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Effects of ethanol volume percent on fluorescein-labeled spinach apo- and holocalmodulin.

We report the effects of EtOH volume percent (0-70%) on spinach apo- and holocalmodulin that have been site-selectively labeled with fluorescein (F). In these experiments, calmodulin (CaM) has one F reporter group attached to Cys-26, and this site is located immediately adjacent to one of the four primary Ca(2+)-binding sites (EF hands). The optimum analytical CaM-F sensitivity to Ca2+ occurs between approximately 10 and 30% EtOH. Our results also show that added EtOH causes changes in CaM and these changes are surprisingly different for apo- and holo-CaM. Apo-CaM-F appears to lose one of its two waters of hydration at approximately 20% EtOH and retains one water of hydration between approximately 20 and 70% EtOH. In apo-CaM-F, the semiangle that describes the range over which the fluorescein reporter group can precess remains essentially constant (42 +/- 2 degrees) between 0 and 70% EtOH. This shows that the fluorescein reporter group precessional freedom in apo-CaM-F is not affected significantly by EtOH. Holo-CaM-F also appears to lose one water of hydration at approximately 20-30% EtOH but then appears to denature as the EtOH volume percent increases. The fluorescein reporter group semiangle within holo-CaM-F decreases from 43 +/- 1 degrees in neat aqueous buffer to 36 +/- 1 degrees at 70% EtOH. This shows that holo-CaM-F is less nativelike and the EF hand "closes down" about the fluorescein reporter group in holo-CaM-F as the EtOH volume percent increases.

Apoproteins↗

Comparison between operative findings on malignant glioma by a fluorescein surgical microscopy and histological findings.

Using a fluorescein surgical microscope that we developed, we performed surgery on 30 cases of malignant glioma. Operative findings and histological findings were then compared in five of these cases. Fluorescein sodium was systemically administered intravenously as a fluorescent dye. About 20 min after intravenous administration, fluorescein activity in the blood decreased, and the fluorescence was observed only in the area lacking the blood-brain barrier function, such as tumors; then, resection of the tumor was started. The fluorescent regions coincided with the enhanced regions on CT and MRI. Fluorescein spread to the surrounding brain edematous region with time, but its intensity was very different from the tumor-associated one. In the histological examination of intensely fluorescent regions, abnormal tumor blood vessels with a thick wall and a small caliber, or with a thin wall and a large caliber, were observed. Dense tumor cells were found in these regions. On the other hand, in regions with weak or no fluorescence, infiltration of tumor cells was scant, and no abnormal blood vessels were found. Fluorescence was not observed in necrotic regions of the tumor center. These histological findings coincided with those obtained in large surgical specimen or autopsy, and tumor-cell rich regions seemed to be removed by resecting abnormal vascular regions (fluorescent regions). Enhanced regions on CT and MRI disappeared after operation by resecting intensely fluorescent regions. These results indicate that the fluorescein surgical microscope developed by us is a useful aid in operations on malignant glioma.

Brain Neoplasms↗

Fluorescein-conjugated bovine albumin; physical and biological properties.

Fluorescein-bovine albumin conjugates have been prepared and found not to differ appreciably in size, shape, and homogeneity from the precursor, bovine serum albumin. Fluorescein has also been conjugated to rat plasma proteins. Their disappearance rates from the circulation of rats correspond with those obtained from the use of isotope labeling. Their sites of localization in rat tissues were shown to be in the cytoplasm but not in the nuclei of Kupffer cells, fixed macrophages, granulocytes, and proximal renal tubules. Adsorption to endothelium was a characteristic finding. Extracellular localizations were predominantly in the lumina of blood vessels and proximal renal tubules (but never in the lumina of collecting tubules), and the interstitial fluid of skeletal and cardiac muscle (but not that of glandular organs such as the adrenals, liver, and spleen). BAC absorption from the skin of rabbits requires days whereas sodium fluorescein absorption is measured in hours, attesting to the persistence of the colloidal state of BAC in vivo. Fluorescein conjugates have been used to visualize the transcapillary passage of circulating proteins in the mesenteric circulation of frogs and rats by direct microscopic observation and found to diffuse slowly in the manner predicted for plasma proteins. The normal cutaneous vessels of the rat are impermeable in the gross to the labeled proteins; second degree burn promptly increases the permeability of these vessels rendering the presence of the label detectable in the gross in the skin. The process of labeling does not render guinea pig albumin antigenic, although slight antigenicity results from labeling whole plasma protein. It is believed that sufficient biological evidence is presented to support the conclusion that fluorescein-conjugated plasma proteins, particularly albumin, behave in vivo like their native precursors.

