Calibration of an in-water multi-excitation fluorometer for the measurement of phytoplankton chlorophyll-a fluorescence quantum yield.

A multi-excitation fluorometer (MFL, JFE Advantech Co., Ltd.), originally designed to discriminate between phytoplankton species present within a population, has been redirected for use in fluorescence quantum yield (FQY) determination. While this calibration for apparent FQY requires no modification of the MFL, it is necessary to have an independent measurement of the spectral absorption coefficient of the subject fluid. Two different approaches to calibration were implemented. The primary method made use of reference fluorescent dye solutions of known quantum yield. The second method made use of acrylic fluorescent plaques and films. The two methods yielded consistent results, except in the 570 and 590 nm LED channels of the MFL. Application of the MFL in FQY determination is illustrated with an in situ Southern Ocean sample.

Aqueous Flow Measured by Fluorophotometry in the Mouse.

PURPOSE: A fluorophotometer designed to measure aqueous flow in murine eyes was tested with artificial fluorescein chambers and in live mice with different anesthesia regimens, aqueous flow suppressants, and an anterior chamber cannulation method. METHODS: Two hours following topical fluorescein application, one group of CD-1 mice was anesthetized with ketamine/xylazine, 2,2,2-tribromoethanol, or ketamine alone. Cornea and anterior chamber fluorescein concentrations were measured periodically for 60 to 90 minutes by fluorophotometric scans to calculate aqueous flow. Later, a subgroup of mice underwent aqueous flow measurement by anterior chamber cannulation. A third group was treated with timolol, dorzolamide, and vehicle in a crossover manner 1 hour prior to fluorophotometric scans. RESULTS: Aqueous flow with ketamine/xylazine anesthesia (0.09 ± 0.05 µL/min, mean ± SD, n = 24) was slower than with tribromoethanol or ketamine alone (P < 0.001). Timolol reduced aqueous flow from 0.20 ± 0.07 µL/min to 0.07 ± 0.03 µL/min (P = 0.001) under tribromoethanol anesthesia and from 0.14 ± 0.03 µL/min to 0.10 ± 0.02 µL/min (P = 0.004) under ketamine anesthesia but not under ketamine/xylazine anesthesia. Dorzolamide reduced aqueous flow from 0.09 ± 0.03 to 0.06 ± 0.03 µL/min (P = 0.04) under ketamine/xylazine anesthesia. Aqueous flow by anterior chamber cannulation (0.20 ± 0.13 µL/min) was greater (P = 0.05) than by fluorophotometry (0.09 ± 0.07 µL/min). CONCLUSIONS: A new noninvasive fluorophotometric method detected effects of general anesthesia and known aqueous suppressants on aqueous flow in mice. Aqueous flow measured by fluorophotometry was slower than by cannulation, and was technically easier with less variability. The mouse fluorophotometer is useful for repeated measurements of aqueous flow in the murine eye making crossover and longitudinal studies possible.

High-throughput quality control of DMSO acoustic dispensing using photometric dye methods.

One high-throughput technology gaining widespread adoption in industry and academia is acoustic liquid dispensing, in which focused sound waves eject nanoliter-sized droplets from a solution into a recipient microplate. This technology allows for direct dispensing of small-molecule compounds or reagents dissolved in DMSO, while keeping a low final concentration of organic solvent in an assay. However, acoustic dispensing presents unique quality control (QC) challenges when measuring the accuracy and precision of small dispense volumes ranging from 2.5 to 100 nL. As part of an effort to develop a rapid and cost-effective QC method for acoustic dispensing of 100% DMSO, we implemented the first high-throughput photometric dual-dye-based QC protocol in the nanoliter volume range. This technical note validates the new photometric 100% DMSO QC method and highlights its cost-effectiveness when compared with conventional low-throughput fluorimetric QC methods. In addition, a potential software solution is described for the analysis, storage, and display of accumulated high-throughput QC data, called LabGauge. As the need for high-throughput QC grows, conventional low-throughput methods can no longer meet demand. Validated high-throughput techniques, such as the dual-dye photometric method, will need to be implemented.

