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A new class of fluorescein/rhodamine hybrids with two spirolactone rings was reported to exhibit dual-output fluorescent behaviors independently. Isolation and characterization for two diastereomers, trans-RhOH and cis-RhOH, have been made and their X-ray crystal structures determined. In a basic environment, the spirolactone ring on the hydroxyl side will be opened to give a fluorescein-like optical output with the lowest absorptions at 485 and 530 nm emission. On the other hand, a rhodamine-like optical output, i.e., 528 nm absorption and 575 nm emission, will be switched on by a H+ or a Hg2+ ion, attributed to the spirolactone ring opening on the amino side. In a methanol-buffer system with different pH values, the corresponding pKa values for the hydroxyl and amino groups were determined as 5.7 and 2.3, respectively. Selective Hg2+-sensing properties have also been discussed, and log Ks values of about 3.60 and 3.73 were determined. Confocal microscopic images of Caenorhabditis elegans incubated with RhOH were found to show enhanced fluorescent intensity with a Hg2+ ion, demonstrating the potential application of RhOH for in vivo biological imaging. [ABSTRACT FROM AUTHOR]

Purpose: Unstable tear film characterized by shorting of tear break-up time (BUT) is associated with discomfort and dryness in contact lens wearers. The glycocalyx is thought to be crucial in maintaining the wettability and lubrication of the ocular surface. We evaluated the ocular surface glycocalyx in soft contact lens (SCL) wearers using a fluorescein-labeled wheat germ agglutinin (F-WGA) as a marker to demonstrate the ocular surface glycoconjugatesin vivo. Methods: Twenty experienced SCL wearers and 20 healthy volunteers with no history of CL wearing (controls) were enrolled in the study. After applying a 5% F-WGA solution to the eyes of study individuals, fluorescent intensities in their respective central corneas were measured by fluorophotometry. The relationship between F-WGA intensity in the corneal surface and clinical parameters associated with contact lens wear were analyzed. Results: F-WGA fluorescence intensity in the SCL group was 418.5 ± 103.3, which was significantly lower than that of the controls (825.0 ± 179.8;p < 0.0001, Mann–Whitney test). F-WGA fluorescence intensity was not correlated with Schirmer’s test values or age, whereas a statistically significant correlation between F-WGA fluorescence intensity and tear film BUT was observed (r = 0.77,p < 0.0001). The decrease in F-WGA fluorescence intensity could be reversed by discontinuation of SCL use. Conclusion: Reduction and/or compositional alteration of ocular surface glycocalyx may be one of the causative factors of SCL-induced eye dryness. [ABSTRACT FROM PUBLISHER]

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively. [ABSTRACT FROM AUTHOR]

A new and facile method for the determination of critical micelle concentration (CMC) of ionic and non-ionic surfactants is proposed in this article. Carbon dots exhibited substantial fluorescence and therefore enhanced the sensitivity of this evaluation. Understanding the formation of surfactant micelles is vital for the applications of biomedicine such as drug fabrication and smart molecular vehicles in delivering therapeutic dosage to various molecular sites. The fluorescence property of carbon dots was utilized for the first time to estimate the critical micelle concentration of surfactants. The central concept of the approach is based on the Stokes shift determination of a system composed of constant amount of carbon dots with varying concentrations of ionic and non-ionic surfactants. The synthesized carbon dots were characterized by FTIR, TEM, XRD, Raman, UV, and fluorescence spectroscope. The carbon dots were excited at 280 nm so as to obtain maximum emission for the Stokes shift measurement. The CMC value of cetyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulfate (SDS), Triton X-100, dodecyldimethyl(3-sulfopropyl)ammonium hydroxide (SB-12) evaluated by this approach was found to be 0.98, 7.3, 0.19, and 3.5 mM, respectively. The signals of spectra were assigned and explained in terms of both electron transitions between specific molecular orbital and the interaction with solvent. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells. [ABSTRACT FROM AUTHOR]

Novel silica gel-immobilized rhodamine (SGR) anchored by hydrazinium hydrate was synthesized. The selectivity and adsorption ability of the SGR for metal cations were investigated with fluorophotometry and atomic absorption spectrophotometery, respectively. The SGR exhibits high selectivity and adsorption capacity for Hg2+. The maximum static adsorption capacity of the SGR for Hg2+was 25.8μmol g−1. The linear response range covers a concentration range of Hg2+from 20 ppb to 2000 ppb and the detection limit is 10 ppb. The determination of Hg2+in real water samples displays satisfactory results, and the SGR can be used repeatedly by treating with a solution of tetrabutylammonium hydroxide. [ABSTRACT FROM AUTHOR]

Study of signal transduction in live cells benefits from the ability to visualize and quantify light emitted by fluorescent proteins (XFPs) fused to different signaling proteins. However, because cell signaling proteins are often present in small numbers, and because the XFPs themselves are poor fluorophores, the amount of emitted light, and the observable signal in these studies, is often small. An XFP's fluorescence lifetime contains additional information about the immediate environment of the fluorophore that can augment the information from its weak light signal. Here, we constructed and expressed in Saccharomyces cerevisiae variants of Teal Fluorescent Protein (TFP) and Citrine that were isospectral but had shorter fluorescence lifetimes, ∼1.5 ns vs ∼3 ns. We modified microscopic and flow cytometric instruments to measure fluorescence lifetimes in live cells. We developed digital hardware and a measure of lifetime called a “pseudophasor” that we could compute quickly enough to permit sorting by lifetime in flow. We used these abilities to sort mixtures of cells expressing TFP and the short-lifetime TFP variant into subpopulations that were respectively 97% and 94% pure. This work demonstrates the feasibility of using information about fluorescence lifetime to help quantify cell signaling in living cells at the high throughput provided by flow cytometry. Moreover, it demonstrates the feasibility of isolating and recovering subpopulations of cells with different XFP lifetimes for subsequent experimentation. [ABSTRACT FROM AUTHOR]

