New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes

The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.     

Physical and biological properties of fluorescent dansylated bile salt derivatives: the role of steroid ring hydroxylation

The hydroxyl groups of bile salts play a major role in determining their physical properties and physiologic behavior. To date, no fluorescent bile salt derivatives have been prepared which permit evaluation of the functional role of the steroid ring. We have prepared five fluorescent cholanoyl derivatives using a dansyl-ethylene diamine precursor linked to the sulfonyl group of taurine; N-(5-dimethylamino-1-naphthalenesulfonyl)-N'-(2-aminoethanesulf onyl)- ethylenediamine. The fluorescent dansyl-taurine was conjugated to the carboxyl group of free bile acids, enabling the labeling of the series: dehydrocholate, ursodeoxycholate, cholate, chenodeoxycholate and deoxycholate. Despite a systematic hydrophobic shift compared with the native bile salts (aqueous solubility and water:octanol partitioning), the influence of steroid ring hydroxylation was retained, with the dehydrocholate and cholate derivatives more water soluble than the dihydroxy derivatives. Similarly, the sequence of HPLC mobilities, reflecting relative hydrophilicity, was identical in the dansyl-taurine derivatives and the native taurine-conjugated bile salts. Cellular uptake of all five steroid derivatives was rapid, and partial inhibition of [3H]taurocholate uptake was observed in isolated hepatocytes. Rates of biliary excretion of the dansylated derivatives by the isolated perfused rat liver correlated closely with hydrophilicity. Collectively, these findings indicate that the influence of the hydroxyl groups is retained in this series of dansylated steroids, and that hydroxylation is a key determinant of their hepatocellular transport and biliary excretion. These fluorescent bile salt derivatives may thus serve as unique probes for investigating structure-function relationships in hepatic processing of steroid-based compounds.     

Protection of neuronal cells against reactive oxygen species by carnosine and related compounds

Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.     

Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA

Interactions of several acridine dyes with DNA from different species were studied by measuring fluorescence lifetimes in the 2–30-nsec range, using the single-photon counting technique, and by measuring fluorescence quantum yields in the steady state. The results confirm the existence of two principal site classes, one in which the dye fluorescence is quenched by interaction with guanine and another in which fluorescence results from the hydrophobic environment of the A·T base pairs. The emitting sites are found, in some cases, to exhibit fluorescent decay curves which can be resolved into two exponential components corresponding to a short and to a long lifetime. The deviation from one exponential component is particularly clear with rivanol, 9-aminoacridine, and quinacrine, with which one component is two or three times longer than the other. The relative proportion of these two components depends only slightly on the DNA base composition and does not depend on the nature of the acridine derivatives. We postulate that this lifetime heterogeneity corresponds to the two discrete steps in the complex formation elucidated by kinetic studies: the first step corresponds to a semi-intercalated, or “external,” dye with a short fluorescence lifetime and the second step corresponds to a totally intercalated dye with a long lifetime. In this model, we assumed that a transient opening of the site near a semi-intercalated dye induces solvent diffusion which in turn is responsible for its short-lived 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.     

Receptor binding of NBD-labeled fluorescent estrogens and progestins in whole cells and cell-free preparations

We have studied the interactions of four fluorescent steroid conjugates with either the estrogen or progesterone receptor, both in whole cells and cell-free receptor preparations. The fluorophore, nitrobenzoxadiazole (NBD), was conjugated with a synthetic progestin, with a steroidal estrogen, a non-steroidal estrogen, and with an antiestrogen. With all compounds, receptor-specific binding could be detected by fluorescence measurements following extraction from the protein into an organic solvent. In the native state, however, the NBD-ligand-receptor complex is essentially non-emissive, although these ligands fluoresce strongly when associated with non-specific binders such as albumin. The binding site concentrations and relative affinities determined by fluorescence (after extraction) correspond well with those determined by [3H]estradiol or [3H]R5020 binding to their respective receptors. In T47D breast cancer cells, the NBD-progestin showed receptor-mediated uptake and nuclear localization. These compounds have provided valuable information about the interactions of low and medium affinity ligands with their receptors; however, the successful use of fluorescent ligands for detecting steroid receptors under native-bound conditions, by "imaging" modalities (fluorescence microscopy and flow cytometry) will require the development of fluorophores that are emissive while receptor bound or assay protocols that enable the environment of ligands associated with the receptor to be controlled.     