[Microemulsion sensitized fluorophotometric determination of protein with probe of phenylfluorone-molybdenum (VI) complex].

The fluorescence spectral behavior of interaction of phenylfluorone(PF)-Mo(VI) and protein was investigated in Triton X-100 microemulsion medium at pH 2.0. A novel method for the determination of protein using phenylfluorone (PF)-Mo(VI) as a fluorescence spectrum probe was developed. Excitation and emission wavelengths are 465 and 525 nm, respectively. The effective factors and the optimum conditions have been studied, and the reducing value of fluorescence intensity is in proportion to the concentration of proteins in the range 0-6.00 microg x L(-1) for bovine serum albumin, 0-4.00 microg x L(-1) for human serum albumin, 0-5.00 microg x L(-1) for ovalbumin, and 0-4.00 microg x L(-1) for lysozyme. The Triton X-100 microemulsion was efficiently used to enhance the sensibility and stability of the system, and the limits of detection are 5.4, 5.2, 1.5 and 8.2 ng x L(-1), respectively. Most of foreign substances do not interfere with the determination, and this method has good selectivity and high sensitivity. It has been applied to the determination of proteins in the urine samples with satisfactory results.

Measuring oxygen tension in the anterior chamber of rabbits.

PURPOSE: Measuring the concentration of oxygen in the aqueous humor without penetrating the eye would provide a new dimension in understanding aqueous humor and corneal dynamics. In this study a preinvasive method was developed for determining the cameral oxygen concentration in anesthetized rabbits by measuring the excited-state lifetime of a phosphorescent dye. METHODS: A scanning ocular fluorometer was designed to excite phosphorescence with a brief flash of light and to measure the decay of luminescence for as long as 1000 microsec after excitation. The measurement window was scanned through the depth of the anterior chamber or fixed at the mid-anterior chamber. A depot of the phosphorescent dye Pd-uroporphyrin was injected into the vitreous of eight pigmented rabbits, and within a few days the dye was measurable in the anterior chamber. The excited-state lifetime of this dye is inversely correlated to oxygen concentration and was calibrated by measuring the lifetime of dye in cuvettes equilibrated with oxygen-nitrogen mixtures. Oxygen tensions were determined from lifetimes measured in the open eye, under a polymethylmethacrylate (PMMA) contact lens, under two oxygen-permeable contact lenses, and immediately after lid closure. RESULTS: Oxygen tension in the mid-anterior chamber before placing a PMMA contact lens was 23 +/- 3 mm Hg (mean +/- SD; n = 6). After 20 minutes of PMMA lens wear, oxygen tension decreased to 4 +/- 2 mm Hg. When the focal diamond was scanned through the anterior chamber, oxygen tension was 24 +/- 5 mm Hg near the corneal endothelium and decreased to 17 +/- 8 mm Hg near the crystalline lens. Under the PMMA contact lens this gradient reversed: Oxygen tensions near the endothelium and lens were 3 +/- 2 mm Hg and 6 +/- 2 mm Hg, respectively. Lid closure for 10 minutes or longer decreased the mid-anterior chamber oxygen tension from 21 +/- 2 mm Hg (n = 19 measurements from seven animals) to 10 +/- 3 mm Hg (n = 15 measurements from five animals). CONCLUSIONS: Measuring excited-state lifetime of phosphorescent dyes in the anterior chamber provides a useful method for determining oxygen concentration in vivo, without penetrating the eye. Cameral oxygen tension under PMMA contact lenses are significantly lower than in the uncovered eye. The profile of oxygen tension through the anterior chamber suggests that oxygen is supplied transcorneally to the aqueous humor.

Aqueous flare in patients with monocular iris atrophy and uveitis. A laser flare and iris angiography study.

