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PubMed2005

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein as a new fluorescent labeling reagent for aliphatic amines in environmental and food samples using high-performance liquid chromatography.

6-Oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl)fluorescein (SAMF), a new fluorescein-based amine-reactive fluorescent probe was well designed, synthesized and used as a pre-column derivatizing reagent for the determination of aliphatic amines in HPLC. It exhibited relatively pH-independent fluorescence (pH 4-9) and excellent photostability. The derivatization was performed at room temperature in 6min. On a C18 column, the derivatives of SAMF with eight aliphatic amines were baseline separated in 28 min with a mobile phase of methanol-water (57:43, v/v) containing 10 mmol l(-1) pH 5.0, H3Cit3-NaOH buffer. With fluorescent detection at lambda(ex)/lambda(em) = 484/516 nm, the detection limit could reach 2-320 fmol (signal-to-noise = 3), which was equivalent to or better than the detection limits obtained from other analytical methods of aliphatic amines. The proposed method has been applied to the determination of the aliphatic amines in environmental and food samples such as lake water, red wine, white wine, and cheese with satisfying recoveries varying from 95 to 106%.

Amines↗

PubMed1975

The use of fluorescein isothiocyanate labelled lectins for immunohistochemical demonstration of saccharides.

Cryostat sections of various tissues from man, rat, mice, hamster, rabbit, guinea pig, sparrow and carp were examined by fluorescence microscopy after incubation with fluorescein isothiocyanate labelled Concanavalin A and Lens culinaris lectin. In the same tissue of various species no basic differences in the pattern of fluorescence were observed. In general, connective tissue structures were stained by both lectins in the same degree, whereas by fluorescein isothiocyanate labelled Concanavalin A additionally a varying staining of the cell surface and of cytoplasmic structures occurred. The results show the general presence of Concanavalin A and Lens culinaris lectin receptors in normal animal tissues.

Animals↗

PubMed1991

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate.

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed2003

Rational design principle for modulating fluorescence properties of fluorescein-based probes by photoinduced electron transfer.

Fluorescence properties of fluorescein-based probes are shown to be finely controlled by the rate of photoinduced electron transfer from the benzoic acid moiety (electron donor) to the singlet excited state of the xanthene moiety (electron acceptor fluorophore). The occurrence of photoinduced electron transfer is clearly evidenced by transient absorption spectra showing bands due to the radical cation of the electron donor moiety and the radical anion of the xanthene moiety, observed in laser flash photolysis experiments. The photoinduced electron transfer rates and the rates of back electron transfer follow the Marcus parabolic dependence of electron transfer rate on the driving force. Such a dependence provides for the first time a quantitative basis for a rational design principle which has high efficiency in modulating fluorescence properties of fluorescein-based probes.

Journal Article↗

PubMed1996

[Effects of fluorescein and indocyanine green angiography on subsequent dark adaptation and the electroretinogram].

BACKGROUND: In clinical practice the electroretinogram (ERG) usually should not be recorded immediately after conventional fluorescein angiography (FLA), because of the bleaching effects of strong light exposure on the course of dark adaptation and on the ERG. Currently it is not known, if indocyaningreen (ICG) angiography using the Heidelberg Retina Angiograph (HRA) has similar effects on the outer retina and if therefore the ERG should also not be recorded after ICG angiography. METHODS: Eight patients with different retinal diseases were examined. After 5 minutes of light adaptation to 160 cd/m2, Ganzfeld ERGs were repeatedly recorded using DTL electrodes during a 30 minutes dark period with a flash luminance of 0.1 cds/.m2. After this dark adaptation period a standardized ERG was recorded according to the ISCEV recommendations. This procedure was followed either by ICG angiography using the HRA or by conventional fluorescein angiography using the Canon CF 60 fundus camera. Within an interval of 5 minutes the above mentioned ERG procedure was repeated. RESULTS: ICG angiography using the HRA did neither affect the course of dark adaptation as measured by increasing b-wave amplitudes during the 30 minutes dark adaptation period, nor the values for each response of the Ganzfeld standard ERG. FLA did neither affect b-wave responses to dim stimuli, nor to stronger stimuli in the scotopic range. However, the course of dark adaptation was significantly prolonged after FLA. The maximum b-wave was not affected. CONCLUSIONS: ICG angiography using the HRA could be performed prior to the recording of the ERG, without any effect on the ERG. In contrast conventional FLA must be performed after ERG recordings, in order not to alter the electrophysiological responses.

Adult↗

PubMed1982

[Studies on the precorneal tear film. First communication: an optical system for demonstrating tear film without fluorescein (author's transl)].