The ability of detecting the subtle variations occurring, among different individuals, within specific DNA sequences encompassed in highly polymorphic genes discloses new applications in genomics and diagnostics. DQB1 is a gene of the HLA-II DQ locus of the Human Leukocyte Antigens (HLA) system. The polymorphisms of the trait of the DQB1 gene including codons 52–57 modulate the susceptibility to a number of severe pathologies. Moreover, the donor-receiver tissue compatibility in bone marrow transplantations is routinely assessed through crossed genotyping of DQB and DQA. For the above reasons, the development of rapid, reliable and cost-effective typing technologies of DQB1 in general, and more specifically of the codons 52–57, is a relevant although challenging task. Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labelling the opposite ends of gene-specific oligonucleotide probes has proven to be a powerful tool to type DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, i.e. time-correlated single-photon counting, which allows working on very diluted template specimens and in the presence of fluorescent contaminants. Here we present a full in-vitro characterization of the fluorescence responses of two probes when hybridized to oligonucleotide mixtures mimicking all the possible genotypes of the codons 52–57 trait of DQB1 (8 homozygous and 28 heterozygous). We show that each genotype can be effectively tagged by the combination of the fluorescence decay constants extrapolated from the data obtained with such probes. [ABSTRACT FROM AUTHOR]

For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca2+-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca2+-free and Ca2+-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca2+-dependent way, unraveling in vitro dissociation constants KD of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca2+-free and Ca2+-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ KD of 180 nM was determined. Thus, quantitative [Ca2+]i recordings were realized, unraveling a reversible dopamine-induced [Ca2+]i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca2+ indicator dye for 2P-FLIM recordings applicable in diverse biological systems. [ABSTRACT FROM AUTHOR]

The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple ‘in solution’ steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (Kd) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction. [ABSTRACT FROM AUTHOR]

We developed genetically-encoded fluorescent sensors based on Förster Resonance Energy Transfer to monitor phosphatidic acid (PA) fluctuations in the plasma membrane using Spo20 as PA-binding motif. Basal PA levels and phospholipase D activity varied in different cell types. In addition, stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed in the leading edge than in the trailing edge of migrating HeLa cells. In MSC80 and OLN93 cells, which are stable cell lines derived from Schwann cells and oligodendrocytes, respectively, a higher ratio of diacylglycerol to PA levels was demonstrated in the membrane processes involved in myelination, compared to the cell body. We propose that the PA sensors reported here are valuable tools to unveil the role of PA in a variety of intracellular signaling pathways. [ABSTRACT FROM AUTHOR]

The Fluorescence2D is free software that allows analysis of two-dimensional fluorescence spectra obtained using the accelerated “triangular” acquisition schemes. The software is a combination of Python and MATLAB-based programs that perform conversion of the triangular data, display of the two-dimensional spectra, extraction of 1D slices at different wavelengths, and output in various graphic formats. [ABSTRACT FROM AUTHOR]

A time-resolved fluorescence (TRF) technique is presented for classifying motor oils. The system is constructed with a third harmonic Nd:YAG laser, a spectrometer, and an intensified charge coupled device (ICCD) camera. Steady-state and time-resolved fluorescence (TRF) measurements are reported for several motor oils. It is found that steady-state fluorescence is insufficient to distinguish the motor oil samples. Then contour diagrams of TRF intensities (CDTRFIs) are acquired to serve as unique fingerprints to identify motor oils by using the distinct TRF of motor oils. CDTRFIs are preferable to steady-state fluorescence spectra for classifying different motor oils, making CDTRFIs a particularly choice for the development of fluorescence-based methods for the discrimination and characterization of motor oils. The two-dimensional fluorescence contour diagrams contain more information, not only the changing shapes of the LIF spectra but also the relative intensity. The results indicate that motor oils can be differentiated based on the new proposed method, which provides reliable methods for analyzing and classifying motor oils. [ABSTRACT FROM AUTHOR]

Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus particles undergo a step of proteolytic maturation, in which the main structural polyprotein Gag is cleaved into its mature subunits matrix (MA), capsid (CA), nucleocapsid (NC) and p6. Gag proteolytic processing is accompanied by a dramatic structural rearrangement within the virion, which is necessary for virus infectivity and has been proposed to proceed through a sequence of dissociation and reformation of the capsid lattice. Morphological maturation appears to be tightly regulated, with sequential cleavage events and two small spacer peptides within Gag playing important roles by regulating the disassembly of the immature capsid layer and formation of the mature capsid lattice. In order to measure the influence of individual Gag domains on lattice stability, we established Förster's resonance energy transfer (FRET) reporter virions and employed rapid kinetic FRET and light scatter measurements. This approach allowed us to measure dissociation properties of HIV-1 particles assembled in eukaryotic cells containing Gag proteins in different states of proteolytic processing. While the complex dissociation behavior of the particles prevented an assignment of kinetic rate constants to individual dissociation steps, our analyses revealed characteristic differences in the dissociation properties of the MA layer dependent on the presence of additional domains. The most striking effect observed here was a pronounced stabilization of the MA-CA layer mediated by the presence of the 14 amino acid long spacer peptide SP1 at the CA C-terminus, underlining the crucial role of this peptide for the resolution of the immature particle architecture. [ABSTRACT FROM AUTHOR]