Animals↗

Sodium fluorescein as a retinal pH indicator?

Retinal neovascularization is a symptom associated with various diseases revealing ocular fundus manifestation. Often, these neovascularizations originate from retinal hypoxia. A concomitant phenomenon of hypoxia is acidosis. To recognise this would permit the identification and treatment of hypoxic fundus areas long before first vascular modifications are seen. Thus, the goal of this investigation was to elucidate whether sodium fluorescein could be used as a retinal pH indicator. Sodium fluorescein solution was diluted in PBS (ratio: 1:150,000). The pH was varied from 6.5 to 8.6 by supplementation of HCl or NaOH, respectively. The fluorescence was excited by a pulsed diode laser (wavelength: 446 nm, pulse width: 100 ps) and detected by time-correlated single photon counting (TCSPC) technique. A least-squares fit of the measured fluorescence decay versus time by an exponential function results in the fluorescence lifetime. Ten measurements were taken at each pH for statistical analysis. The dependence of the fluorescence lifetime on the temperature and the concentration of sodium fluorescein was investigated in the same way. The fluorescence lifetime was found to rise from 3.775 ns to 4.11 ns with increasing pH (6.5 to 8.6). However, the gradient decreases with increasing pH. We found highly significant differences (Student's t-test, P<0.0005) of the fluorescence lifetimes for pH values with a mean difference of 0.125 at pH<7.65 whereas the differences were still significant (P 7.65 and mean pH differences of 0.2. The fluorescence lifetime was independent of the temperature (22 degrees C to 37 degrees C) and the concentration of sodium fluorescein (dilution 1:150,000 to 1:2000). The fluorescence lifetime of sodium fluorescein depends on the pH but not on temperature and concentration. Thus, the discrimination of areas with retinal acidosis should be possible by combination of the TCSPC technique with scanning laser ophthalmoscopy. Further investigations have to clarify whether the accuracy of the measurement at the fundus in vivo is sufficient.

Acidosis↗

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.

DNA Probes↗

Studies on the nature of the flap/bed interaction in rodents--Part II: Morphological and fluorescein kinetic data.

The current study in rats concerns two aspects of the flap/bed interaction: fluorescein kinetics and histological finding. In the control group of animals in the fluorescein kinetics experiment, flaps were raised and then reattached. In one experimental group, the flaps were raised and placed on a sterile polyethylene film. In two other groups, the flaps were raised and then the pedicle was cut, either immediately or at specific intervals. At intervals up to 24 hours several animals were injected with 5% fluorescein. In the control group fluorescein penetration increased with time, while it decreased in the polyethylene-film group. There was virtually no fluorescein staining in both cut-pedicle groups. To study histological events, the animals were divided into groups with flaps and groups in which the distal end of each flap was severed to form a contiguous skin graft. Other conditions were varied. All flaps were cultured, and biopsy specimens were obtained from three areas at several time intervals. Histological sections from the distal flap were markedly different from the corresponding skin grafts. These results corroborate previously noted survival rates.

Animals↗

The photography of fluorescein.

The last few years have seen a number of new flaps described and a renewed interest in the use of fluorescein, but there have been few photographs of the fluorescein effect, because special light sources were required with the filters that were employed. The realization that fluorescein can be excited by electromagnetic radiation in the visible range allows a simplified technique in which an ordinary electronic flash unit may serve as the only light source. The photography of fluorescein is not difficult to perform, and since minimal additional equipment is required, all workers who use fluorescein should begin to document their work more accurately and dramatically.

Fluoresceins↗

Comparison of fluorescein angiography and optical coherence tomography for patients with choroidal neovascularization after photodynamic therapy.

PURPOSE: To investigate retinal morphology by means of fluorescein angiography (FA) and optical coherence tomography (OCT) in patients who had undergone photodynamic therapy (PDT) with verteporfin at their 3-month-interval examination. METHODS: Sixty patients with predominantly classic choroidal neovascularization (CNV) secondary to age-related macular degeneration were evaluated with FA and OCT 3 months after their last PDT. FA images were evaluated in a masked fashion for staining of and leakage from the lesion and also for cystoid loculation of fluorescein in the macula. OCT was used to evaluate foveal thickness and the presence of subretinal fluid or cystoid spaces within the retina, also in a masked fashion. RESULTS: The median age of the 60 patients was 78 years, and the median visual acuity of the eyes examined was 20/100. The median number of previous PDT sessions was 2. Fluorescein staining was seen in 57 eyes (95%), and fluorescein leakage was seen in 50 eyes (83%). Cystoid loculation of fluorescein was seen in 21 eyes (35%). By OCT, cystoid spaces in the macula were seen in 42 patients (70%), and subretinal fluid was seen in 15 patients (25%). Leakage seen shown by FA was correlated with the OCT finding of cystoid spaces but not with the OCT finding of subretinal fluid. Some patients had leakage during FA that did not have any observable induced OCT abnormality attributable to fluid accumulation. CONCLUSIONS: After PDT leakage from CNV seen during FA is associated with intraretinal fluid, often seen in loculated cystoid spaces, but not with subretinal fluid.

