Different chromatin fractions of tomato (Solanum lycopersicum L.) and related species

Conventional chromosome staining has suggested that more than 75% of the tomato chromosomes are constituted by heterochromatin. In order to determine whether more deeply stained proximal regions are classic heterochromatin, the distributions of C-bands and chromomycin A₃ (CMA) bands, and the prophase condensation patterns, were analysed in tomato. In this and most other species of the tomato clade, the 5S and 45S rDNA sites were also localised. In tomato, CMA banding was similar to C-banding. After conventional staining, all species displayed large condensed heteropycnotic regions that did not correspond to C-bands or CMA bands. Analyses of the CMA banded karyotypes revealed a low heterochromatin content. Around 12–17% of the chromatin of tomato was CMA⁺ and 1/4 to 1/5 of this heterochromatin corresponded to 45S rDNA. In other species, the CMA⁺ heterochromatin showed extensive variation (8–35%), but was never near the values found in the literature for tomato. These data suggest the existence of three principal fractions of chromatin in tomato and related species: the late condensed euchromatin corresponding to the terminal regions of the chromosomes, the precocious condensed euchromatin that occupies the major part of the chromosomes and the constitutive heterochromatin that represents those regions revealed by C-bands.     

Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.     

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water–glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.     

时间分辨光谱法用于离体人肺鳞癌,腺癌及正常组织的荧光寿命测量

用Ｎｄ：ＹＡＧ倍频光５３２．０ｎｍ泵浦ＤＣＭ染料输出的６３２．８ｎｍ激光作激发源，通过时间分辨法测得离体人肺腺癌，鳞癌及正常组织的特征峰荧光寿命分别为５．１，５．６和６．３ｎｓ实验结果表明：肿瘤组织的荧光寿命总小于正常组织的荧光寿命，这种方法将有可能用于精确诊断和定位肿瘤。     

Continuous Fluorescence Depletion Anisotropy Measurement of Protein Rotation

Protein rotation in viscous environments can be measured by fluorescence depletion anisotropy (FDA) which combines long lifetimes of chromophore triplet states with the sensitivity of fluorescence excitation and detection. FDA achieves sensitivity well beyond that attainable by the more common technique of time-resolved phosphorescence anisotropy (TPA). We have now combined benefits of both time-domain and frequency-domain FDA into a single continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repeating waveform. Fluorescence signals excited from triplet-forming fluorescent probes are digitized over recurring waveform periods by a high-speed signal averager. CFDA experiments typically involve substantial ground state depletion. Thus signals, unlike those of TPA, are not linear in the exciting light intensity and simple data analysis based on such linearity is not appropriate. An exact solution of the coupled diffusion and triplet production/decay equation describing CFDA within individual data points has been combined with simulated annealing optimization to extract triplet and anisotropy decay kinetics from experimental data. Related calculations compare possible excitation waveforms with respect to rotational information provided per fluorescence photon. We present CFDA results for the model system of eosin conjugates of carbonic anhydrase, BSA and immunoglobulin G in 90% glycerol at various temperatures and initial cellular results on eosin-IgE bound to 2H3 cell Type I Fcε receptors. We explore how CFDA reflects rotational parameters of heterogeneous systems and discuss challenges of extending this method to single cell microscopic measurements.     

A Streak Camera System for Time Resolved Fluorimetry

Presently, there are several techniques for measurement of fluorescence lifetimes of organic molecules. These techniques, reviewed by Ware,¹ can be divided into two basic groups, those based upon pulsed sources and those based on modulated sources and phase shift measurement. In the pulsed methods, repetitive, short pulse width, intense excitation pulses excite the fluorophor and the fluorescence decay is measured; the source temporal response must be deconvoluced from the fluorescence decay in order to evaluate the fluorescence signal and lifetime. Typical sources of excitation include nanosecond flashlamps and more recently nanosecond N₂-laser (with or without a dye laser) and mode-locked lasers with picosecond pulse widths^2-6. The modulated source phase shift methods,¹ involve sinusoidal excitation of the fluorophor and measurement of the phase shift between the modulated excitation source and the modulated flourescence. Because modulation frequencies are limited to approximately 20 MHz, fluorescence lifetimes are limited to ～.1 ns and above. In addition, in the phase shift methods, only “one point” lifetimes are obtained, i.e., the entire fluorescence decay curve is not obtained.     

Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individual chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during cell cycle and apoptosis. Thus, detection of DNA which was partially denatured in situ provided a means to image areas of condensed chromatin. DNA denaturation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies of denaturability of cellular DNA utilized flow cytometry and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in individual chromosomes. For instance, it was not possible to detect the initial points of chromosome condensation in G2-phase of the division cycle or in apoptosis. In order to achieve this goal we have recently extended these studies by applying confocal microscopy. We investigated DNA denaturability in normal human fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and apoptosis. Following removal of RNA and partial denaturation of DNA with acid cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. Blind deconvolution was used to further improve the quality of 3D images. Photobleaching of AO fluorescence was minimized and a correction for chromatic aberration and register shift was implemented. Nuclei of interphase cells exhibited predominantly green fluorescence representing AO binding to ds DNA. Punctuate areas of red fluorescence representing AO binding to denatured DNA and most likely associated with local regions of condensed chromatin were also present in all interphase nuclei. The proportion of denatured DNA increased in cells entering mitosis. In prophase individual condensing chromosomes exhibited varied proportions of green and red fluorescence indicating different content of denatured chromatin. In some chromosomes bands of denatured and denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase chromosomes exhibited red fluorescence along all length of their arms indicating the highest and uniform susceptibility to denaturation. In telophase chromosomes contained predominantly denaturation-resistant DNA again and denaturated regions were significantly less abundant. At cytokinesis some decondensing chromosomes were still resolved. At this stage almost all regions of denatured DNA were located close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condensation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding central regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin.     

Study of the dissipation of vibrational energy excess using pulse fluorometry

The dependence of the emission anisotropy decay of dye fluorescence versus excitation wavelength was measured in order to study the relaxation of the vibrational energy excess. The results of these measurements allow the conclusion (in accordance with earlier findings) that the heat transfer in the solvent is a fast enough process not to influence Brownian rotations of dye molecules.     

偶氮染料掺杂高分子薄膜的荧光光谱特性

利用旋涂法制备了新的偶氮染料掺杂薄膜，在室温下测试了该薄膜的吸收光谱，稳态荧光光谱和时间分辨荧光光谱，研究了荧光衰减动力学过程，得到了反式和顺式异构体的激光发单线态（S1）的荧光寿命。     

Solvent Effects on the Spectroscopic Properties of Styrylquinolinium Dyes Series

The study on the relationship between the structure and spectroscopic properties of styrylquinolinium dyes were carried out by measuring the electronic visible absorption, steady-state and time-resolved fluorescence spectra of quinoline based hemicyanine dyes. The influence of the solvent on absorption and emission spectra and the solvatochromic properties, observed for both ground and first excited states, for all the dyes were applied for the evaluation of their excited state dipole moments. The ground state dipole moments of dyes under the study were established by applying ab initio calculations. The measured, using solvatochromic methods, excited state dipole moments of tested hemicyanines are in the range from 5.38 to 18.90 D and the change in the dipole moments caused by excitation were found to differ from 1.88 to 6.64 D. It was observed that for all tested dyes the dipole moments of the excited states were higher than those of a ground states. The fluorescence lifetime measurements with picosecond resolution was performed for entire series of hemicyanine dyes possessing different dialkylamino groups attached to the phenyl ring. The average lifetimes of the dye fluorescence, determined from the measured data by multi-order exponential decay curve fitting, were in the range from about 120 to 1200 ps at the fluorescence peak wavelength. The fluorescence lifetime measurements were performed for dyes in ethyl acetate solutions. The time-resolved fluorescence spectra measurements allowed to propose the mechanism of the dyes excited states deactivation.     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

Lifetimes of rovibrational levels of the long-range I′ 1Πg state of H2 and D2: experiment and theory

The lifetimes of the lowest lying rovibrational levels of the outer well I′ ¹Π_g state of molecular hydrogen were measured for both H₂ and D₂. The measurements were made by direct observation of the time-dependent decay of the fluorescence. The observed lifetimes depend on isotopomer and increase with vibrational excitation. The predominant decay route for these levels is fluorescence. Previously published ab initio lifetimes calculated for these states, which accounted for non-adiabatic interactions [J. Chem. Phys. 92, 7461 (1990)], are in good agreement with experiment for H₂ but are too long by four or five orders of magnitude for D₂. We present new ab initio results at the adiabatic level for the fluorescence lifetimes. The current results are in reasonable agreement with the experimental lifetimes for both H₂ and D₂. We explain the isotopomer and vibrational dependence of the lifetimes and discuss the neglected interactions and decay pathways.     

Fluorescence Characteristics of Isolated Dye Molecules within Silicalite-1 Channels

Fluorescence characteristics of hemicyanine dye molecules isolated from neighboring molecules and strongly restricted inside nanosized pores of zeolite (silicalite-1) crystal were investigated. For samples in which the molecules were sufficiently far away from the others, the fluorescence decay lifetime of the molecules was about 2.2?ns. As the intermolecular distance was reduced, the steady-state fluorescence peak shifted toward the longer wavelength and the fluorescence efficiency decreased markedly. The fluorescence decay lifetime also decreased to 0.8?ns for a sample with the smallest intermolecular distance of 2.1?nm. These results were explained in terms of a dipole-dipole interaction between pairs of dye molecules. From the relation between the intermolecular distances and the fluorescence decay lifetimes of the molecules, the radius of energy transfer of hemicyanine donor-acceptor pair in zeolite matrix was determined to be 2.2?nm, in fair agreement with the calculated F?rster radius between dye molecules of the same species.     

Radiative lifetimes of superradiant populated Ba Rydberg levels

Radiative lifetimes of 6s7f¹F₃, as8f¹F₃, and 6s9d¹D₂ Ba Rydberg levels were measured in an atomic beam by time resolved recording of the exponential decay of the fluorescence. The excitation was performed by use of two pulsed dye lasers and a superradiant emission or a superradiant cascade controlled by a cavity.     