Interpretation and Optimization of Absorbance and Fluorescence Signals from Voltage-sensitive Dyes

Voltage-sensitive dyes produce absorbance and fluorescence changes that can be used to image voltage. The present study develops a systematic approach to the optimization of these signals. A mathematical analysis assesses the dye optical density (OD) that optimizes the signal-to-noise ratio in absorbance and fluorescence measurements. The signal-to-noise ratio is maximal for a dye OD of 2 (natural logarithm) in absorbance and ~1 in fluorescence. The fluorescence result is approximate because, in contrast to absorbance, the optimal dye OD varies with the amount of scattering and intrinsic absorbance of the tissue. The signal-to-noise ratio of absorbance is higher in thick preparations such as brain slices; fluorescence is superior in thin preparations such as cell culture. The optimal OD for absorbance and fluorescence, as well as the superiority of absorbance, were confirmed experimentally on hippocampal slices. This analysis also provided insight into the interpretation of signals normalized to resting light intensities. With both absorbance and fluorescence, the normalized signal (I/I) varies with OD, and does not reflect the change in dye absorbance. In absorbance this problem is remedied by dividing I/I by the dye OD to obtain the absorbance change. For fluorescence a correction is possible, but is more complicated. Because this analysis indicates that high levels of stain optimize the signal-to-noise, dyes were tested for pharmacological actions and phototoxicity. The absorbance dye RH155 was found to have pharmacological action at high staining levels. The fluorescent dye RH414 was phototoxic. Adverse effects could not be detected with the absorbance dye RH482.     

New fluorescent macrolide derivatives for studying interactions of antibiotics and their analogs with the ribosomal exit tunnel

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.     

Recovery of components of fluorescence spectra of mixtures by intensity- and anisotropy decay-associated analysis: the bacterial luciferase intermediates

Reaction of FMNH2 and O2 with bacterial luciferase followed by blue light irradiation results in a product previously claimed to have the same fluorescence spectral distribution as the bioluminescence. Preparations of this "high fluorescence" intermediate, however, contain two fluorescent components, one from the intermediate and the other its breakdown product, FMN. Since the intermediate has a fluorescence lifetime of around 10 ns and a rotational correlation time in the range of 100 ns, compared to 5.0 and 0.15 ns, respectively, for the FMN, the two components can be successfully resolved from the total fluorescence by an anisotropy decay- and fluorescence decay-associated analysis employing simultaneous global computational methods. The fluorescence spectra of the intermediates from two types of luciferase were analyzed in this way; one luciferase was from Vibrio harveyi and the other was from an unusual type of V. fischeri that had an in vivo bioluminescence maximum at 505 nm, a wavelength almost 20 nm longer than that of the V. harveyi bioluminescence. For V. harveyi the true fluorescence of the intermediate is distinct from the bioluminescence, being found at a wavelength about 10 nm longer. For the type of V. fischeri examined, any difference in the two spectra is less certain. A control experiment with the dye 8-amino-1- naphthalenesulfonate bound to BSA and mixed with FMN recovered the original spectrum of the bound dye accurately.     

Universal guidelines for the conversion of proteins and dyes into functional nanothermometers

In the last decade, technological advances in chemistry and photonics have enabled real‐time measurement of temperature at the nanoscale. Nanothermometers, probes specifically designed to relay these nanoscale temperature changes, provide a high degree of temperature, temporal, and spatial resolution and precision. Several different approaches have been proposed, including microthermocouples, luminescence and fluorescence polarization anisotropy‐based nanothermometers. Anisotropy‐based nanothermometers excel in terms of biocompatibility because they can be built from endogenous proteins conjugated to dyes, minimizing any system perturbation. Moreover, the resulting fluorescent proteins can retain their native structure and activity while performing the temperature measurement, allowing precise temperature recordings from the native environment or during an enzymatic reaction in any given experimental system. To facilitate the future use of these nanothermometers in research, here we present a theoretical model that predicts the optimal sensitivity for anisotropy‐based thermometers starting with any protein or dye, based on protein size and dye fluorescence lifetime. Using this model, most proteins and dyes can be converted to nanothermometers. The utilization of these nanothermometers by a broad spectrum of disciplines within the scientific community will bring new knowledge and understanding that today remains unavailable with current techniques.     