Objective--To evaluate aqueous humor flow rate in the eyes of clinically normal cats by use of a noninvasive technique successfully used in other species. Animals--20 domestic shorthair cats. Procedures--1 drop of 10% fluorescein sodium was instilled into both eyes of 5 cats every 5 minutes until 3 drops had been administered. Fluorophotometry was performed at 2, 4, 5, 6, 7, 8, 9, and 10 hours after fluorescein application to monitor fluorescein removal and determine aqueous humor flow rate. The 3-drop protocol was used for the remaining 15 cats, and fluorophotometry was performed at 5, 6, 7, and 8 hours after fluorescein application. Aqueous humor flow rates were calculated manually by use of established equations with minor adjustments to constant values to reflect feline anatomic features. Correlation coefficients and slope ratios were calculated to assess the legitimacy of the flow rate data. Paired t tests were calculated to assess for differences between the right and left eyes. Results--Mean ± SD calculated aqueous humor flow rate in the right, left, and both eyes of the 20 cats was 5.94 ± 2.30 µL/min, 5.05 ± 2.06 µL/min, and 5.51 ± 2.21 µL/min, respectively. Correlation coefficients and slope ratios revealed that the aqueous humor flow rates were accurate. No significant differences in values for the right and left eyes were detected. Conclusions and Clinical Relevance--Accurate aqueous humor flow values for cats can be determined by use of the fluorophotometric technique evaluated in this study. [ABSTRACT FROM AUTHOR]

Abstract: The luminescence of thenoyltrifluoroacetonate (TTA) coordination complexes of trivalent europium ion (Eu(III)) in aqueous solutions and in solid-state polymeric films is probed upon single- and two-photon preresonant excitation with Ti:sapphire femtosecond laser. Particularly, diamine-liganded Eu(III)(TTA)3 and poly(oxyethylene phosphate)tris(β-diketonate)Eu(III) complexes are examined aiming their possible applications as luminescent labels for sensing and imaging of biological molecules. Even at a pre-resonance, the excitation of these compounds with high-intensity, broadband light of frequency-doubled Ti:sapphire femtosecond laser centered around 400nm results in a luminescence response suitable for fluorometric applications. [Copyright &y& Elsevier]

AIM: This study was performed to independently assess the turnover rates of aqueous and lipid layers of the tear film. METHODS: Two fluorescent dyes, fluorescein sodium and 5-dodecanoylaminofluorescein (DAF), which is a free-fatty-acid conjugate of fluorescein, were applied to the right eye of 12 healthy volunteers. Fluorescent intensity of the precorneal tear film was measured at the central cornea every minute for 10 min for fluorescein sodium, and every 5 min for 50 min for DAF. The turnover rate was calculated by plotting fluorescent intensity against time in a semilog plot and expressed as %/min. RESULTS: Turnover rates of fluorescein sodium and DAF were 10.3 (SD 3.7)%/min and 0.93 (0.36)%/min, respectively. The turnover rate of DAF was significantly lower than that of fluorescein sodium (p<0.05, Mann–Whitney test). The turnover rate of DAF positively correlated with that of fluorescein sodium (r = 0.93, p<0.05). CONCLUSION: Our results indicate that the turnover of lipids in tears is much slower than the aqueous flow of tears, and that this lipid turnover is associated with the aqueous flow of tears in healthy adults. [ABSTRACT FROM AUTHOR]

Abstract: The luminescence of four europium thenoyltrifluoroacetonate complexes containing diamine ligands is studied in solutions. The compounds are resonantly excited in the ligand absorption band by means of high-power nanosecond UV laser pulses. Additionally, a continuous light source is also used for resonant, as well as pre-resonant excitation of the complexes. The luminescence response characterizes the synthesized europium coordination complexes as emissive compounds suitable for fluorometric applications. Related to the possible usefulness of the diamine-liganded Eu complexes as luminescent labels for sensing and imaging of biological molecules, the luminescence response of these compounds upon UV excitation was probed also in aqueous media. [Copyright &y& Elsevier]

Purpose: The use of biopolymers in peribulbar injection for controlled drug delivery provides alternative options to eyedrops and intravitreal or surgical methods. Polymerizable biopolymers are especially likely to have a role because of their particular properties. In liquid form, they can be easily injected into the target site and, after polymerization, they provide a prolonged and controlled release of the drug. This study was undertaken to demonstrate the suitability of a thermopolymerizable biopolymer poloxamer (Lutrol F127) for peribulbar injections and controlled drug release. Methods: The toxicity of injected poloxamer compounds was evaluated by visual inspection and histological and immunohistochemical tissue evaluation. The release of marker substances such as 5(6)-carboxyfluorescein (376 Da) or fluorescein isothiocyanate-dextran (FITC-dextran) (4−40 kDa) from poloxamer was used to simulate drug release and penetration into the eye using in vivo fluorometry. Results: According to our clinical and pathological analyses, poloxamer was well tolerated in peribulbar injections and did not cause acute toxicity at the site of injection. The marker compounds were released from the site of injection during the first 24 hours. Conclusions: Although poloxamer appears to be suitable for peribulbar injections, a more prolonged period of dissolution is desirable for clinical purposes. [ABSTRACT FROM AUTHOR]

