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At least 19 recordsLinked to original sources
PubMed2005

Evolution of fluorescein as a platform for finely tunable fluorescence probes.

Fluorescence imaging is the most powerful technique currently available for continuous observation of dynamic intracellular processes in living cells. Suitable fluorescence probes are naturally of critical importance for fluorescence imaging, but only a very limited range of biomolecules can currently be visualized because of the lack of flexible design strategies for fluorescence probes. At present, design is largely empirical. Here we show that the carboxylic group of traditional fluorescein dyes, formerly considered indispensable, has been replaced with other substituents, affording various kinds of new fluoresceins. Further, by breaking out of the traditional structure of fluorescein, we developed the first and totally rational design strategy for novel fluorescence probes based on a strict photochemical basis. The value of this approach is exemplified by its application to develop a novel, highly sensitive, and membrane-permeable fluorescence probe for beta-galactosidase, which is the most widely used reporter enzyme.

Biosensing Techniques↗
PubMed1987

Location of T-cell leukaemia cells in a model rat system by means of a fluorescent probe.

Fluorescence probes for the active centre of an enzyme associated with tumour cells have been used to locate leukaemia cells in a model rat system. These fluorescent techniques are inexpensive and rapid to carry out. The leukaemic cells can be located by fluorescence microscopy in frozen sections, wax embedded sections and resin embedded sections. The technique is illustrated with reference to sections of leukaemic rat kidney, epididymis and testis. These studies confirm earlier histological findings employing conventional staining techniques and have the advantage that individual leukaemia cells can be detected in leukaemic animals undergoing drug therapy. The evidence suggests that these techniques will be of value in further studies of the design of drugs directed to leukaemia cells.

Aminacrine↗
PubMed2004

A FRET-based analysis of SNPs without fluorescent probes.

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.

Benzothiazoles↗
PubMedDate not supplied

[Electrostatic potential on the surface of low density lipoproteins from the plasma of patients with ischemic heart disease: estimation using a fluorescent probe].

Fluorescent probe I-aniline-8-naphthalene sulfonate was used to evaluate the LDL surface potential values in patients with coronary artery atherosclerosis. The more negative charge was found on the LDL surface in patients with atherosclerosis of three coronary arteries as compared with that of healthy persons.

Adult↗
PubMedDate not supplied

[Study of the effect of tegalide and bithionol on biological membranes using fluorescent probes].

Fluorescent probes were used to study structural changes in different membranes affected by gamma-radiation, protonophores and radioprotective agents, tegalide and bithionol. The preparations of the defined concentrations decreased the microviscosity of membranes, lowered the peaks and changed the temperature of phase transitions in liposomes from dipalmitoyl lecithin, and induced the output of Ca2+ from mitochondria. The effects depended on the radiation dose, the structure, concentration and lipophilicity of the preparation; protonophores produced a specific effect.

Animals↗
PubMedDate not supplied

In vivo tomographic imaging of near-infrared fluorescent probes.

Fluorescence imaging is increasingly used to probe protein function and gene expression in live animals. This technology could enhance the study of pathogenesis, drug development, and therapeutic intervention. In this article, we focus on three-dimensional fluorescence observations using fluorescence-mediated molecular tomography (FMT), a novel imaging technique that can resolve molecular function in deep tissues by reconstructing fluorescent probe distributions in vivo. We have compared FMT findings with conventional fluorescence reflectance imaging (FRI) to study protease function in nude mice with subsurface implanted tumors. This validation of FMT with FRI demonstrated the spatial congruence of fluorochrome activation as determined by the two techniques.

Animals↗
PubMed1985

Evidence for plasma membrane impermeability to small ions in acrosome-intact mouse spermatozoa bound to mouse zonae pellucidae, using an aminoacridine fluorescent pH probe: time course of the zona-induced acrosome reaction monitored by both chlortetracycline and pH probe fluorescence.