Studies of Interaction Between Cyanine Dye T-284 and Fibrillar Alpha-Synuclein

A key feature of Parkinson’s disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the “binding channel” running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different “populations” due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.     

Fluorescence intensity and anisotropy decays of the DNA stain Hoechst 33342 resulting from one-photon and two-photon excitation

We examined the steady-state and time-resolved fluorescence spectral properties of the DNA stain Hoechst 33342 for one-photon (OPE) and two-photon (TPE) excitation. Hoechst 33342 was found to display a large cross section for two-photon excitation within the fundamental wavelength range of pyridine 2 and rhodamine 6G dye lasers, 690 to 770 and 560 to 630 nm, respectively. The time-resolved measurements show that intensity decays are similar for OPE- and TPE. The anisotropy decay measurements of Hoechst 33342 in ethanol revealed the same correlation times for TPE as observed for OPE. However, the zero-time anisotropies recovered from anisotropy decay measurements are 1.4-fold higher for TPE than for OPE. The anisotropy spectra of Hoechst 33342 were examined in glycerol at ?20°C, revealing limiting values close to the theoretical limits for OPE (0.4) and TPE (0.57). The steady-state anisotropy for OPE decreases in the shorter-wavelength region (R6G dye laser, 280–315 nm), but the two-photon anisotropy for 560 to 630-nm excitation remains as high as in the long-wavelength region (690–770 nm). This result suggests that one-photon absorption is due to two electronic, but only one transition contributes to the two-photon absorption over the wavelength range from 580 to 770 nm. Our demonstration of these favorable two-photon properties for Hoechst 33342, and the high photostability of the dye reported by other laboratories, suggests that this dye will be valuable for time-resolved studies of DNA with TPE and for two-photon fluorescence microscopy.     

稀土配合物的时间分辨荧光光谱法研究(Ⅱ)——Eu,Sm-DBM-TOPO体系的荧光衰减动力学特性和激光诱导时间分辨荧光光谱

本文以Nd:YAG泵浦的染料激光器为激发光源,门控光学多道分析仪为检测器,自制的触发器和荧光盒组装成一台激光诱导时间分辨光谱装置。采用此装置研究了在表面活性剂Triton X-100存在下铕、钐-二苯甲酰甲烷-三正辛基氧化膦(Eu、Sm-DBM-TOPO)体系的激光诱导发光光谱特性和荧光衰减动力学特性。讨论了荧光发射过程中的能量传递机制。拟定出测定痕有铕和钐的时间分辨光谱方法,用于高纯稀土氧化物等样品中痕量铕和钐的测定,结果满意。     

Spectroscopic and photophysical investigations on the nature of localization of rhodamine-123 and its dibromo derivative in different cell lines

Steady-state and time-resolved spectroscopic properties of rhodamine-123 (rh123) and 4,5-dibromorhodamine methyl ester (dbr123) bound to different cell lines are evaluated. Studies are also performed on the dye bound to extracted mitochondria. Results are compared with those obtained in homogeneous and microheterogeneous media. Results suggest that these dyes can specifically bind only with cell mitochondria. As a result of binding, excitation and emission spectra are red shifted by 10 to 12 nm. The fluorescence decay of these dyes bound to mitochondria shows two lifetimes. Values are about 4.0 and 2.0 ns forrh123 and about 1.9 and 0.5 ns fordbr123. Detailed global analysis of emission wavelength and dye concentration dependences of the fluorescence decay is performed. Results indicate that these dyes are bound to two different binding sites at mitochondria. The decay-associated fluorescence spectrum for the species corresponding to each binding site is recovered. Species1, corresponding to the longer lifetime, is found to be more red shifted compared to species2. The fluorescence of species2 is heavily quenched. The origin of this quenching is explained in terms of resonance energy transfer between donor species2 and acceptor species1. The possible nature of the two binding sites is also discussed.     

Localized excitation effects in (E,E)-2,5-<Emphasis Type="Italic">bis</Emphasis>[2-(4-N,N-dipropylaminophenyl) ethylenyl]-3,6-dimethylpyrazine and generation of its different forms in solution

Dipole moments of (E,E)-2,5-bis[2-(4-N,N-dipropylaminophenyl)ethylenyl]-3,6-dimethylpyrazine (VS365) quadrupolar dye in 1,4-dioxane and cyclohexane in addition to its instantaneous fluorescence spectra and fluorescence lifetimes in dichloromethane (pure and with added trifluoroacetic acid) were measured. Quantumchemical calculations show that the π − π conjugated system is localized in the flattest part of the molecule due to disorder in the ground-state geometry of the dye molecule and that this part is the one responsible for light absorption. The effect of localized excitation of the dye causes a considerable change in its dipole moment Δ^aμ. Various fluorescent forms of the dye that feature considerably spaced emission spectra and different fluorescence lifetimes originate by adding trifluoroacetic acid to the solution of oligophenylenevinylene in dichloromethane.