Abstract: Background:: Systemic antihistamines such as loratadine are efficacious in the treatment of many allergic conditions. However, their use has been associated with drying effects, particularly of the ocular surface. Objective: The purpose of this study was to compare the ocular drying effects of topical treatment with epinastine hydrochloride ophthalmic solution 0.05 % twice daily and systemic treatment with loratadine 10 mg once daily in patients with seasonal allergic conjunctivitis through the use of several standard clinical tests for dry eye. Methods:: This was a single-center, 4-visit, open-label, investigator-masked crossover study in healthy adult volunteers with a history of seasonal allergic conjunctivitis. Participants received 4 days of treatment with topical epinastine administered as 1 drop per eye twice daily and 4 days of treatment with systemic loratadine once daily, with a 10-day washout between treatments. Fluorophotometry was conducted at the baseline visit, at the crossover visit, and after 4 days of each treatment to assess tear volume, tear flow, and tear turnover rate. Decreases from baseline in tear volume, tear flow, or tear turnover rate were considered indicative of a drying effect. Keratitis, global (corneal and conjunctival) fluorescein staining (scale from 0–20 points), and tear film break-up time (TFBUT) were assessed after fluorophotometry at each visit. An increase in keratitis (as measured by global fluorescein staining) and conjunctival lissamine green staining, and a decrease in TFBUT were considered further evidence of a drying effect. Results:: Eighteen subjects were enrolled in and completed the study (78% female; 94% white; age range, 18–55 years). After 4 days of twice-daily use, topical epinastine was associated with no significant changes in tear volume, tear flow, or global fluorescein staining. After 4 days of once-daily use, systemic loratadine was associated with a 33.7% reduction in tear volume (mean reduction, 4.5 μL; range, −7.53 to 22.36 μL; P < 0.05), a 35.0% reduction in tear flow (mean reduction, 0.93 μL/min; range, −2.57 to 2.73 μL/min; P < 0.05), and a 21.7% increase in global fluorescein staining (mean increase, 0.74 point; range, −2.5 to 6.5 points; P < 0.05). No significant change in TFBUT was observed with either treatment. Conclusions:: These healthy adult volunteers with a history of seasonal allergic conjunctivitis had no clinical signs of ocular drying after 4 days of twice-daily treatment with topical epinastine. After 4 days of once-daily dosing, systemic loratadine was associated with clinical signs of ocular dryness, including decreased tear volume and tear flow. Use of loratadine was also associated with an increase in global fluorescein staining, indicating an increase in ocular surface damage. [Copyright &y& Elsevier]

Abstract: Lens autofluorescence originates from an accumulation of fluorescent substances such as the tryptophan-derived residues and protein aggregations, which are associated with the preclinical progress of cataractogenesis, diabetes and lens aging. Our purpose is to determine if pregnancy alters the typical constituents of the lens autofluorescence. Fifteen healthy pregnant women (22 eyes) who were in their third trimester of pregnancy and 23 age-matched healthy controls (37 eyes, non-pregnant females). Lens autofluorescence, lens transmission and corneal autofluorescence were studied with fluorophotometry. The lens autofluorescence values were 358±151ngml−1 in the control group and 201±110ngml−1 in the pregnants women. The difference was significant (p=0.0074). Lens transmission values were 0.93±0.02ngml−1 in the control group and 0.94±0.02ngml−1 in the pregnants women: the difference was not significant. Corneal autofluorescence values were 21.9±7.5ngml−1 in the control group and 18.2±5.8ngml−1 in the pregnant women. The difference was not significant. Our study showed a significant decrease in lens autofluorescence in pregnant women compared to a normal population. The decrease can be partly attributed to the aqueous component of the lens that increases significantly during the final trimester of pregnancy and that this provokes a dilution of the fluorescent substances. [Copyright &y& Elsevier]