PURPOSE: To study aqueous flare longitudinally in patients with Fuchs' heterochromic uveitis (FHU) and for comparison in another group with iris atrophy (non FHU). METHODS: A new laser flare meter was used. Iris angiography was performed in most FHU patients. RESULTS: The flare values in the affected eyes in the FHU group were always higher compared to the normal eyes and rather constant over time. Cataract surgery did not permanently change the flare values. Systemic - but not local steroid therapy caused normalization of the flare. Iris angiography displayed leakage in all FHU eyes. Leakage from original vessels dominated although some newly-formed leaking vessels were observed. CONCLUSIONS: The results support the notion that FHU is a low grade, chronic, stable disease. Leaking iris vessels, original or newly-formed, are probably the source of the increased flare. Our hypothesis is that inflammation plays a major part. Atrophy per se does not enhance flare.

Fluorophotometry with a small animal adapter.

The usefulness of the Fluorotron Master fluorophotometer fitted with a small animal adapter was evaluated with tree shrews. We also determined that the optimal measurement parameters for these animals, which are regarded as promising for experimental use in ophthalmic research, are: fluorescein-Na concentration, 0.5 x 10(-9) to 1.0 x 10(-6) g/mL; fluorescein-Na dosage, 2 mg/kg; measurement time, 30 minutes after injection of fluorescein-Na into the ocular compartments. Results indicated that ultrafiltration for the measurement of protein-unbound fluorescein can be done with a hematocrit tube and a minimal blood sample, thereby reducing the impact of the sampling on the animal's general condition. The Fluorotron Master with the small animal adapter can offer advantages in estimating the blood-ocular barrier permeability using disease models in tree shrews.

Evaluation of albumin concentration in rabbit anterior chamber with laser flare-cell meter.

To find an appropriate way of using Kowa's laser flare-cell meter in evaluating the anterior chamber albumin concentration, we characterized the relationship among the flare intensity determined with the flare-cell meter in terms of photon count/ms (flare value), flare value converted to albumin concentration, and anterior chamber albumin concentration in drug-treated rabbit eyes. Flare measurement was done in rabbits that received intravenous administration of fluorescein isothiocyanate-labeled albumin and instillation of prostaglandin E1, pilocarpine, or tropicamide. Flare value, anterior chamber fluorescence, plasma fluorescence, and plasma albumin concentration were determined. Anterior chamber albumin concentration was calculated from the plasma albumin concentration and the corrected aqueous/plasma fluorescence ratio. The correlation between the flare value converted to albumin concentration (Albequiv) and the anterior chamber albumin concentration (Albac) had two phases: 1) Linear correlation was found when the flare value was below 100 photon count/ms: Albac = 0.53 Albequiv - 0.006, r = 0.98; 2) When the flare value was above 100 photon count/ms, the correlation was lower and had a different slope. In this condition, the Albac was correlated better with the flare value itself: Albac = 0.0042 [flare value] + 0.17, r = 0.88. The results suggested that anterior chamber albumin concentration is evaluated quantitatively from the converted flare value when the flare value is below 100 photon count/ms and from the flare value itself when the flare value is above that level.

Ocular fluorometry: standardization and instrumentation development. A Concerted Action in the European Community (EUROEYE).

