[Elements of functional organic fluorometry. VII. Fluorometry of pyridine derivatives].
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Fluorometry↗
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Results for “FLUOROMETRY”
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Serum quinidine concentrations as measured by direct fluorometry, double-extraction fluorometry, and enzyme immunoassay.
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Humans↗
Non-invasive assessment of the donor corneal endothelium using ocular redox fluorometry.
AIMS: To investigate the usefulness of ocular redox fluorometry for evaluating donor corneal endothelial viability. METHODS: Corneas from 42 recipients of penetrating keratoplasty and four donor corneas were examined by ocular redox fluorometry. Autofluorescence from reduced pyridine nucleotides (PN) and oxidised flavoproteins (Fp) of the human corneal endothelium were measured non-invasively, and the PN/Fp ratio was used as a tissue metabolic indicator. Specular microscopy and electron microscopy were also performed. RESULTS: Both the quality of specular microscopic image and the PN/Fp ratio were significantly correlated with the degree of corneal endothelial damage determined by histological examination. Corneas with poor specular microscopic image showed significantly decreased PN/Fp ratio compared with corneas with good or fair specular images (p = 0.041 and 0.027, respectively). The PN/Fp ratio increased in corneas with mildly damaged endothelium but decreased in corneas with severely damaged endothelium determined by histological examination. Evaluation of corneal endothelium by combination of specular microscopy and ocular redox fluorometry showed excellent association with that of histopathological examination (p < 0.0001). CONCLUSION: Ocular redox fluorometry is useful for assessing donor corneal endothelial viability. Combination of ocular redox fluorometry and specular microscopy may increase the ability of donor cornea selection.
Adult↗
Doppler ultrasound, laser Doppler, and perfusion fluorometry in bowel ischemia.
Improved accuracy and objectivity in the evaluation of intestinal viability has been reported by some investigators using Doppler ultrasound, and more recently laser Doppler velocimetry and perfusion fluorometry. To compare the sensitivity and clinical applicability of these techniques, intestinal viability was evaluated by each method in nine 15- to 50-cm loops of small bowel prepared by division of the mesenteric vasculature in five anesthetized dogs. The sensitivity of Doppler ultrasound was 86%, of laser Doppler flow velocity 85%, of laser Doppler index 94%, and of perfusion fluorometry 95%. Though the sensitivity of Doppler ultrasound is significantly less than that of laser Doppler and perfusion fluorometry, this is not unexpected since the latter two techniques are more quantitative than Doppler ultrasound. Clinically, Doppler ultrasound compares favorably with laser Doppler and perfusion fluorometry, and its low cost and simplicity suggest its adjunctive use in the operative setting.
Animals↗
Improved salvage of complicated microvascular transplants monitored with quantitative fluorometry.
Quantitative fluorometry has been used to monitor circulation in transplanted toes and cutaneous flaps in our unit since 1982. Analysis of 177 uncomplicated transplants monitored by quantitative fluorometry shows that this technique has low false indication rates for arterial occlusion (0.6 percent of patients) and venous occlusion (6.2 percent of patients). None of these patients was reexplored because of a false monitor reading, and except for single abnormal sequences, monitoring appropriately indicated intact circulation throughout the postoperative period. Quantitative fluorometry has correctly indicated vascular complications in 21 (91.3 percent) of 23 transplants over an 8-year period. The salvage rate (85.7 percent) of the fluorescein-monitored reexplored transplants was significantly higher than the salvage rates of similar reexplored transplants not monitored with fluorescein and of reexplored muscle flaps (which cannot be monitored with the fluorometer used at this unit). These clinical data indicate that quantitative fluorometry is a valid and useful postoperative monitor for transplanted toes and cutaneous flaps.
Fluorometry↗
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.
Eye Diseases↗
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.
Anterior Eye Segment↗
Detection of hindered rotations of 1,6-diphenyl-1,3,5-hexatriene in lipid bilayers by differential polarized phase fluorometry.
Differential polarized phase fluorometry has been used to investigate the depolarizing motions of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the isotropic solvent propylene glycol and in lipid bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC), dipalmitoyl-L-alpha-phosphatidylcholine (DPPC), and other phosphatidylcholines. Differential phase fluorometry is the measurement of differences in the phase angles between the parallel and perpendicular components of the fluorescence emission of a sample excited with sinusoidally modulated light. The maximum value of the tangent of the phase angle (tan Delta(max)) is known to be a function of the isotropy of the depolarizing motions. For DPH in propylene glycol the maximum tangent is observed at 18 degrees C, and this tangent value corresponds precisely with the value expected for an isotropic rotator. Additionally, the rotational rates determined by steady-state polarization measurements are in precise agreement with the differential phase measurements. These results indicate that differential phase fluorometry provides a reliable measure of the probe's rotational rate under conditions where these rotations are isotropic and unhindered.Rotational rates of DPH obtained from steady-state polarization and differential phase measurements do not agree when this probe is placed in lipid bilayers. The temperature profile of the tan Delta measurements of DPH in DMPC and DPPC bilayers is characterized by a rapid increase of tan Delta at the transition temperature (T(c)), followed by a gradual decline in tan Delta at temperatures above T(c). The observed tanDelta(max) values are only 62 and 43% of the theoretical maximum. This defect in tanDelta(max) is too large to be explained by any degree of rotational anisotropy. However, these defects are explicable by a new theory that describes the tan Delta values under conditions where the probe's rotational motions are restricted to a limiting anisotropy value, r(infinity). Theoretical calculations using this new theory indicate that the temperature dependence of the depolarizing motions of DPH in these saturated bilayers could be explained by a rapid increase in its rotational rate (R) at the transition temperature, coupled with a simultaneous decrease in r(infinity) at this same temperature. The sensitivity of the tan Delta values to both R and r(infinity) indicates that differential phase fluorometry will provide a method to describe more completely the depolarizing motion of probes in lipid bilayers.