Several non-invasive techniques for dry eye diagnosis have been developed in the past decade. These include quantitative assessment of tear volume, tear film stability, tear dynamics, and integrity of ocular surface epithelium. A combination of meniscometry and interferometry is useful for proving focal dry eye, by confirming whether or not tears at the meniscus have an effect on the ocular surface. Interferometer is also useful to evaluate tear dynamics on soft contact lenses. Fluorophotometry is useful for assessing the severity of dry eye from the view point of corneal epithelial barrier function and measuring the tear turnover rate. Both video-meibography and meibometry are useful for screening meibomian gland dysfunction. The advances in these techniques accumulate knowledge regarding pathophysiology of dry eye and allow precise diagnosis of dry eye. More targeted treatment will become feasible based on the clearer pathophysiology. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Recent clinical studies show that a single intravitreal injection of the corticosteroid triamcinolone acetonide (TAA) may reduce edematous retinal swelling and improve visual acuity in patients with diabetic macular edema (DME). In addition, clinical and experimental studies strongly suggest that blood–retinal barrier breakdown in diabetes is induced by vascular endothelial growth factor (VEGF). These results suggest that corticosteroids may modulate VEGF-mediated responses in vivo. To test this hypothesis directly, the current study evaluated the effects of TAA and dexamethasone (DEX) in a newly developed rabbit model of VEGF-induced blood–retinal barrier and blood–aqueous (iris) barrier breakdown. Methods: VEGF165 or vehicle was injected intravitreally in female Dutch Belt rabbits, and scanning ocular fluorophotometry was used to non-invasively measure fluorescein leakage from retinal and iris vasculature. VEGF-induced retinal vasculopathy was further assessed with fundus imaging, fluorescein angiography, and ocular coherence tomography. For pharmacologic studies, rabbits were treated with either DEX (2mgkg-1 daily, s.c.), TAA (2 or 4mg, intravitreal), indomethacin (20mgkg-1 daily, s.c.), or vehicle (s.c. or intravitreal). Human umbilical vein endothelial cells (HUVEC) were loaded with the fluorescent Ca2+ indicator dye fluo-4 and treated with dexamethasone (0·1–10μm) or vehicle for either 2 or 24hr prior to stimulation with 10ngml-1 VEGF165. Results: VEGF injected intravitreally induced a time and dose-dependent breakdown of the blood–retinal and blood–aqueous barriers. Maximal vascular leakage was measured at 48hr after intravitreal injection with a dose of 500ng VEGF. Other effects of VEGF included prominent retinal vasodilation, vessel tortuousity, fluorescein leakage from retinal vessels, and inner retinal edema. These VEGF-mediated responses are transient and approach baseline by 1 week. VEGF-induced blood–retinal and blood–aqueous barrier breakdown was completely blocked by the corticosteroid DEX administered systemically for 3 days. In contrast, the non-steroidal anti-inflammatory drug, indomethacin, had no effect. In a separate study with VEGF injected intravitreally at six different time points over 5 months, a single intravitreal 2mg dose of TAA completely blocked VEGF-induced retinal and iris leakage for 45 days. VEGF/VEGF receptor-2-mediated Ca2+ mobilization in endothelial cells was not affected by 2 or 24hr pretreatment with dexamethasone. Conclusion: These results indicate that one mechanism by which corticosteroids block blood–ocular barrier breakdown and edema is via their modulation of signaling or effector proteins downstream of the VEGF receptor. [Copyright &y& Elsevier]

Abstract: We conducted a series of surgical studies (n=155) to find out the possible utility of the Dutch Belted rabbits as an ocular test model. Following either vitrectomy (n=59) or lensectomy (n=96) studies using either BSS® or BSS® Plus, we characterized the corresponding magnitude and duration of inflammatory response of selected endpoints over a one-week period. Preoperative Dutch Belted rabbits served as controls for baseline determination (n=27). Inflammatory endpoints included clinical inflammation, blood aqueous barrier (BAB) changes measured by particle-scatter and fluorophotometry, corneal edema, and prostaglandin-E2 (PGE2). Topical ocular 0·1% dexamethasone (MAXIDEX®) served as a positive treatment group. We compared the inflammatory features (treated and untreated) to determine significance. Using either irrigating solution, the endpoints (n=101) were shown increased in both models. While the clinical scores were similar following both types of surgery (ns; p=0·51), the lensectomy study caused a more marked effect on corneal edema (p=0·0004) and PGE2 production (p=0·002) compared with the vitrectomy study. After the lensectomy procedure, BSS Plus® (n=52) compared with BSS® (n=24) showed a significant improvement (p=0·004) of clinical score during the recovery phase. Further improvement was gained over BSS Plus® (n=52) using MAXIDEX® treatment. Topical MAXIDEX® (lensectomy, n=20/group; vitrectomy, n=12/group) reduced clinical score (p<0·001), decreased BAB breakdown to fluorescein (p<0·01), lessened particle flare (p<0·05), inhibited aqueous PGE2 levels (p<0·001), and reduced corneal edema (p=0·01) in the lensectomy group. The use of the rabbit model offers a convenient test to identify therapeutic agents that could lessen ocular complications after these common ocular surgeries. [Copyright &y& Elsevier]

Purpose: Tear turnover rate (TTR) is defined as the percent decrease of fluorescein concentration in the tears per minute after the instillation of fluorescein. The purpose of this study was to examine differences in TTR in a sample with symptoms of dry eye and an asymptomatic control sample using the Fluorotron Master™ Fluorophotometer. Methods: TTR was measured using the OcuMetrics Fluorotron Master™. It measures the decay of the fluorescence of high molecular weight fluorescein FITC Dextran instilled into the tear film. Twenty participants (post-menopausal women) were enrolled in the study (10 asymptomatic (age 64.7±6.99) and 10 symptomatic (age 61.5±7.98)). Participants were grouped according to either a positive (symptomatic) or negative (asymptomatic) McMonnies Dry Eye Questionnaire, i.e., an indication of self-reported ocular dryness and the use of rewetting/lubricating drops (questions 4 and 5). TTR was measured in the afternoon only. Measurements were made on the right eye with a controlled blink rate (15 blinks/min), for up to 30 min, post-insertion of 2 μl of 2% FITC Dextran (MW 9500). The scan data were used to construct a graph of log fluorescein concentration (ng/ml) as a function of time and the TTR calculated (%/min=(1−ln (slope))×100). Results: There was a significant difference in the TTR between symptomatic and asymptomatic subjects. Mean TTR (symptomatic) was 4.89±2.74%/min (range, 2.04–11.81) and mean TTR (asymptomatic) was 11.85±3.31%/min (range, 5.76–16.45) (P<0.0001). Conclusions: Fluorophotometry may be used to demonstrate differences in the tear turnover rate in this post-menopausal group of women, with patients experiencing symptoms of dry eye having a lower TTR than the normals. [Copyright &y& Elsevier]