Imaging of Mitochondrial and Non-Mitochondrial Responses in Cultured Rat Hippocampal Neurons Exposed to Micromolar Concentrations of TMRM

Tetramethylrhodamine methyl ester (TMRM) is a fluorescent dye used to study mitochondrial function in living cells. Previously, we reported that TMRM effectively labeled mitochondria of neurons deep within mouse brain slices. Use of micromolar concentration of dye, which was required to get sufficient staining for two-photon imaging, resulted in typical fluctuations of TMRM. With prolonged exposure, we recorded additional responses in some neurons that included slow oscillations and propagating waves of fluorescence. (Note: We use the terms “fluctuation” to refer to a change in the fluorescent state of an individual mitochondrion, “oscillation” to refer to a localized change in fluorescence in the cytosol, and “wave” to refer to a change in cytosolic fluorescence that propagated within a cell. Use of these terms does not imply any underlying periodicity.) In this report we describe similar results using cultured rat hippocampal neurons. Prolonged exposure of cultures to 2.5 µM TMRM produced a spontaneous increase in fluorescence in some neurons, but not glial cells, after 45–60 minutes that was followed by slow oscillations, waves, and eventually apoptosis. Spontaneous increases in fluorescence were insensitive to high concentrations of FCCP (100 µM) and thapsigargin (10 µM) indicating that they originated, at least in part, from regions outside of mitochondria. The oscillations did not correlate with changes in intracellular Ca²⁺, but did correlate with differences in fluorescence lifetime of the dye. Fluorescence lifetime and one-photon ratiometric imaging of TMRM suggested that the spontaneous increase and subsequent oscillations were due to movement of dye between quenched (hydrophobic) and unquenched (hydrophilic) compartments. We propose that these movements may be correlates of intracellular events involved in early stages of apoptosis.     

Synthesis of thiol-reactive, long-wavelength fluorescent phenoxazine derivatives for biosensor applications

Two environmentally sensitive, long-wavelength fluorescent phenoxazine derivatives, INR and IANR, were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins. The linkers were designed based on the attachment sites at two different positions on the phenoxazine, which were chosen in order to study the orientation of the dye with respect to the binding protein. Conjugation of the dyes to the S337C maltose binding protein (MBP) mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities. The dye INR gave a 3-fold (+200%) change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant (K(d)) of 435 microM. The fluorescence change for IANR was only 20% and the K(d) was 1.4 mM. Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye, resulting in little change in environment between the open and the closed-form conformations. The linker in INR, on the other hand, showed restricted motion, which placed the dye in different environments in the open and closed forms of the protein. Thus, design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.     

Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells

The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 M), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K⁺. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.     

Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.     

<Emphasis Type="Italic">N,N</Emphasis>-Dialkylaminostyryl dyes: specific and highly fluorescent substrates of peroxidase and their application in histochemistry

Fluorescent labeling of immuno-bound or endogenous peroxidase (PO) activity has been achieved to date by means of phenol derivatives with a low substitution degree. Here it is demonstrated that N,N-dialkylamino-styryl dyes can also act as fluorescent substrates of PO. They undergo enzymatically cross-linking reactions to surrounding cell constituents in an analogous manner thus permitting highly fluorescent and permanent labeling. This approach is narrowly related to the catalyzed reporter deposition (CARD) technique based on tyramine conjugates and the recently described catalytic cross-linking approach of hydroxystyryl derivatives. The substitution patterns for optimal cross-linking capability and the spectral properties of obtained specific reaction products were studied using an iterative semi-empirical approach. The best staining performance is achieved with N,N-dimethylaminoaryl derivatives. Their N,N-dialkyl homologues as well as the primary aryl amine pendants failed as PO substrates. Due to their basic character, novel substrates occasionally tend to unspecific interactions (staining nuclei, mast cells, or keratin). Centering this side specificity and repressing the staining capability of PO was achieved by chemical modification of the respective dye leading to new specific probes for keratin and cytoplasmatic RNA. In conclusion, catalytic cross-linking of heterocyclic 4-N,N-dimethylamino-styryl dyes represents a promising approach for the permanent fluorescent staining of PO in fixed cells and tissues, complementing the CARD technique. In contrast to CARD-related approaches, new substrates are characterized by a broad excitation and emission range of fluorescence and the outstanding spatial resolution of specific fluorescence signaling known so far from their 4-hydroxystyryl analogues. They currently represent the smallest fluorescent substrates of PO. Histochemical and immuno-histochemical applications share several outstanding features: High detection sensitivity, spatial resolution of fluorescence signaling, and photo stability. 4-N,N-dimethylamino-styryl substrates are compatible with their phenol and phenol-ester analogues. Their combination facilitates the trichromatic immuno-histochemical demonstration of three different targets simultaneously at one excitation wavelength in a conventional epi-fluorescence microscope.     