Aged↗

Quantitative fluorimetric determination of cell-surface glycoconjugates with fluorescein-substituted lectins.

Fluorescein-substituted lectins, which can be used to visualize cell surface glycoconjugates, are shown to be usable in the quantitative determination of the number of receptor sites and of their association constant. The fluorescence measurements of the fluorescein-substituted lectins released from the cell surface with the related inhibitor, give quantitative data in a large range of fluorescein-substituted lectin concentration (0.1 to 100 microgram/ml). Using fluorescein-substituted concanavalin A or [3H]acetyl-concanavalin A, it was found that baby hamster kidney cells (BHK 21, wild-type) bind 10 +/- 2 x 10(6) lectin molecules per cell with an apparent association constant of 1.8 or 1.7 x 10(6) 1 x mol-1, respectively. Using the fluoresceinyl and [3H]acetyl-substituted wheat germ agglutinin, we found 40 +/- 5 x 10(6) sites per cell with an apparent binding constant of 1 and 1.3 +/- 0.3 x 10(6) 1 x mol-1, respectively. When fluorescein-substituted succinyl wheat germ agglutinin was used instead of the unsuccinylated wheat germ agglutinin derivatives, the number of binding sites was reduced 7 times, while the binding constant was very slightly lowered. Concanavalin A derivatives gave monotonic Scatchard plots; on the opposite, wheat germ agglutinin derivatives gave biphasic Scatchard plots suggesting that wheat germ agglutinin binds to two classes of receptors.

Animals↗

Anterior segment fluorescein angiography in inflammatory diseases of the cornea.

To study the vascular changes in inflammatory diseases of the cornea 22 patients with various corneal inflammations were examined by means of anterior segment fluorescein angiography. Simple avascular central and marginal corneal ulcers stained with fluorescein in the late phase of angiography. An inflamed limbus and an early microscopic pannus adjacent to the ulcer were seeen in simple corneal ulcers. Progressive pannus with pronounced fluorescein leakage was observed in chronic corneal ulcer, disciform keratitis, Mooren's ulcer, and complicated acute keratoconus. In sclerokeratouveitis and in gutter associated with rheumatoid arthritis the corneal vessels showed less leakage. The iris vessels showed fluorescein leakage as a sign of irritative iritis during the active stage of simple and chronic corneal ulcers, in disciform keratitis, Mooren's ulcer, and in graft rejection. It is concluded that anterior segment fluorescein angiography gives valuable information of the vascular architecture, flow and leakage in inflammatory diseases of the cornea.

Adult↗

Binding of fluorescein to vitreous in vitro.

When the blood-retina barrier is estimated by fluorophotometry, it is usually assumed that fluorescein is not bound in the vitreous. If a certain amount of fluorescein is not free it will influence the precise estimation of the permeability. To investigate a possible binding of fluorescein, vitreous from 25 eyes was aspirated post mortem, and an indirect estimate of the free fraction of fluorescein was made. The mean ratio between free and total amount of fluorescein was found to be 0.78 +/- 0.15 SD. The implication of the present result on previously published data on the blood-retina barrier is that the values for permeability may have been estimated erroneously. The direction of the error on the estimated permeability can not be simply predicted.

Blood-Retinal Barrier↗

Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.

BACKGROUND: The early microbiological diagnosis of corneal infections may prevent the condition from worsening. AIM: To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. METHODS: Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. RESULTS: The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). CONCLUSIONS: The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.

Acanthamoeba↗

Measurement of retinal blood flow with fluorescein leucocyte angiography using a scanning laser ophthalmoscope in rabbits.