Abstract. Purpose: Corneal fluorescence is believed to be caused by advanced glycation end products formed by non-enzymatic glycation on corneal proteins. The purpose of the present twin study was to examine whether the process is related to genetic or environmental factors. Methods: Corneal fluorescence was measured in 59 monozygotic and 54 dizygotic twin pairs. The influences of genetic and environmental factors were estimated using structural equation modelling. Results: Interindividual variation in corneal fluorescence was attributable to environmental factors, whereas the effect of genetic factors was of little or no significance. Corneal fluorescence correlated significantly with smoking habits (r = 0.38) and the 2-hour oral glucose tolerance test response (r = 0.27), and increased with age (p < 0.0001). Conclusion: Fluorophore accumulation in the cornea was attributable to age and environmental effects, of which smoking was the most conspicuous identifiable factor, although glucose was also of relevance. However, the greater part of interindividual variation in corneal fluorescence remains unexplained. [ABSTRACT FROM AUTHOR]

Abstract. Purpose: To investigate whether a nitric oxide donor given as a single oral dose is able to modify aqueous humour flow in healthy volunteers. Methods: Ten healthy volunteers participated in a randomized, double-masked and placebo-controlled cross-over study. Aqueous humour flow was assessed by fluorophotometry after intake of isosorbide-5-mononitrate (ISMN), 10 mg. Topical timolol maleate, which is known to reduce aqueous humour flow, was used as a positive control. Intraocular pressure (IOP) was measured by applanation tonometry and blood pressure was registered. Results: The basal rate of aqueous humour flow did not change significantly after a single oral dose of ISMN. The aqueous humour flow in the timolol-treated eye was reduced as compared to the contralateral control eye (p = 0.002). Mean IOP 6 hours after placebo and ISMN intake did not differ significantly. Timolol lowered IOP by 4 mmHg (p < 0.001). ISMN did not lower systolic blood pressure, but diastolic blood pressure was reduced by 4 mmHg (p = 0.048). Conclusion: A single oral dose of 10 mg ISMN had no significant effect on aqueous humour flow in healthy volunteers. [ABSTRACT FROM AUTHOR]

The aim of the study was to develop a mucoadhesive ocular insert for the controlled delivery of ophthalmic drugs and to evaluate its efficacy in vivo. The inserts tested were based either on unmodified or thiolated poly(acrylic acid). Water uptake and swelling behavior of the inserts as well as the drug release rates of the model drugs fluorescein and two diclofenac salts with different solubility properties were evaluated in vitro. Fluorescein was used as fluorescent tracer to study the drug release from the insert in humans. The mean fluorescein concentration in the cornea/tearfilm compartment as a function of time was determined after application of aqueous eye drops and inserts composed of unmodified and of thiolated poly(acrylic acid). The acceptability of the inserts by the volunteers was also evaluated. Inserts based on thiolated poly(acrylic acid) were not soluble and had good cohesive properties. A controlled release was achieved for the incorporated model drugs. The in vivo study showed that inserts based on thiolated poly(acrylic acid) provide a fluorescein concentration on the eye surface for more than 8 h, whereas the fluorescein concentration rapidly decreased after application of aqueous eye drops or inserts based on unmodified poly(acrylic acid). Moreover, these inserts were well accepted by the volunteers. The present study indicates that ocular inserts based on thiolated poly(acrylic acid) are promising new solid devices for ocular drug delivery. [Copyright &y& Elsevier]

: PurposeTo evaluate the effects of a lipophilic analog of superoxide dismutase in the treatment of persistent epithelial defects of the cornea.: DesignInterventional case series.: MethodsNineteen eyes of 19 patients with noninfectious corneal ulcers were enrolled. Patient profiles consisted of seven cases with Mooren-type peripheral ulcers, three cases with chemical or thermal burns, two cases with vernal keratoconjunctivitis, four cases with cicatricial keratoconjunctivitis, and three cases with sterile ulcers after corneal surgery. Patients consisted of 11 men and 8 women, with a mean age of 51.5 ± 17.9 years (range, 14–77). Only patients who did not respond to conventional therapy were recruited for the study. Lecithinated superoxide dismutase was applied for 2 weeks, and sketches and/or fluorescein slit micrographs were recorded before and at the end of the study. The main outcome measure was decrease in ulcer size.: ResultsAs a group, the average ulcer size relative to the entire cornea decreased from 18.2 ± 15.1% to 10.0 ± 15.0% after 2 weeks (P < .05). A dramatic effect (>90% decrease in ulcer size) was observed in eight cases (100% in six cases), whereas no apparent change was found (<30% decrease in ulcer size) in five cases. The presence of polymorphonucleocytes within the ulcerated tissue was confirmed by brush cytology.: ConclusionLecinthinated superoxide dismutase was effective in decreasing persistent epithelial defect size in a subset of patients with sterile corneal ulcers refractive to conventional therapy. A large-scale clinical investigation is merited. [Copyright &y& Elsevier]

