS and UBM.

Fluorescence Lifetime Based Corneal Metabolic Imaging

We are developing a time-gated fluorescence lifetime imaging microscopy (FLIM) instrument for in vivo metabolic imaging of corneal tissues, based on the fluorescence of metabolic co-factor FAD. Here we report on the first results of this project, namely on ex vivo measurements done with a time correlated single photon counting FLIM instrument, and on the first measurements done with the time-gated microscope prototype. Ex-vivo measurements with rat corneas show that it is possible to image FAD fluorescence from corneal epithelial layer and to obtain information from its fluorescence decay parameters that correlates to their metabolic activity. Measurements with test-targets and fluorescence lifetime standards show that the prototype of the time-gated fluorescence lifetime microscope for in vivo FAD imaging has the precision and the accuracy, as well as the timing and lateral spatial resolutions, required for such measurements.

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.

Beta-amyloid oligomerisation monitored by intrinsic tyrosine fluorescence

Aggregation of the peptide beta-amyloid is known to be associated with Alzheimer's disease. According to recent findings the most neurotoxic aggregates are the oligomers formed in the initial stages of the aggregation process. Here we use beta-amyloid's (Aβ's) intrinsic fluorophore tyrosine to probe the earliest peptide-to-peptide stages of aggregation, a region often merely labelled as a time lag, because negligible changes are observed by the commonly used probe ThT. Using spectrally resolved fluorescence decay time techniques and analysis we demonstrate how the distribution of 3 rotamer conformations of the single tyrosine in Aβ tracks the aggregation across the time lag and beyond according to the initial peptide concentration. At low Aβ concentrations (≤5 μM), negligible aggregation is observed and this is mirrored by little change in the fluorescence decay parameters, providing a useful baseline for comparison. At higher concentrations (≈50 μM), and contrary to what is generally accepted from ThT studies the rate of aggregation can be described by an exponential growth to a plateau in terms of the relative contributions of two of the three rotamers, with a characteristic aggregation time of ≈33 h.

The Sll0606 Protein Is Required for Photosystem II Assembly/Stability in the Cyanobacterium Synechocystis sp. PCC 6803

An insertional transposon mutation in the sll0606 gene was found to lead to a loss of photoautotrophy but not photoheterotrophy in the cyanobacterium Synechocystis sp. PCC 6803. Complementation analysis of this mutant (Tsll0606) indicated that an intact sll0606 gene could fully restore photoautotrophic growth. Gene organization in the vicinity of sll0606 indicates that it is not contained in an operon. No electron transport activity was detected in Tsll0606 using water as an electron donor and 2,6-dichlorobenzoquinone as an electron acceptor, indicating that Photosystem II (PS II) was defective. Electron transport activity using dichlorophenol indolephenol plus ascorbate as an electron donor to methyl viologen, however, was the same as observed in the control strain. This indicated that electron flow through Photosystem I was normal. Fluorescence induction and decay parameters verified that Photosystem II was highly compromised. The quantum yield for energy trapping by Photosystem II (F(V)/F(M)) in the mutant was less than 10% of that observed in the control strain. The small variable fluorescence yield observed after a single saturating flash exhibited aberrant Q(A)(-) reoxidation kinetics that were insensitive to dichloromethylurea. Immunological analysis indicated that whereas the D2 and CP47 proteins were modestly affected, the D1 and CP43 components were dramatically reduced. Analysis of two-dimensional blue native/lithium dodecyl sulfate-polyacrylamide gels indicated that no intact PS II monomer or dimers were observed in the mutant. The CP43-less PS II monomer did accumulate to detectable levels. Our results indicate that the Sll0606 protein is required for the assembly/stability of a functionally competent Photosystem II.

Assessing protein–surface interactions with a series of multi-labeled BSA using fluorescence lifetime microscopy and Förster Energy Resonance Transfer

Reliably measuring the physicochemical properties of protein thin layers deposited on surfaces is critical to understanding the surface chemistry, biocompatibility, and performance of implanted biomaterials. Here we apply a series of multi-fluorophore labeled Bovine Serum Albumin (BSA) proteins as model probes to investigate surface-induced conformational changes of BSA by the use of a confocal Fluorescence Lifetime Imaging Microscopy and Förster Resonance Energy Transfer (FLIM–FRET) method. In this FLIM–FRET approach we study six different constructs where the BSA is covalently linked to one (BSA-F1) or five (BSA-F5) fluorescein molecules, one (BSA-T1) or seven (BSA-T7) rhodamine molecules, and hetero labeled with both (BSA-F4-T2 and BSA-F6-T1). The fluorescence intensity and decays were simultaneously measured at two different emission regions (green and red channels) of the labeled BSA deposited on substrates of different hydrophilicity and hydrophobicity. To generate reliable data, several different regions (10 4 μm 2 in each case) of the surfaces were scanned for each measurement. The amplitude-weighted lifetimes, obtained from the fluorescence decay parameters, are discussed based on the average distance between the conjugated fluorophores acting as a donor and acceptor pair in the Energy Transfer framework. The number of probes conjugated has significant effects on the fluorescence emission intensity and lifetimes in solution and on surfaces. The BSA-F4-T2 constructs showed a significant ability to differentiate using lifetime the hydrophilicity and hydrophobicity of the surfaces, by detecting local expansion and contraction of protein structure in the deposited layers. Using these multiple labeled BSA probes in conjunction with FLIM–FRET can provide a way to assess structural changes in proteins induced by variations in surface chemistry of biomaterials.

Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.

Subnanosecond spectrofluorimetry of new indolocarbazole derivatives in solutions and protein complexes and their dipole moments

The spectral and temporal characteristics of new 6,12-dimethoxyindolo[3,2-b]carbazole, 5,11-dimethyl-6,12-dimethoxyindolo[3,2-b]carbazole, and 5,11-dihexyl-6,12-di(hexyloxy)indolo[3,2-b]carbazole fluorescence probes in organic solvents and protein complexes are studied. The dipole moments of indolocarbazoles in 1,4-dioxane were measured by electrooptical absorption method. The measured dipole moments have values within the range of (3.1–3.6) × 10−30 C m in the equilibrium ground state and increase to (4.8–5.6) × 10−30 C m after excitation. The excited state lifetime of indolocarbazole derivatives increases with increasing polarity and viscosity of the environment. The binding of indolocarbazoles with trypsinogen and human serum albumin increases the fluorescence intensity, changes the intensity ratio of fluorescence bands, and increases the average excited state lifetime of indolocarbazoles. The analysis of the instantaneous fluorescence spectra and fluorescence decay parameters at different wavelengths revealed the existence of several types of probe binding sites in proteins. It is found that the fluorescence characteristics of indolocarbazole derivatives depend on the conformation rearrangements of trypsinogen due to its thermal denaturation.