Microfluorometric estimates of proteins associated with murine hepatocyte and thymocyte nuclei, residual structures, and nuclear matrix derivatives

Isolated diploid hepatocyte and thymocyte nuclei and their derivatives ("residual structures" and nuclear matrices, as defined by Kaufmann et al. 1981) were evaluated by microfluorometry following reaction with the following fluorochromes: brilliant sulfaflavine (BSF) used at pH 2.8 for the demonstration of total protein; acridine orange (AO) used at pH 9.0 to reveal acidic groups of proteins; and 3-(4-maleimidylphenyl)-7-diethylamino-4-methylcoumarin (CPM) used under conditions required to demonstrate the sum of sulfhydryl (SH) and disulfide (SS) groups of proteins. The results suggested that the proteins reacting with AO and CPM differed from each other and from those revealed by fluorochroming with BSF. In every comparison, hepatocyte nuclei and their derivatives were more fluorescent than the respective populations of thymocyte nuclei and their derivatives. In material fluorochromed with BSF and AO, nuclear matrices were less fluorescent than residual structures, which, in turn, were less fluorescent than intact nuclei. In contrast, nuclear matrices fluorochromed with CPM were less fluorescent than intact nuclei but more fluorescent (paradoxically) than residual structures. The ratios of the total fluorescence values of hepatocyte and thymocyte nuclei fluorochromed with BSF changed significantly during extractions required to produce residual structures and nuclear matrices, while comparable ratios in material fluorochromed with AO or CPM did not change significantly. Comparisons of the ratios of the fluorescence values of intact nuclei and their derivatives in a variety of combinations yielded complex and variable results.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of novel taspine diphenyl derivatives as fluorescence probes and inhibitors of breast cancer cell proliferation

Two novel taspine diphenyl derivatives (Ta‐dD) were designed and synthesized by introducing different coumarin fluorescent groups into the basic structure of Ta‐dD. The main advantage of these two compounds is that they can be used as fluorescence probes and inhibitors simultaneously. In the present study, the fluorescent properties of the probes were measured and their inhibition of four breast cancer cell lines was tested. Different concentrations of the fluorescence probe were added to MCF‐7 breast cancer cells for fluorescence imaging analysis under normal conditions. The results suggested that both of the new compounds have not only fluorescence but also the ability to inhibit effects on different breast cancer cell lines, which indicates their possible further use as dual functional fluorescence probes in tracer analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.     

Binding of a cationic phenazinium dye in anionic liposomal membrane: a spectacular modification in the photophysics

Interaction of a cationic phenazinium dye, phenosafranin (PSF), with the anionic liposomal vesicle/bilayer of dimyristoyl-l-α-phosphatidylglycerol (DMPG) has been demonstrated using steady state and time resolved fluorescence and fluorescence anisotropy techniques. The charge transfer emission spectrum of PSF shows a dramatic modification in terms of fluorescence yield together with an appreciable hypsochromic shift in the lipid environment. The blue shift indicates a lowering in polarity inside the vesicle as compared to that in bulk water. The fluorescence and fluorescence quenching studies and micropolarity determination reveal that the cationic fluorophore has a profound binding interaction with the anionic DMPG membrane. Anisotropy study indicates the imposition of a motional restriction on the probe inside the bilayer. The electrostatic interaction between the cationic dye and the anionic lipid membrane has been argued to be the reason behind all these observations. The results could be useful in analyzing membrane organization and heterogeneity in natural membranes exploiting PSF or alike compounds as fluorescent probes.