Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13

AIMS: To assess the extent to which DNA and RNA bacterial content contributes to fluorescent response of SYTO 13. METHODS AND RESULTS: RNA and DNA of Escherichia coli 536 cells were extracted and fluorimetrically quantified to compare the different contents, throughout a 24 h culture, with their SYTO 13 fluorescence emission when analysed by the cytometer. SYTO 13 fluorescence varied depending on the stage of bacterial growth and in accordance with both DNA and RNA content. RNA content accounted for at least two-thirds of the total fluorescence of a cell. Escherichia coli cells were treated with chloramphenicol to improve their RNA content. With this treatment, both nucleic acids remained constant but there was a clear improvement in fluorescent emission. SYTO 13 fluorescence was also studied in E. coli X-1488 minicells. CONCLUSIONS: Although both nucleic acids are implicated, RNA accounts for a major part of SYTO 13 fluorescence. The fluorescence cannot be considered as a direct reflection of nucleic acid content. Other factors, such as topology or supercoiling, need to be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm the efficacy of SYTO 13 for labelling bacteria and for assessing the distinct physiological status. A better knowledge of the parameters implicated in its fluorescence emission has been achieved.     

Quantum dot-containing polymer particles with thermosensitive fluorescence

Composite polymer particles consisting of a solid poly(acrolein-co-styrene) core and a poly(N-vinylcaprolactam) (PVCL) polymer shell doped with CdSe/ZnS semiconductor quantum dots (QDs) were fabricated. The temperature response of the composite particles was observed as a decrease in their hydrodynamic diameter upon heating above the lower critical solution temperature of the thermosensitive PVCL polymer. Embedding QDs in the PVCL shell yields particles whose fluorescence is sensitive to temperature changes. This sensitivity was determined by the dependence of the QD fluorescence intensity on the distances between them in the PVCL shell, which reversibly change as a result of the temperature-driven conformational changes in the polymer. The QD-containing thermosensitive particles were assembled with protein molecules in such a way that they retained their thermosensitive properties, including the completely reversible temperature dependence of their fluorescence response. The composite particles developed can be used as local temperature sensors, as carriers for biomolecules, as well as in biosensing and various bioassays employing optical detection schemes.     

Characteristics of Fluorescence and Delayed Light Emission from Green Photosynthetic Bacteria and Algae

Green photosynthetic bacteria exhibit variations in the intensity of their fluorescence during illumination. The initial intensity of fluorescence, measured at the onset of illumination, has a spectrum in which the major pigment Chlorobium chlorophyll predominates. The minor pigment bacteriochlorophyll predominates in the spectrum of the time-varying part of the fluorescence. The spectrum of delayed light emission is identical to that of the time-varying fluorescence. The variations in fluorescence also resemble the delayed light in their kinetics and in their dependence on exciting light intensity. Similar results are obtained for the kinetics of prompt and delayed light emission in the algae Chlorella and Anacystis. These findings raise the possibility that the variations in fluorescence actually represent a fast component of delayed light emission, of intensity comparable to the intensity of fluorescence. In Anacystis there is an outburst of light emission that develops after the exciting light has been turned off, reaching a maximum intensity after 1 to 3 seconds. This emitted light has the spectrum of chlorophyll fluorescence. It appears to be a novel example of bioluminescence with singlet excited chlorophyll as the emitter.     

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies – one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength – have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET).The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ²). An increase in range for κ² with increasing FRET efficiency has been observed, with average κ² values different from the dynamic random average of 2/3. These observations call for the need of κ² determination in proximity measurements, where the donor and acceptor orientations are not predictable.An increasing range of κ² with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a “fluidity gradient” exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy.Two new quantities – the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the “dissymmetry index” of the polarized FRET efficiency components – have also been introduced for the characterization of the orientational dynamics of the excited state during FRET.     

Selective Staining by the Fluorochrome, 5,5'-Diphenyl-9-ethyl-DiOC2(3). I. Physicochemical Studies of Dye-Dye and Dye-Tissue Interactions

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC₂(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein β-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.     

Fluorescence-based soft-sensor for monitoring beta-lactoglobulin and alpha-lactalbumin solubility during thermal aggregation

A soft-sensor for monitoring solubility of native-like alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) and their aggregation behavior following heat treatment of mixtures under different treatment conditions was developed using fluorescence spectroscopy data regressed with a multivariate Partial Least Squares (PLS) regression algorithm. PLS regression was used to correlate the concentrations of alpha-LA and beta-LG to the fluorescence spectra obtained for their mixtures. Data for the calibration and validation of the soft sensor was derived from fluorescence spectra. The process of thermal induced aggregation of beta-LG and alpha-LA protein in mixtures, which involves the disappearance of native-like proteins, was studied under various treatment conditions including different temperatures, pH, total initial protein concentration and proportions of alpha-LA and beta-LG. It was demonstrated that the multivariate regression models used could effectively deconvolute multi-wavelength fluorescence spectra collected under a variety of process conditions and provide a fairly accurate quantification of respective native-like proteins despite the significant overlapping between their emission profiles. It was also demonstrated that a PLS model can be used as a black-box prediction tool for estimating protein aggregation when combined with simple mass balances.     

Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.     