Tear film is usually studied by using fluorescein. An optical system is described which despenses with the use of fluorescein. It mainly consists in an equal diffuse illumination of the cornea, provided through an opaque truncated cone that is illuminated from behind. The cone is situated as close as possible to the front of the eye. The cornea and its surface can be observed through a hole at the narrow end of the cone. Interferences in the lipid layer of the tear film are recognizable as "colors of thin layers". The physical background to interferences in thin, convex layers is discussed. Break-ups are visualized by using a supplementary "Schlieren" method.

Biomechanical Phenomena↗

PubMed1992

Immunophotodiagnosis of colon carcinomas in patients injected with fluoresceinated chimeric antibodies against carcinoembryonic antigen.

Based on previous experiments in nude mice, showing that fluoresceinated monoclonal antibodies against carcinoembryonic antigen localized specifically in human carcinoma xenografts and could be detected by laser-induced fluorescence, we performed a feasibility study to determine whether this immunophotodiagnosis method could be applied in the clinic. Six patients, with known primary colorectal carcinoma, received an i.v. injection of 4.5 or 9 mg of mouse-human chimeric anti-carcinoembryonic antigen monoclonal antibody coupled with 0.10-0.28 mg of fluorescein (molar ratio 1/10 to 1/14). The monoclonal antibody was also labeled with 0.2-0.4 mCi of 125I (1 Ci = 37 GBq). Photodetection of the tumor was done ex vivo on surgically resected tissues for the six patients and in vivo by fluorescence rectosigmoidoscopy for the sixth patient. Upon laser irradiation, clearly detectable heterogeneous green fluorescence from the dye-antibody conjugate was visually observed on all six tumors; almost no such fluorescence was detectable on normal mucosa. The yellowish tissue autofluorescence, which was emitted from both tumor and normal mucosa, could be subtracted by real-time image processing. Radioactivity measurements confirmed the specificity of tumor localization by the conjugate; tissue concentrations of up to 0.059% injected dose per g of tumor and 10 times less (0.006%) per g of normal mucosa were found. The overall results demonstrate the feasibility of tumor immunophotodiagnosis at the clinical level.

Antibody Affinity↗

PubMed1986

Cell surface antigen identification by a modified fluorescein immunosphere method.

Fluorescein immunospheres may be used in an indirect technic to label lymphocyte cell surface antigens. Wright's counter-staining allows simultaneous identification of fine morphology and surface antigen properties. Studies designed to determine methods for separating cells with bound immunospheres from free immunospheres revealed that removal of unbound immunospheres by flotation over cold protein solution creates excessive numbers of false negatives. Glutaraldehyde cross-linking of the primary and secondary antibodies linking immunospheres to the lymphocyte stabilizes the complex. This modification of the fluorescein immunosphere method compares favorably with flow cytometry.

Adult↗

PubMed1987

Medium for the simultaneous detection of pyocyanin and fluorescein pigments of Pseudomonas aeruginosa.

To help simplify the identification of Pseudomonas aeruginosa by clinical microbiology laboratories, the authors developed a new medium, pyocyanin-fluorescein agar (PFA), which enhances the production of both Pseudomonas pigments simultaneously. Production of pigments on PFA was equivalent to production on a pyocyanin agar (P agar) and a fluorescein agar (F agar) used in combination and was superior to either P agar or F agar used alone. The medium is simple to prepare and it detected pigment in 94% of P. aeruginosa isolates tested.

Culture Media↗

PubMed2003

Size exclusion and diffusion of fluoresceinated probes within collagen fibrils.

The diffusion of fluoresceinated probes inside single collagen fibrils was investigated by imaging the migration of fluorescence along the fibrils in oil and by monitoring fluorescence recovery after photobleaching (FRAP). Probes were excluded from the fibrils according to their size. Probes that were not excluded diffused in the fibrils, but FRAP occurred 6 x 10(-4) times more slowly than in water due to binding interactions between collagen and the probes. The dissociation constant of the fluorescein-collagen complex was determined (K(D)=1.8+/-0.1 microM).

Journal Article↗

PubMed1975

Auxin transport inhibitors: fluorescein and related compounds.

Fluoresceins are shown to be effective inhibitors of indoleacetic acid transport as measured by the receiver agar block technique, eosin having the same order of activity as 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid, with fluorescein less effective. It is suggested that many of their characteristic effects on plants, especially those which involve auxin, are at least partially due to their effects on auxin transport.

Journal Article↗

PubMedDate not supplied

Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein.

In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.

Algorithms↗

PubMed1971

Specific staining of wall mannan in yeast cells with fluorescein-conjugated concanavalin A.