AIMS: To measure blood flow in the rabbit retinal circulation with fluorescein leucocyte angiography using a scanning laser ophthalmoscope. METHODS: Blood was withdrawn from the ear vein of a rabbit (New Zealand White), mixed with fluorescent dye in a test tube and centrifuged. The yellow-brown layer containing fluorescein stained leucocytes was collected and injected into the ear vein of the same rabbit while performing fluorescein angiography with a scanning laser ophthalmoscope. The image of retinal angiography displaying circulating fluorescent leucocytes was recorded on video tape. From each frame of the video tape, the consecutive positions of fluorescein stained leucocytes were digitised using an image analysis system and the velocity of blood flow was calculated. RESULTS: Fluorescent leucocytes were clearly visualised in the retinal arteries, capillaries, and veins which allowed measurement of blood flow. The mean capillary velocity was 0.69 (SD 0.21) mm/s. The mean velocities of leucocytes measured in different sized vessels were as follows: 5.83 (2.42) mm/s in arteries over 50 microns, 3.33 (0.62) mm/s in those 35-50 microns, and 2.42 (1.08) mm/s in arteries under 35 microns, 3.08 (1.56) mm/s in veins over 50 microns, 2.79 (1.49) mm/s in those 35-50 microns, and 1.21 (0.50) mm/s in veins under 35 microns. Blood flow pulsation occurs in arteries, arterioles, veins, and venules but not capillaries. CONCLUSION: Fluorescein leucocyte angiography can be used for simultaneous measurement of the blood flow in retinal arteries, veins, and capillaries.

Animals↗

Colour Doppler imaging and fluorescein filling defects of the optic disc in normal tension glaucoma.

AIM: To investigate the relation between blood flow parameters of the retrobulbar vessels measured by means of colour Doppler imaging (CDI) and fluorescein filling defects of the optic nerve head in patients with normal tension glaucoma (NTG) and control subjects. METHODS: 29 patients with NTG and 29 age and sex matched control subjects were included in this study. Blood flow velocities-peak systolic velocity (PSV), end diastolic velocity (EDV), and resistive indices (RI) of the ophthalmic artery (OA), the central retinal artery (CRA), and of the temporal and nasal short posterior ciliary arteries (TPCA, NPCA)-were measured with CDI. Fluorescein angiograms were performed with a scanning laser ophthalmoscope. The extent of absolute fluorescein filling defects of the optic nerve head in relation to the optic nerve head was assessed. RESULTS: The PSV of the OA, the PSV and EDV of the CRA, and of the TPCA and NPCA were significantly reduced in NTG (p<0.05). The RI of the CRA, the TPCA and NPCA were significantly increased in NTG (p<0.01). The optic nerve head fluorescein filling defects were significantly larger in NTG (p<0.01). The filling defects were significantly negatively correlated (p<0.05) with the PSV and EDV of the CRA (PSV(CRA): r = -0.41; EDV(CRA): r = -0.34), with the PSV and EDV of the NPCA (PSV(NPCA): r = -0.34; EDV(NPCA): r = -0.38), and with the EDV of the TPCA (r = -0.29). A significant positive correlation (p<0.05) was found with the RI of both PCAs (RI(NPCA): r = 0.28; RI(TPCA): r = 0.29). CONCLUSION: Patients with NTG had reduced blood flow velocities and higher resistive indices in most retrobulbar vessels. Optic nerve head fluorescein filling defects were larger compared to controls. The filling defects were correlated with end diastolic velocities and resistive indices of the PCAs and with blood flow velocities of the CRA. Capillary loss of the optic nerve head may be related to higher downstream resistance and reduced blood flow velocities of the retrobulbar vessels.

Blood Flow Velocity↗

Bioavailability of fluorescein from a new drug delivery system in human eyes.

AIM: To assess the ocular bioavailability of fluorescein from a novel drug delivery system compared with one single preservative free eye drop. METHODS: Part A: In a randomised study 10 volunteers applied the lyophilisate to one eye and a conventional fluorescein eye drop to the fellow eye. Fluorophotometry was performed before and every 2 minutes up to 30 minutes after application in the cornea and anterior chamber. Part B: Another 10 volunteers applied each form of the application. Fluorophotometry was performed before, +2 minutes, and at +8, +10, +12 hours. The dose corresponding to a single fluorescein dose of the lyophilisate was 68 l microg fluorescein SE 0.17%. RESULTS: Part A: During the first 30 minutes after administration of the preservative free eye drop of 40 microg the corneal and anterior chamber concentration means were up to 16 times higher in eyes treated with the lyophilisate. Part B: 8-12 hours after application the mean fluorescein concentration in the cornea of the lyophilisate group was two times higher than at baseline. Eyes treated with eye drops had baseline values at +8, +10 and +12 hours. CONCLUSION: A significantly better bioavailability was achieved in human eyes by using lyophilisate compared with the same dose from a conventional eye drop. Lyophilisates are a favourable alternative to conventional eye drops since they have no preservatives, higher long term stability, no pH adjustment, and easy handling.

Adult↗

Dynamic and quantitative analysis of choroidal neovascularization by fluorescein angiography.

