Fluorescence intensity and lifetime distribution analysis: toward higher accuracy in fluorescence fluctuation spectroscopy

Fluorescence fluctuation methods such as fluorescence correlation spectroscopy and fluorescence intensity distribution analysis (FIDA) have proven to be versatile tools for studying molecular interactions with single molecule sensitivity. Another well-known fluorescence technique is the measurement of the fluorescence lifetime. Here, we introduce a method that combines the benefits of both FIDA and fluorescence lifetime analysis. It is based on fitting the two-dimensional histogram of the number of photons detected in counting time intervals of given width and the sum of excitation to detection delay times of these photons. Referred to as fluorescence intensity and lifetime distribution analysis (FILDA), the technique distinguishes fluorescence species on the basis of both their specific molecular brightness and the lifetime of the excited state and is also able to determine absolute fluorophore concentrations. The combined information yielded by FILDA results in significantly increased accuracy compared to that of FIDA or fluorescence lifetime analysis alone. In this paper, the theory of FILDA is elaborated and applied to both simulated and experimental data. The outstanding power of this technique in resolving different species is shown by quantifying the binding of calmodulin to a peptide ligand, thus indicating the potential for application of FILDA to similar problems in the life sciences.     

Size analysis of residual host cell DNA in cell culture-produced vaccines by capillary gel electrophoresis

Residual host cell DNA poses potential safety concerns for cell culture-derived vaccines or other biological products. In addition to the quantity of residual DNA, the size distribution is an important measure for determination of its associated risk factor. We have developed a new method for residual DNA size analysis, based on capillary gel electrophoresis (CGE) technology with sensitive laser induced fluorescence detection (LIF). The performance of this method was optimized through empirical selection of appropriate testing conditions and optimized conditions are presented. Examples are given to demonstrate the successful employment of this method for residual DNA size analysis of cell culture-produced vaccine samples.     

High pressure static fluorescence to study macromolecular structure-function

Through some typical examples, the high pressure static fluorescence method is described. The potentiality of the intrinsic and extrinsic fluorescence probes are analyzed for structural characterizations. Special attention is given to the use of fluorescence to understand the behavior of enzymatic reactions under high pressure. The application of fluorescence polarization is also presented together with some relevant spectroscopic problems inherent in data interpretation.     

Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.     

Designing matrix models for fluorescence energy transfer between moving donors and acceptors.

A recipe is given for designing theoretical models for donor-acceptor systems in which fluorescence energy transfer and motion takes place simultaneously. This recipe is based on the idea that a system exhibiting both motion and fluorescence energy transfer can be modeled by specifying a number of "states" and the rates of transitions between them. A state in this context is a set of specific coordinates and conditions that describe the system at a certain moment in time. As time goes on, the coordinates and conditions for the system change, and this evolution can be described as a series of transitions from one state to the next. The recipe is applied to a number of example systems in which the donors and/or acceptors undergo either rotational or translational motion. In each example, fluorescence intensities and anisotropies for the donor and acceptor are calculated from solutions of eigensystems. The proposed method allows for analyzing time-resolved fluorescence energy transfer data without restrictive assumptions for motional averaging regimes and the orientation factor. It is shown that the fluorescence quantities depend on the size of the motional step (i.e., on the number of states), only if fluorescence energy transfer occurs. This finding indicates that fluorescence energy transfer studies may reveal whether the dynamics of a system (e.g., a protein) is better described in terms of transitions between a relatively small number of discrete states (jumping) or a large number of dense states (diffusion).     

叶绿素延迟荧光的发生及其在光合作用研究中的应用

叶绿素延迟荧光主要由绿色植物中光系统Ⅱ的天线色素产生,光系统Ⅱ反应中心色素P680接受天线色素吸收的光能后转变为激发态的P680,P680回到基态时释放出一个电子传给原初电子受体,随后电子沿光合电子传递链向PSI传递。当进入电子传递链的电子发生电荷重组时会使P680再次激发形成P680,P680将激发能传递给天线色素后,激发能以荧光的形式释放出来,即为延迟荧光。延迟荧光的检测和分析技术为无损测定植物光合机构的结构与功能变化提供了新的方法。利用该方法可以获得丰富的光合机构信息,如光系统Ⅱ受体侧及供体侧的伤害程度、跨类囊体膜质子梯度的大小等。本文介绍了延迟荧光的产生原理和测定方法,并且举例说明了延迟荧光测定技术在光合作用研究中的应用。     

Measurement of rotational motion in membranes using fluorescence recovery after photobleaching.