Purpose: To determine the amount of pupillary constriction to four different concentrations of pilocarpine in normal human subjects and to determine if pupillary constriction correlates with bioavailability of the instilled concentrations. The amount of pupillary constriction to dilute pilocarpine is utilized as a diagnostic test for Adie tonic pupil as distinguished from a normal pupil response.Design: Twenty healthy volunteers had automated binocular infrared pupillography in the dark after instillation of four different concentrations of dilute pilocarpine. Ocular penetration of eye drops was also assessed using 2% fluorescein sodium as a tracer.Methods: Prospective institutional double-masked study of both eyes of twenty healthy volunteers, ten with brown irides, ten with blue irides, between the ages of 20-40 years.Results: A pilocarpine dose-dependent curve showed decreased pupil size within 15 minutes, peaking at 30-60 minutes. No difference was noted between right and left eyes, iris color, or corneal permeability.Conclusions: Normal pupils constrict to dilute concentrations of pilocarpine (0.25% or 0.125%), but constrict insignificantly to concentrations of 0.0313% or 0.0625%. Pupil constriction with 0.0625% pilocarpine should distinguish an Adie pupil from normal. This confirms the utility of this simple office diagnostic procedure. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the loss of lens transparency incurred by patients undergoing trabeculectomy. Design: A prospective cohort study. Participants and Controls: Data corresponding to 33 eyes of 33 consecutive patients with primary open-angle glaucoma (POAG) subjected to trabeculectomy (group 1) were compared with those corresponding to 12 eyes of 12 patients with POAG receiving topical antiglaucomatous treatment (group 2). Intervention: Lens fluorophotometry was performed on the group 1 patients before and 12 months after surgery. In group 2, fluorophotometry was conducted at the onset of the study and at 12 months of follow-up. Main Outcome Measures: Starting and final lens autofluorescence and transmittance values corresponding to each subject group were compared. Results: In group 1, starting and final autofluorescence was 556.3 ± 184.3 and 691.1 ± 179.3 Eq ng/ml, and starting and final transmittance was 0.78 ± 0.11 and 0.67 ± 0.14, respectively. Respective values for group 2 were 574.3 ± 94.8 and 595.2 ± 107.0 Eq ng/ml and 0.72 ± 0.17 and 0.71 ± 0.16. The mean change between final and initial autofluorescence was statistically different between groups (134.7 ± 123.7, group 1, 20.9 ± 25.1 Eq ng/ml, group 2; P < 0.001). Similarly, a significant difference (P < 0.001) in transmittance change was observed between the surgery and control groups (-0.11 ± 0.072, group 1; 0.02 ± 0.008, group 2) Conclusions: It was demonstrated by lens fluorophotometry that trabeculectomy in POAG leads to a loss in lens transparency. [ABSTRACT FROM AUTHOR]

Purpose: To examine the long-term outcome of vitreous fluorophotometry including the predictability of retinopathic proliferative development in diabetic subjects. Methods: 21 diabetic subjects were examined by long-term kinetic vitreous fluorophotometry and their barrier properties, as revealed by a permeability-index, were compared to a group of normal subjects. 13 diabetics were classified as having a high permeability-index, while 8 diabetics were classified as having a low permeability-index. The two groups were followed for 5 years with regard to development of proliferative retinopathy. Results: 6 of 13 diabetics with a high permeability-index developed proliferative retinopathy within the 5-year follow-up period. This was in contrast to the low permeability-index group where none of 8 diabetics developed proliferative retinopathy. Conclusion: An increased permeability-index points towards an increased risk of development of proliferative retinopathy. [ABSTRACT FROM AUTHOR]

PURPOSE. The buffering action of human tears is thought to be important to keep its pH constant. We measured the change in pH in the precorneal tear film in vivo when the acidic solution is challenged, using a fluorophotometric technique. METHODS. Twelve eyes from 6 healthy subjects were entered in this study. Each subject was pretreated with either one drop of 0.4% oxybuprocaine for once (light anesthesia), three times (deep anesthesia), or none (controls). The measurement was initiated by instilling 20 μl of 0.067 M phosphate buffer at pH 5.5 containing 2 mM bis-carboxyethyl-carboxyfluorescein free acid, a pH sensitive dye, into the subject's eye. The pH was determined by the ratio of fluorescent intensities at two excitation wavelengths (490 and 430 nm). pH recovery time (PHRT) as defined by the time required for pH to reach 95% of pH at equilibrium was used for the marker of tear buffering action. Tear turnover rate was also determined using the fluorescent decay curve at 430 nm, which was independent of pH, but dependent on dye concentration. RESULTS. Immediately after the instillation, the pH value in the tear film was around 6.0–6.5 in all cases. The tear film rapidly became more alkaline, reaching its normal value in 2.3 ± 0.5 min in untreated eyes. The pretreatment with 0.4% oxybuprocaine retarded the neutralization process. A single regression analysis revealed that the PHRT had a significant negative correlation with the tear turnover rate (r = -0.78). Conclusions. Our results suggest that the neutralization process of tears largely depends on the tear turnover rate. The buffering action of tears in vivo consists of the tear turnover as well as its chemical buffering capacity. [ABSTRACT FROM AUTHOR]