PURPOSE: In this study, the authors sought to develop and characterize techniques for measuring changes in choroidal neovascularization (CNV) lesion size and fluorescence over time for quantitative analysis of fluorescein angiograms. METHODS: Initial assessment of the quantitative technique was made by retrospectively analyzing digital fluorescein angiograms taken before and 3 months after photodynamic therapy (PDT) for CNV (6 patients, group 1). The method was then applied prospectively to digital fluorescein angiograms (baseline and day 71) obtained on 12 patients taking part in a clinical trial investigating the effect of vascular endothelial growth factor (VEGF) Trap in CNV (group 2). Two masked observers, with the use of image processing, measured the area of hyperfluorescence and fluorescence intensity above background. Values for each image were plotted against time after dye injection to generate curves, and each area under the curve (AUC) was calculated. RESULTS: The physician who treated the patients in group 1 judged the condition of three patients to be improved and of three to be worse 3 months after PDT. Masked retrospective grading of fluorescein angiograms showed an 11% decrease in AUC for fluorescence area and a 32% decrease in AUC for fluorescence intensity in the three patients whose conditions clinically improved but increases of 131% and 292% in the three patients whose conditions clinically worsened. In group 2, a 38% decrease in AUC for fluorescence intensity and a 19% decrease in AUC for fluorescence area were observed in patients who received VEGF Trap compared with increases of 66% (P = 0.004, Mann-Whitney U test) and 21% (P = 0.07) for patients who received placebo. Macular volume decreased by 11% in VEGF Trap-treated patients and increased by 10% in placebo-treated patients (P = 0.03). CONCLUSIONS: This study reports a technique for analysis of change in fluorescence area and intensity over time during fluorescein angiography (FA) using a continuous scale and its application in a clinical setting and a clinical trial. Compared with previous techniques making use of categorical scales, this approach provides an advantage for evaluating responses to treatment that may improve the value of FA as an outcome measure in clinical trials.

Choroidal Neovascularization↗

Fluorescein: a rapid, sensitive, nonlethal method for detecting skin ulceration in fish.

There is a need to develop simple, rapid, and accurate methods for assessing health in fish populations. In this study we demonstrate that use of fluorescein, a nontoxic fluorescent dye, can rapidly and easily detect the presence of skin ulcers in all fish tested, including rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), goldfish (Carassius auratus), and hybrid striped bass (Morone saxatilis male X M. chrysops female). Exposure of fish to as little as 0.10 mg fluorescein per milliliter of water for 3 minutes was sufficient to identify experimentally induced lesions, even pinpoint ulcerations. Such lesions were not visible to the naked eye but were clearly demarcated with fluorescein treatment. Examination of fish that appeared clinically normal often revealed the presence of focal ulcerations, which might have been a consequence of damage during capture, but it also might suggest that skin ulceration may be common even in "clinically normal" fish. Exposure of either nonulcerated or experimentally ulcerated hybrid striped bass to an excessively high concentration of fluorescein had no apparent effect on health or survival. Our studies suggest that fluorescein may be a highly useful tool for rapid health screening in fish populations.

Animals↗

Identification of intranasal cerebrospinal fluid leaks by topical application with fluorescein dye.

The purpose of this paper is to describe a safe new technique for intraoperative identification of the site of cerebrospinal fluid rhinorrhea. Cerebrospinal fluid (CSF) rhinorrhea after intracranial or intranasal surgery is a known potential complication with significant morbidity and mortality. It is currently accepted that endoscopic intranasal management of CSF rhinorrhea is the preferred method of surgical repair, with higher success rates and less morbidity than intracranial surgical repair in selected cases. Accurate identification of the site of CSF leakage is necessary for a successful endoscopic surgical repair. Computer tomography (CT) with or without intrathecal contrast and preoperative nasal endoscopy are frequently used to preoperatively localize the site of the leak. Intrathecal fluorescein administered immediately before surgery has aided in the intraoperative identification of the site of CSF leak in 25-64% of patients undergoing endoscopic repair of CSF rhinorrhea in whom preoperative CT scanning and nasal endoscopy had not identified the site of CSF leak. Intrathecal fluorescein, however, has been associated with severe complications, such as lower extremity weakness, numbness, generalized seizures, opisthotonus, and cranial nerve deficits. We present three cases of CSF rhinorrhea in which fluorescein was applied intranasally during the endoscopic surgical repair. Ten percent fluorescein was applied to the nose with a cotton swab. Under endoscopic visualization the fluorescein changed its fluorescent color from amber/yellow to a dark green and was found streaming from high in the nasal cavity, which led to accurate identification of the site of the CSF leak.

Administration, Topical↗