A method has been developed for the measurement of the rotational motion of membrane components. In this method fluorescent molecules whose transition dipole moments lie in a given direction are preferentially destroyed with a short intense burst of polarized laser radiation. The fluorescence intensity, excited with a low intensity observation beam of polarized laser radiation, changes with time as the remaining fluorescent molecules rotate. The feasibility of the method has been demonstrated in a study of the rotation of the fluorescent lipid probe, dil ([bis,-2-(N-octadecyl-3,3-dimethyl-1-benzo[b]pyrrole]-trimethincyanine iodide) incorporated into membranes composed of distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC) and 0.20 mol% cholesterol, below the main chain-melting transition temperatures of the phosphatidylcholines. Rotation times in the 0.6-800 s range were observed. The fluorescence recovery (or decay) curves are in satisfactory agreement with theoretical calculations.     

Bayesian localization microscopy based on intensity distribution of fluorophores

Super-resolution microscopy techniques have overcome the limit of optical diffraction. Recently, the Bayesian analysis of Bleaching and Blinking data (3B) method has emerged as an important tool to obtain super-resolution fluorescence images. 3B uses the change in information caused by adding or removing fluorophores in the cell to fit the data. When adding a new fluorophore, 3B selects a random initial position, optimizes this position and then determines its reliability. However, the fluorophores are not evenly distributed in the entire image region, and the fluorescence intensity at a given position positively correlates with the probability of observing a fluorophore at this position. In this paper, we present a Bayesian analysis of Bleaching and Blinking microscopy method based on fluorescence intensity distribution (FID3B). We utilize the intensity distribution to select more reliable positions as the initial positions of fluorophores. This approach can improve the reconstruction results and significantly reduce the computational time. We validate the performance of our method using both simulated data and experimental data from cellular structures. The results confirm the effectiveness of our method.     

Design and synthesis of ICT-based fluorescent probe for high-sensitivity protein detection and application to rapid protein staining for SDS-PAGE

A novel fluorescent molecular probe possessing styryl, sulfonyl, and cyanopyranyl moieties that was termed compound 1 was designed and synthesized to detect proteins through noncovalent bonding. Compound 1 did not produce fluorescence emission in the absence of proteins. However, its fluorescence spectrum showed a dramatic increase in the fluorescence intensity and strong orange emission after the addition of BSA. These changes were caused by intramolecular charge transfer (ICT). The fluorescence intensities of compound 1 were plotted as a function of the protein concentrations. A good linear relationship was observed up to a protein concentration of 325 mug/mL, and the detection limit was 70 ng/mL under the given assay conditions; this detection limit was higher than that of previously reported compounds. To demonstrate the application of compound 1, proteins in an SDS-PAGE gel were stained with compound 1 and were successfully imaged with a higher sensitivity and shorter staining operation time as compared to those of the silver staining method and SYPRO Ruby staining method. Thus, easy and high-sensitivity protein detection can be performed with the fluorescent probe, and this probe is ideally suited to proteomic applications.     

Deconvolution of single photon counting data with a reference method and global analysis

A method based on quenched references and global analysis was used to deconvolute timeresolved single photon counting data. The results from both computer simulated data and real experiments showed that highly accurate and reliable deconvolutions were possible. Fluorescence lifetimes and Stern-Volmer quenching constants for quenching with NaI were determined for the reference substances para-terphenyl, PPO (2,5-diphenyloxazol), POPOP (1,4-bis-(5-phenyl-2-oxazolyl)-benzene), and dimethyl-POPOP, all in ethanol. The fluorescence from a mixture of POPOP, anthracene, and diphenylanthracene in ethanol at different wavelengths was successfully resolved into the known relative contributions from the species at each wavelength. Fluorescence intensity decays of tryptophan in solution were studied at different wavelengths and globally analyzed with the method. Also, fluorescence anisotropy described by isotropic and anisotropic rotations in homogeneous and heterogeneous emitting systems were simulated and successfully deconvoluted. The method was applied to real fluorescence anisotropy data of diphenylanthracene and POPOP in paraffin oil, as well as to data from experiments on the blue copper-containing protein stellacyanin and its apo-form. In these cases, the method both corrected for errors due to, for example, the wavelength-dependent transit-times in the photomultiplier, and realized global deconvolutions of the total, parallel, and perpendicular components of the fluorescence. General algorithms for arbitrary fluorescence impulse responses are given.A preliminary account of this work was presented at the NATO ASI in Acireale, Italy (Löfroth 1985a, in press)     

On-line measurements of culture fluorescence: Method and application

Summary In this paper a new probe allowing the measurement of NAD(P)H-dependent culture fluorescence in a bioreactor is presented. This sterilizable probe can be inserted in every bioreactor using a standard fitting of 25 mm. Under well defined conditions high specificity and sensitivity as well as high stability are further advantages of this probe. Application examples are given to demonstrate the operation possibilities of this fluorescence probe. In batch growth the culture fluorescence can be used for on-line estimation of biomass concentration. Metabolic alterations due to substrate of oxygen deficiency can easily be detected by fluorometric measurements. In kinetic studies the fluorescence probe is of great use because of a very small time delay.     

Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

A novel approach to improve specificity of algal biosensors using wild-type and resistant mutants: an application to detect TNT

A new genetic approach was developed for increasing specificity of microalgal biosensors. This method is based on the use of two different genotypes jointly to detect a given pollutant: (i) a sensitive genotype to obtain sensitivity; and (ii) a resistant mutant to obtain specificity. The method was tested by the development of a microalgal biosensor for the detection of the explosive 2,4,6-trinitrotoluene (TNT) using a wild-type strain (DcG1wt) of Dictyosphaerium chlorelloides (Chlorophyceae) as the sensitive organism, and a TNT-resistant mutant, obtained from DcG1wt strain by a modified Luria-Delbrück fluctuation analysis. The inhibition of chlorophyll a fluorescence of PSII by TNT was used as the biological signal. Significant differences in maximal fluorescence of light-adapted algae (F'(m)) between wild-type DcG1wt cells and TNT-resistant mutants, were observed in all the TNT concentrations tested (from 0.5 to 31.3 mg l(-1)) after only 3 min of exposure. Resistant mutants always exhibited significant higher F'(m) values in the presence of TNT than wild-type cells. These results suggest that the use of two different genotypes (sensitive and resistant to a given pollutant) jointly is a useful method to improve microalgal biosensors specificity.     

Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence.     

Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules

Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239-247) with an aromatic ring system responsible for an intense absorption band in the 400-480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7-8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N, N'-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method.     

Slower step of the polycyclic aromatic hydrocarbons metabolism: kinetic data from microspectrofluorometric techniques

A microspectrofluorometer has been used for kinetic studies of the decrease of benzo(a)pyrene and benzo(k)fluoranthene fluorescence. This decrease is observed for single living cells: L cells and human peripheral blood monocytes, after their incubation which culture medium containing these compounds and washing the petri dish with fresh medium. The entire fluorescence spectra is recorded at given time intervals in order to watch at some eventual spectral modification. The fluorescence decrease is monoexponential and its parameters are computed with a program based on the least squares fit method. Such determination shows no difference between the calculated rate constants of metabolisation for B(a)P and B(k)F and, as long as we consider L cells with a similar morphological shape, only statistical fluctuations of the rate constants of metabolism are observed. As compared, monocytes show faster kinetics of the decrease of the B(a)P intracellular fluorescence due to B(a)P metabolism, and also a more reached dispersion of the values of the rate constant than the one observed for L cells indicating some heterogeneity in the monocyte population of each donor.     

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes

Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.     

Mechanisms of chromosome banding

Prior studies on subfractions of mouse and Kangaroo rat DNA have suggested that variations in base concentration within a given genome may not be great enough to account for Q-banding. To examine this with another species, calf DNA was subfractionated by CsCl ultracentrifugation into GC-rich satellites and the main band DNA was further fractionated into AT-rich, intermediate and GC-rich portions. The effect of varying concentrations of these DNAs on quinacrine and Hoechst 33258 fluorescence was examined. Although with both compounds there was less fluorescence in the presence of the GC-rich satellites than main band fractions, these results per se did not answer the question of whether the variation in base composition alone was adequate to account for chromosome banding. To answer this the fluorescence observed in the presence of DNA of a given base composition was related to the fluorescence observed in the presence of DNA of 40% GC content (F/F40). This allowed the derivation of a term B which indicated the relative change in fluorescence per 1% change in base composition of DNA. To determine the percent change in fluorescence observed in Q-banding, the photoelectric recordings of Caspersson et al. (1971) were used. From these data we conclude: 1. Quinacrine is twice as sensitive to changes in base composition as Hoechst 33258. 2. Variation in the base content of DNA along the chromosome is sufficient to account for most Q-banding, except possibly for some of the extremes of quinacrine fluorescence. This was further examined with daunomycin. Even though daunomycin gives good fluorescent banding, DNAs varying in base composition from 100 to 40% GC content all resulted in the same relative fluorescence of 0.03. However, in the presence of poly (dA-dT) the relative fluorescence was 0.85, indicating a great sensitivity to very AT-rich DNA. This suggests that with daunomycin and possibly other fluorochromes, stretches of very AT-rich DNA may be more important in fluorescent banding than simple variation in mean base composition.