Previous studies have shown that capacitated mouse spermatozoa bind to zonae pellucidae of mouse eggs with acrosomes apparently intact. The question addressed in this study was the following: are the membrane permeability barriers of the apparently acrosome-intact sperm still retained or is there a preliminary stage of the acrosome reaction in which these barriers are lost and the intracellular space becomes accessible to extracellular substrates? The experimental approach was to use the fluorescent pH probe 9-amino-3-chloro-7-methoxyacridine, which accumulates in intracellular spaces of lower pH than the suspending medium with the result that the cells become fluorescent. Freshly capacitated mouse spermatozoa bound to isolated zonae showed uniform fluorescence over the head and midpiece with this fluorescent probe at early times of binding. The fluorescence was abolished by NH4+ and nigericin, agents that equilibrate H+ across cell membranes. At these early times of binding, the acrosomes were fully intact as judged by chlortetracycline fluorescence pattern, which itself was unaffected by either N4+ or nigericin. The time course of the loss of this chlortetracycline pattern characteristic of acrosome-intactness was closely paralleled by loss of fluorescence of 9-amino-3-chloro-6-methoxyacridine over the first 90 min; thereafter, loss of the chlortetracycline pattern was somewhat more rapid. This result shows that acrosome-intact sperm bound to zonae pellucidae retain the permeability barriers of the plasma membrane to small cations; no evidence was found for an early stage of membrane "leakiness" preceding the acrosome reaction. The ionophore A23187 induced a very rapid acrosome reaction in sperm bound to isolated zonae, as judged with both fluorescence probes. This rapid reaction was partially inhibited by 3-quinuclidinyl benzilate, which, in the absence of ionophore, completely blocks the occurrence of the acrosome reaction in sperm bound to zonae. This suggests involvement of a specific calcium entry mechanism in the acrosome reaction of mouse sperm induced by mouse zonae pellucidae.

Acrosome↗
PubMed1978

Study on calcium transport by sarcoplasmic reticulum vesicles using fluorescence probes.

Fluorescence changes of 1-anilino-8-naphthalenesulfonic acid and 3,3'-dipropyl-2,2'-thiadicarbocyanine during Ca transport by sarcoplasmic reticulum vesicles were studied. The fluorescence of both probes is enhanced corresponding to the rapid initial Ca uptake. The enhancement could be interpreted in terms of increased binding of the former (anionic) dye and decreased binding of the latter (cationic) dye to the vesicles, suggesting a change in the surface charge of the membranes associated with Ca transport. Under limited conditions where Na, K, and Mg were not added exogenously, Ca could be transported without concomitant counter-transport of these cations.

Adenosine Triphosphate↗
PubMed1978

A study of H+ transport in gastric microsomal vesicles using fluorescent probes.

Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (deltapH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the deltapH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to the accumulation in response to deltapH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that that K+-stimulated ATPase is responsible for the transport of H+ into the vesicles and thus develops a deltapH across the membrane. The deltapH generated by the K+-ATPase has a definite requirement for internal K+. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H+ and K+ ionophores. On the other hand, the sole use of the K+ ionophore, valinomycin, stimulates the ATP-induced formation of deltapH by increasing the availability of K+ to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl-. Analysis of the Cl- requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated deltapH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K+-stimulated ATPase drives a K+/H+ exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.

Acridines↗
PubMed2003

Novel fluorescent colloids as a DNA fluorescence probe.

Fluorescent perylene colloids in the 80-90 nm size range have been prepared by the reprecipitation method. These nanoparticles were modified by cetyltrimethylammonium bromide (CTAB) which inhibited their growth. The nanoparticles also readily interacted with DNA. The fluorescence emission was measured at lambda(ex)/ lambda(em)=400/565 nm. The fluorescence decrease of colloid-CTAB in aqueous solution was measured in the presence of nucleic acids. Under the optimum conditions, the ratio of fluorescence intensity in the absence and presence of nucleic acids was proportional to the concentration of nucleic acids over the range 0.02-5.1 micro g mL(-1) for FS (fish sperm) DNA or CT (calf thymus) DNA. The detection limits were 0.01 micro g mL(-1) for FS DNA and 0.012 micro g mL(-1) for CT DNA, respectively. Based on this approach, a new quantitative method for DNA assay is presented in this paper.

Buffers↗
PubMed1985

Interaction between the subunits of human erythrocyte spectrin using a fluorescence probe.