In vivo Chlorophyll Fluorescence Monitoring with Solid State Photosensors

Solid state photosensors with their many advantages have not been utilized for chlorophyll fluorescence research. One reason is their reputed low photosensitivity compared to photomultiplier tubes. A photodarlington sensor has a relatively low photosensitivity and a nonlinear current output versus light intensity. However, when incorporated into an apparatus described in this paper it can respond to fluorescence signals from leaves using light excitation as low as 500 μW/cm². The sensitivity of another solid state device (photovoltaic photodiode) was compared to five photomultipliers using a monochromator-microphotometer testing apparatus. This solid state sensor proved to have equal or greater sensitivity to chlorophyll fluorescence. An apparatus incorporating the photovoltaic photodiode is discussed.     

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of fluoroprobes for measuring intracellular pH

We evaluated four different fluoroprobes to determine their capabilities and limitations in measuring intracellular pH by the fluorescent indicator technique. In vitro, carboxyfluorescein, dimethylcarboxyfluorescein, biscarboxyethyl carboxyfluorescein, and 4-methylumbelliferone (4MU) all showed comparably intense fluorescence and excellent pH sensitivity near their respective pKa values. Major differences were found between 4MU and the fluoresceins in terms of protein binding, concentration effects, bleach rates, and the retention time within cells. Both fluorescence and a fluorescence ratio at pH-sensitive/pH-insensitive excitation wavelengths increased with pH for all compounds, and the ratio completely corrected for large changes in the excitation light intensity. In contrast, the ratio showed large artifactual changes as dye concentration increased because of self-quenching effects and spectral shifts. Protein interactions likewise caused spectral shift and ratio aberrancies, but calcium, magnesium, and oxygen had no effect on the fluorescence ratios. We conclude that measurements of cell pH by fluorescence techniques are subject to artifacts induced by self-quenching and protein binding. Use of the fluorescence ratio technique does not necessarily correct for these artifacts, and in particular the ratio technique does not correct for changes in fluoroprobe concentration. Because the major artifacts cause the ratios for 4MU and for the fluoresceins to move in opposite directions, an experimental maneuver can be shown to cause a true change in pH if the fluorescence and ratios change in the same direction for these two classes of fluoroprobes.     

Direct evidence for intracellular divalent cation redistribution associated with platelet shape change

Chlortetracycline was used as a fluorescent probe to monitor shifts in divalent cation distribution when blood platelets were induced to change shape. Human platelets in their native plasma were incubated at 25°C with 50 μM chlortetracycline. It was found that when platelet shape change was stimulated by ADP or the divalent cation ionophore A 23187, a significant decrease in platelet-chlortetracycline fluorescence occurred. This fluorescence shift was consistent with the time course for the change in platelet shape. ATP, which inhibits ADP-induced shape change, also inhibited the decrease in platelet-chlortetracycline fluorescence.     

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.     

Heat-induced changes in the membrane fluidity of Chinese hamster ovary cells measured by flow cytometry.

Flow cytometry was used to measure the fluorescence polarization of the lipid probe trimethylammonium-diphenylhexatriene as an indicator of plasma membrane fluidity of Chinese hamster ovary (CHO) cells heated under various conditions. Fluorescence polarization was measured at room temperature about 25 min after heating. When cells were heated for 45 min at temperatures above 42 degrees C, fluorescence polarization decreased progressively, signifying an increase in plasma membrane fluidity. The fluorescence polarization of cells heated at 42 degrees C for up to 55 h was nearly the same as for unheated control populations, despite a reduction in survival. The fluorescence polarization of cells heated at 45 degrees C decreased progressively with heating time, which indicated a progressive increase in membrane fluidity. The fluorescence polarization distributions broadened and skewed toward lower polarization values for long heating times at 45 degrees C. Thermotolerant cells resisted changes in plasma membrane fluidity when challenged with subsequent 45 degrees C exposures. Heated cells were sorted on the basis of their position in the fluorescence polarization distribution and plated to determine survival. The survival of cells which were subjected to various heat treatments and then sorted from high or low tails of the fluorescence polarization histograms was not significantly different. These results show that hyperthermia causes persistent changes in the membrane fluidity of CHO cells but that membrane fluidity is not directly correlated with cell survival.     

The trace and subgrouping of lymphocyte activation by dynamic fluorescence intensity and polarization measurements.

Cell activation involves conformational changes of cytosolic enzymes, and/or their regulatory proteins, as well as intracellular matrix re-organization. In this work, these changes were monitored by dynamic measurements of fluorescence polarization in single cells incubated with or without phytohaemagglutinin (PHA), using the Cellscan mark S (CS-S) cytometer. This instrument and the procedure used proved to be a powerful tool for distinguishing subpopulations of cells. Grouping of cells by their staining rates (the time rate of change of the fluorescence intensity) yielded three major subgroups. For each subgroup, the fluorescence depolarization (FDP) induced by the incubation with PHA was measured. The kinetics of the subgroups indicate that the major FDP is contributed by the cells with the lowest staining rate. This FDP is approximately 1.5 times greater than that of a bulk population. It is believed that the analysis of kinetic probing might yield an important and more sensitive method for functional marking of subgroups of cells by their response characteristics.     

Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution.

The present paper describes a new experimental scheme for following diffusion and chemical reaction systems of fluorescently labeled molecules in the nanomolar concentration range by fluorescence correlation analysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction partners, the selectivity to investigate the temporal evolution of reaction product is significantly increased compared to ordinary one-color fluorescence autocorrelation systems. Here we develop the theoretical and experimental basis for carrying out measurements in a confocal dual-beam fluorescence correlation spectroscopy setup and discuss conditions that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-correlation amplitude.     

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.     

New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA-specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X-100. Dye VL772 exhibits yellow fluorescence and little DNA-RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue-fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.     

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.