A Beer-Lambert analysis of fluorophotometric scans of ocular lenses has been carried out. It is shown that an assumption of a single flourophor allows us to calculate the distribution of this fluorophor along the central lens axis. For the great majority of lenses in our study, covering a wide range of ages, our results are in essential agreement with published results on excised lenses. For a small subset of lenses in the study the assumption of a single fluorophor is found to be unsatisfactory. In these cases a reasonable symmetry assumption allows us to estimate the distribution of an additional agent which removes light from the entering or exiting beams without contributing to the fluorescence. The fact that this agent is restricted to the lens nucleus and that it occurs in lenses in which nuclear changes are clinically evident suggests that it is an expression of nuclear cataract. [ABSTRACT FROM AUTHOR]

The article explores the genome size of Porphyra yezoensis using fluorometry, and assesses the potential of this alga for further analysis at the genomic level. The genome size of P. yezoensis was estimated to be 260 Mbp, which is the lowest value on the genus Porphyra. Genome size of unicellular alga, Cyanidioschyzon merolae is smaller than that of multicellular alga, Porphyra. The size of the genome of P. yezoensis is between that of Arabidopsis thaliana or Chlamydomonas reinhardtii and Oryza sativa. This result indicates that P. yezoensis is an appropriate species for genome analysis and that it is comparable with other model organisms because of its compact-size genome.

Background: The aim was to investigate the efficacy of mechanical meibomian gland squeezing combined with eyelid scrubs and warm compresses in participants with moderate and severe meibomian gland dysfunction (MGD).Methods: In this prospective, uncontrolled, open label, intervention study, 32 eyes of 32 participants with moderate and severe MGD were treated with mechanical squeezing of meibomian glands in combination with eyelid scrubs and warm compresses. We evaluated tear film break-up time (TBUT), corneal and conjunctival fluorescein staining scores, biomicroscopic examination of lid margins and meibomian glands, Ocular Surface Disease Index (OSDI) questionnaire and tear film lipid layer thickness determined via an interferometer before initiating treatment and after one month of treatment.Results: There were significant improvements in TBUT, corneal and conjunctival fluorescein staining scores, lid margin abnormality, meibum quality and expressibility, OSDI and MGD stage after mechanical meibomian gland squeezing combined with eyelid scrubs and warm compresses (p < 0.001 for TBUT, corneal fluorescein staining scores, Dry Eye Workshop score, Oxford staining score, lid margin abnormality, meibum quality, expressibility, OSDI and MGD stage and p = 0.001 for conjunctival fluorescein staining scores). There were no significant differences in lipid layer thickness or interferometer-derived parameters before treatment and after one month of treatment. Palpebral conjunctival erosion detected after the squeezing treatment resolved spontaneously in all participants.Conclusions: Mechanical squeezing of meibomian glands combined with eyelid scrubs and warm compresses can provide clinical benefits without serious adverse events. [ABSTRACT FROM AUTHOR]

The specific and sensitive detection of peroxynitrite (ONOO – /ONOOH) in biological systems is a great challenge due to its high reactivity towards several biomolecules. Herein, we validated the advantages of using fluorescein-boronate (Fl-B) as a highly sensitive fluorescent probe for the direct detection of peroxynitrite under biologically-relevant conditions in two different cell models. The synthesis of Fl-B was achieved by a two-step conversion synthetic route with high purity (> 99 %) and overall yield (∼ 42%). Reactivity analysis of Fl-B with relevant biological oxidants were performed. The rate constant for the reaction of peroxynitrite with Fl-Bwas 1.7 x 10 6 M -1 s -1 , a million times faster than the rate constant measured for H 2 O 2 (k = 1.7 M -1 s -1 ) and 2,700 faster than HOCl (6.2 x 10 2 M -1 s -1 ) at 37° C and pH 7.4. The reaction of Fl-B with peroxynitrite was significant even in the presence of physiological concentrations of CO 2 , a well-known peroxynitrite reactant. Experimental and simulated kinetic analyses confirm that the main oxidation process of Fl-B takes place with peroxynitrite itself via a direct bimolecular reaction and not with peroxynitrite-derived radicals. Fl-B was successfully applied for the detection of endogenously- generated peroxynitrite by endothelial cells and in macrophage-phagocyted parasites. The generated data allowed estimating the actual intracellular flux of peroxynitrite, e.g., ionomycin-stimulated endothelial cells generated peroxynitrite at a rate of ∼ 0.1 μM s -1 , while immunostimulated macrophages do so in the order of ∼ 1 μM s -1 inside T. cruzi -infected phagosomes. Cellular peroxynitrite detection was achieved by conventional laboratory fluorescence-based methods including flow cytometry and epi-fluorescence microscopy. Fl-B was shown to be more sensitive than the coumarin boronate due to a higher molar absorption coefficient and quantum yield. Overall, our results show that Fl-B is a kinetically selective and highly sensitive probe for the direct detection of cell-derived peroxynitrite. Moreover, the data allowed to tailor boronate-derivatives targeted to identify subcellular sites and rates of peroxynitrite generation; indeed, ongoing work is focused on the generation and characterization of new organelle-targeted boronate-based probes. [ABSTRACT FROM AUTHOR]