Fluorescence labeling of spectrin subunits was performed with N-(1-anilinonaphthyl-4)maleimide (ANM) to study the interaction between alpha and beta subunits. The fluorescence anisotropy of both ANM alpha and ANM beta increased linearly with the addition of nonfluorescent beta or alpha subunit, and saturated at a protein ratio about 1, indicating that 1 mol alpha subunit binds to 1 mol beta subunit with high affinity in vitro. Furthermore, this binding seemed to be reversible, because the anisotropy value decreased when an excess fo nonfluorescent alpha was added to the ANM alpha/beta mixture. The anisotropy of ANM alpha attained a maximum level within l min after addition of the same quantity of nonfluorescent beta at 12 degrees C, and the anisotropy of this mixture decreased rapidly when an excess of nonfluorescent alpha was added. These findings suggested that both the binding process of beta to ANM alpha and the dissociation step of ANM alpha from the ANM alpha-beta complex were quite rapid. The results obtained here imply that dynamic interaction between alpha and beta subunits of spectrin should be taken into account in understanding the role of the spectrin molecule in the cytoskeletal mesh.

Electrophoresis, Polyacrylamide Gel↗
PubMed2001

Rational design of fluorescein-based fluorescence probes. Mechanism-based design of a maximum fluorescence probe for singlet oxygen.

Fluorescein is one of the best available fluorophores for biological applications, but the factors that control its fluorescence properties are not fully established. Thus, we initiated a study aimed at providing a strategy for rational design of functional fluorescence probes bearing fluorescein structure. We have synthesized various kinds of fluorescein derivatives and examined the relationship between their fluorescence properties and the highest occupied molecular orbital (HOMO) levels of their benzoic acid moieties obtained by semiempirical PM3 calculations. It was concluded that the fluorescence properties of fluorescein derivatives are controlled by a photoinduced electron transfer (PET) process from the benzoic acid moiety to the xanthene ring and that the threshold of fluorescence OFF/ON switching lies around -8.9 eV for the HOMO level of the benzoic acid moiety. This information provides the basis for a practical strategy for rational design of functional fluorescence probes to detect certain biomolecules. We used this approach to design and synthesize 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) as a singlet oxygen probe and confirmed that it is the most sensitive probe currently known for (1)O(2). This novel fluorescence probe has a 9,10-dimethylanthracene moiety as an extremely fast chemical trap of (1)O(2). As was expected from PM3 calculations, DMAX scarcely fluoresces, while DMAX endoperoxide (DMAX-EP) is strongly fluorescent. Further, DMAX reacts with (1)O(2) more rapidly, and its sensitivity is 53-fold higher than that of 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs), which are a series of fluorescence probes for singlet oxygen that we recently developed. DMAX should be useful as a fluorescence probe for detecting (1)O(2) in a variety of biological systems.

Fluorescein↗
PubMed1983

The behavior of the fluorescent probe diS-C3-(5) in membrane and aqueous media.

Based on an analysis of back fluorometric titration data a partition coefficient, Kp = (5.70 +/- 0.95) x 10(4), and partition constant, K = (2.37 +/- 0.43) x 10(6), were found for the probe diS-C3-(5) in egg lecithin vesicle suspension. The relative probe quantity in an aqueous medium and in liposomes was calculated using these parameters. The number of chromophore states in this system was computer-analysed and it was shown that the probe fluorescence could be described by two fluorescing dye forms, aqueous and membrane monomers. The dependence of fluorescence intensity on the probe concentration was studied in various salt media, and a dimerization (association) constant Ka = 5 x 10(4) mol -1 . l in the buffer, and Ka = (8.1 +/- 1.5) x 10(4) mol-1 . l in 0.1 or 0.2 mol/l salt medium (KCl or NaCl) was found. From the fluorescence and absorption data critical concentrations of the onset of large probe aggregate formation were calculated for various aqueous media. The concentration dependence of the probe fluorescence in the membrane phase was calculated. The critical concentration of interaction characterizing the efficiency of the fluorescence concentration quenching processes (CCI) was found to be approx. 5-6 mol probe per 1000 mol lipid. The top probe concentration in a membrane (the "saturation" concentration) was estimated from the slope of the initial linear parts of the back fluorometric titration curves, and was found to be equal to (59 +/- 13) mol probe per 1000 mol lipid.