A procedure is given for the coupling of fluorescein isothiocyanate to concanavalin A, a protein which specifically combines with a variety of polysaccharides, and for the subsequent isolation of the reactive conjugate. This fluorescent conjugate stains Saccharomyces cerevisiae but not Schizosaccharomyces pombe or Rhodotorula glutinis. The cell walls of the latter two organisms do not contain branched homopolymers of alpha-linked mannose. Furthermore, the staining of S. cerevisiae is competitively inhibited by either unlabeled concanavalin A or methyl-alpha-d-manno-pyranoside. On the basis of this evidence, it is concluded that the staining of S. cerevisiae results from the specific interaction of the fluorescein-concanavalin A conjugate with the alpha-mannan present in the cell wall of this yeast.

Antigens↗

PubMed1967

Evaluation of fluorescein isothiocyanate-labeled whole antiserum in the immunofluorescent identification of microorganisms.

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 mug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 mug of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 mug of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.

Bacteriological Techniques↗

PubMed1979

Hen fluorescein-labeled gonococcal lipopolysaccharide antibody in the delayed fluorescent antibody technique for the confirmation of Neisseria gonorrhoeae.

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.

Animals↗

PubMed1985

Physiological and pathobiological significance of ocular glycoproteins. I. Studies using fluorescein labelled glycine max.

Cell surface carbohydrates play an important role in several biological, immunological, and neoplastic phenomena including development, growth regulation, cellular locomotion, receptor activation, and tumour metastasis. Fluorescein labelled lectins which bind to specific carbohydrate residues in glycoproteins and glycolipids are being increasingly used as chemical probes to study cell components. Several different preparations of ocular tissues from human, rabbit, and rat were examined for the distribution of N-acetyl-D-galactosamine (D-gal NAc) by means of fluorescein-labelled lectin from soybean (glycine max). A very strong fluorescence was observed in the corneal epithelium; Descemet's membrane and corneal endothelium were also strongly fluorescent. The conjunctival epithelium similarly showed a strong reaction, as did the goblet cells. The iris epithelium and the dilator pupillae were only weakly fluorescent, but the ciliary body showed strong fluorescence, as did the blood vessels. As compared with lens fibres the lens epithelium was strongly fluorescent. The outer retina, that is, the photoreceptors, the pigment epithelium, and Bruch's membrane, showed a very strong reactivity. The optic nerve showed moderate fluorescence, but reaction with extraocular muscles was variable. The skin of the upper and lower eyelids, hair follicles, and blood vessels showed strong lectin binding. Sections of retinoblastoma and malignant melanoma showed no reaction. The physiological and pathological significance of these findings is discussed.

Acetylgalactosamine↗

PubMed2002

Capillary density and retinal diameter measurements and their impact on altered retinal circulation in glaucoma: a digital fluorescein angiographic study.

AIM: Normal pressure glaucoma (NPG) patients exhibit prolonged retinal arteriovenous passage times in fluorescein angiography and colour Doppler imaging suggests increased resistance downstream from the central retinal and posterior ciliary arteries. The aim of the study was to elucidate the morphological source of decreased perfusion and increased resistance of the ocular circulation in NPG. METHODS: Retinal arteriovenous passage time (AVP) and peripapillary arterial and venous diameters were measured in digital scanning laser fluorescein angiograms. For estimation of retinal capillary density the area of the foveal avascular zone (FAZ) and the perifoveal intercapillary area (PIA) was quantified. 36 patients with NPG (mean age 57 (SD 13) years) and 21 healthy subjects (mean age 51 (13) years) were enrolled in the comparative study. RESULTS: In NPG patients the AVP (2.55 (1.1) seconds) was significantly prolonged (p<0.001) when compared with healthy subject data (AVP: 1.70 (0.39) seconds). No differences for arterial or venous diameter, FAZ, and PIA were observed in NPG patients compared with healthy subjects. FAZ, PIA, arterial and venous diameter were not correlated with visual field indices (except venous diameter with PSD, r=0.35 (p<0.05)) or cup to disc ratios. AVP was significantly correlated (p<0.05) with the size of the optic nerve head (r=-0.28), visual field indices (MD: r=-0.3; PSD: r=0.3; CPSD: r=0.3), and contrast sensitivity (r=-0.34). CONCLUSION: AVP times are significantly prolonged in NPG. The slowing of the retinal transit does not result from capillary dropout, or changes of peripapillary arterial or venous diameters with increased vascular resistance.

Blood Flow Velocity↗

PubMedDate not supplied

Retinal fluorescein angiographic evidence for atheromatous microembolism. Demonstration of ophthalmoscopically occult emboli and post-embolic endothelial damage after attacks of amaurosis fugax.

There is evidence that microemboli responsible for amaurosis fugax may be atheromatous but it can elude ophthalmoscopic confirmation, because such emboli quickly fragment and disappear from view in the retinal vessels. This report documents 2 patients in whom fluorescein angiography of the retina provided evidence of microembolization after an attack of amaurosis although the fundus appeared normal. In one patient the angiogram revealed intraluminal material; in the other it showed fluorescein leakage from patent arteriolar bifurcations.

Aged↗