A Concerted Action on Ocular Fluorometry, stressing standardization and instrumentation development has been funded by the European Community. Agreement was reached on harmonization of protocols. The results obtained show that the protocols proposed for Clinical Ocular Fluorometry were generally appropriate and may be followed closely, with reproducible and meaningful results. In each group, areas for improvement could, however, be detected, particularly regarding facility of use of the newly developed softwares. The success of the ECNOF was very rewarding and every effort is being made to consolidate this success in the publication and dissemination of the agreed guidelines and results. The field of Ocular Fluorometry appears to have even more potential than was apparent at the beginning of this Concerted Action. The needs for instrumentation development have been clarified and four main directions where progress has been achieved are identifiable: spectral fluorescence analysis of naturally occurring ocular fluorophores, light scattering analysis of ocular structures and fluids, improved axial resolution for better quantification of ocular permeabilities and, finally, development of simple routine clinical instrumentation. Corneal and lens natural fluorescence appear extremely promising as indicators of disease status, particularly in diabetes. Blood-retinal barrier permeability has the potential to become a screening test isolating the eyes at risk for developing diabetic blindness and, therefore needing closer follow-up and earlier treatment. Light scattering methodologies particularly in association with ocular fluorometry, may allow improved monitorization of chronic inflammation, better therapeutical management of a variety a sight-threatening diseases.

[Future developments in ocular fluorophotometry instrumentation]. / Futuros desenvolvimentos na instrumentação em fluorofotometria ocular.

The scope of ocular fluorometry is to monitor exogenous and endogenous fluorophores in ocular tissues, in relation with ophthalmic and systemic diseases using the unique optical prospectives of the eye. The elderly population and the incidence of blindness are increasing rapidly due to more cases of diabetes, glaucoma, cataract and age-related macular degeneration. Monitoring changes in specific fluorophores in the eye may help identify the high risk groups in these diseases. New developments in instrumentation include differential fluorometry and introduction of confocal optics. Differential fluorometry has already achieved significant progress for the study of the autofluorescence of the lens and cornea and measurements in the aqueous. Improved spatial resolution obtained with improved optics opens interesting possibilities like measurement of corneal endothelial permeability and retinal vascular permeability. The results already obtained will be presented with particular incidence on measurements of lens fluorescence (normals--336.2 +/- 56.3; diabetes--659.9 +/- 123.9; age group--40-50 y) and corneal endothelial permeability (normals--3.14 +/- 60.10(-1) cm-1).

[Functional evaluation of the corneal endothelium using anterior segment fluorophotometry]. / Evaluation fonctionnelle de l'endothélium cornéen par la fluorophotométrie du segment antérieur.

The AA have studied the permeability of the corneal endothelium by anterior segment fluorophotometry using instillation of fluorescein. The Fluorotron Master TM has been modified with the introduction of a new slit (58.4 microns) and of the voltage output (6.5 v) has also been increased. The resolution has been improved from 2.39 mm to 0.59 mm. Of this study, we have examined 20 patients submitted to extracapsular cataract extraction with intraocular lens implantation (ECCE + IOL). The patients have been divided in two groups, according to the surgical technic used: "can-open" and intracapsular. Each patient have been submitted before surgery and 1 week after to an ophthalmologic examination, paquimetry and anterior segment fluorophotometry. The permeability coefficient (PAC) increases with the duration of surgery and when using "can-opener" instead of endocapsular.

Ocular fluorometry: principles, fluorophores, instrumentation, and clinical applications.

Ocular fluorometry is rapidly evolving as a versatile technique for research and diagnosis in ophthalmology. The main reasons for this increasing success are 1) the ideal characteristics of the eye as an optical device for excitation of tissue fluorescence and for the detection of the fluorescent emission; 2) the development of novel fluorometric techniques, including differential and time-resolved fluorescence spectroscopy; and 3) the increasing use of coupling geometries with high-resolution and high spatial selectivity. Both endogenous and exogenous fluorophores are of interest to ocular fluorometry. The most significant among endogenous fluorophores are the fluorescing pigments of the lens and of the retinal pigment epithelium (RPE). The nature, topography, and fluorescence properties of such pigments depend on age and pathology and on the level of light exposure. Exogenous fluorophores of interest are both intentionally induced and unintentionally accumulated drugs (some of which are phototoxic). Laser-based fluorometric techniques play a leading role in ocular fluorometry. The peculiar properties of the laser for the excitation of fluorescence make this source a favorite candidate for ocular fluorometry both in vitro and in vivo.