Benzothiazoles↗
PubMed1981

Intrinsic molecules in fluid phospholipid bilayers. Fluorescence probe studies.

Fluorescence probe data using 1,6-diphenyl-1,3,5-hexatriene for various concentrations of intrinsic molecules (cholesterol, gramicidin A amd cytochrome oxidase) within fluid lipid bilayers have been examined. The polarization value increases with increasing concentration of intrinsic molecule and then approaches a limiting value. Empirical curve-fitting of the experimental data, change of polarization with concentration, shows that each system can be fitted approximately by an exponential curve. A theory has been constructed based upon the assumption that only one intrinsic molecule need be adjacent to a fluorescent probe molecule to affect its motion drastically. The change in probe motion then depends upon the probability p of all positions next to a lipid chain being free of intrinsic molecules. The value of the probability p has been calculated and it is shown that (formula: see text) depending on whether the intrinsic molecule spans the lipid bilayer or not. The approximation p = e-Mx gives a good fit to the data for all x, thereby explaining the observed phenomenological fit. The fluorescent probe data is interpreted to show that protein-protein contacts increase as the intrinsic protein concentration increases within the lipid bilayer. An apparent dichotomy between the results from the fluorescence probe and from the deuterium magnetic resonance is explained in terms of a dominant affect on the probe being its hindrance to motion by interaction with the intrinsic molecule (protein) whilst individual C2H2 groups of the chain may exhibit greater disorder.

Cholesterol↗
PubMed1976

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes.

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe N-phenyl 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrene probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.

1-Naphthylamine↗
PubMed2002

Two-photon excitation imaging of pancreatic islets with various fluorescent probes.

Various fluorescent probes were assessed for investigating intact islets of Langerhans using two-photon excitation imaging. Polar fluorescent tracers applied on the outside rapidly (within 3 min) penetrated deep into the islets via microvessels. Likewise, an adenovirus carrying a Ca(2+)-sensitive green fluorescent protein mutant gene, yellow cameleon 2.1, was successfully transfected and enabled ratiometric cytosolic Ca(2+) measurement of cells in the deep layers of the islets. Interestingly, FM1-43, which is lipophilic and does not permeate the plasma membrane, also rapidly reached deep cell layers of the islets. In contrast, lipophilic fluorescent probes that permeate the plasma membrane (for example, fura-2-acetoxymethyl and BODIPY-forskolin) accumulated in the superficial cell layers of the islets, even 30 min after application. Thus, two-photon excitation imaging of pancreatic islets is a promising method for clarifying signaling mechanisms of islet cells, particularly when it is combined with membrane-impermeable probes. In addition, our data suggest that membrane-permeable antagonists may affect only the superficial cell layers of islets, and so their negative effects should be interpreted with caution.

Animals↗
PubMed2003

A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral.

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.

Cell Membrane Permeability↗
PubMed2006

Tunable design strategy for fluorescence probes based on 4-substituted BODIPY chromophore: improvement of highly sensitive fluorescence probe for nitric oxide.

4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) is a well-known fluorophore, with a high molar extinction coefficient and high fluorescence quantum efficiency (Phi(fl)). Furthermore, its structure can be modified to change its excitation and emission wavelengths. However, little work has been done on the structural modification of fluorines at the B-4 position with other functional groups. We synthesized 4-methoxy-substituted BODIPY derivatives in satisfactory yields, and found that they exhibited improved solubility in aqueous solution. Moreover, their oxidation and reduction potentials were greatly decreased without any change in their absorbance and fluorescence properties. These features of 4-substituted BODIPYs may be useful for developing novel fluorescence probes based on the intramolecular photoinduced electron transfer (PeT) mechanism, because it is possible to optimize the PeT process precisely by modulating the electrochemical properties of the fluorophore. The value of this approach is exemplified by its application to the development of a highly sensitive and pH-independent fluorescence probe for nitric oxide.

Boron Compounds↗