Membranes, Artificial↗
Time-resolved fluorometry in nucleic acid hybridization and western blotting techniques.
Time-resolved fluorometry is now used extensively for immunological assays and to a lesser extent in other research areas. In this review I describe applications of time-resolved fluorometry in nucleic acid hybridization and in blotting techniques, including Southern and Western blotting. Clearly, time-resolved fluorometry has potential for playing a major role in techniques other than immunological assays.
Bacterial Proteins↗
Quantitative fluorometry as a monitor for microvascular surgery.
There is a clear need for effective methods of monitoring for postoperative occlusion of vessels in microvascular surgery. We have evaluated one technique, quantitative fluorometry, in the laboratory and clinically. Our laboratory study used rat abdominal flaps under conditions of controlled occlusion. We found accurate detection of vascular occlusion within 20 minutes, but we were unable to differentiate arterial from venous occlusion. Our clinical review of 34 microvascular cases (14 free flaps and 20 replantations) that employed fluorometry revealed corroboration of occlusion (indicated by another monitoring technique) in six cases and a diagnosis at variance with other monitoring methods in one case, thus preventing an operative exploration. We recommend the use of quantitative fluorometry as a primary or adjunctive method of monitoring when patency is in question and have outlined a protocol for clinical use.
Animals↗
Laparoscopic fluorometry: a new minimally invasive tool for investigation of the intestinal microcirculation.
The aim of this study was to develop and establish a new system of laparoscopic fluorometry for the purpose of investigating the intestinal microcirculation. In 25 pigs (German Landrace, 16-25 kg body weight), ischemia was established in two segments (A, irreversible; B, reversible ischemia; C, internal control) of the small intestine by a laparoscopic technique. Microcirculation in the segments was assessed by laparoscopy at a second-look operation 24 h later by means of the fluorescence system Endoscan. The fluorescence of the three bowel segments was measured by arbitrary dye fluorescence units (DFU) 15 min after starting reperfusion, before and after injection of sodium fluorescein (NaFlu, 0.25 mg/kg body weight). The dividing line between viable and nonviable bowel tissue was established from the inflow and outflow rates of NaFlu with the aid of ROC (receiver operating characteristic) curves. The specificity and sensitivity of the new method were evaluated by correlating the results with the viability of each intestinal segment as predicted by three laparoscopically experienced surgeons and by histological examination. By means of the calculated separation sharpness (fluorescence index at 2 min >0.5, outflow factor of NaFlu at 10 min >20%), the overall predictions of intestinal viability in all 25 animals achieved a sensitivity of 93.5% and a specificity of 94.1% by laparoscopic fluorometry, versus a sensitivity of 70.8% and a specificity of 87.5% for the prediction of bowel viability by ordinary laparoscopic technique. Used as an adjunct to conventional laparoscopy, laparoscopic fluorometry brought significant gains in sensitivity and specificity in the distinction between reversible and irreversible intestinal ischemia.
Animals↗
Effect of hemolysis on serum retinol as assessed by direct fluorometry.
To assess the effect of hemolysis on serum retinol concentrations determined by direct fluorometry, we assayed 196 blood samples from children 6-72-mo of age with various grades of hemolysis for serum retinol by both fluorescence and HPLC. Mean serum retinol concentrations determined by HPLC did not differ significantly according to hemolysis grade; however, fluorometric values did. Additionally, serum retinol concentrations obtained from HPLC and those obtained from direct fluorometry were significantly different in samples with severe hemolysis. Multivariate-regression analysis showed that hemolysis grade was a significant predictor of the difference in mean serum retinol values determined by the two methods. Although severe hemolysis interfered with determinations of serum retinol by direct fluorometry, this method is still a viable choice for field studies of vitamin A status.
Child↗
Burn depth evaluation with fluorometry: is it really definitive?
Clinical evaluation of burn depth soon after injury is subjective, based on gross visual assessment. Previous investigators have quantified this process using fluorometry. Their studies show fluorescein levels in full-thickness burns to be far below control levels and partial-thickness burns to be about 60% of nonburned skin. In both rat and human models, 59 burn sites (eight rats) and 37 burn sites (seven patients) were assessed. Readings were taken for three hours on the rats and one hour on the patients during the first 48 hours, and the procedure was repeated for five days postburn. Maximum values during these periods were determined for burn and nonburn sites, and background levels were subtracted from these values. The rate of fluorescein uptake and the peak times for burn and nonburn sites were then compared. Actual depth of burn was determined by whether or not healing had occurred. The results showed no significant difference between partial-thickness and full-thickness burns using fluorometry, as standard deviations in both models for both depths of burn were large. Therefore, fluorometry did not provide a definitive evaluation of burn depth. These results differ from those reported by previous investigators.
Animals↗
Vascular island skin-flap tolerance to warm ischemia: an analysis by perfusion fluorometry.
Fiberoptic perfusion fluorometry and assessment of ultimate viability were used to analyze the tolerance to warm ischemia of rat vascular island skin flaps. Both acute flaps and flaps raised 24 hours previously and then reraised were subjected to 0 (control), 6, 8, 10, and 12 hours of vascular pedicle clamping. Following clamp release, serial fluorometry documented the progressive delay in effective reflow resulting from extended periods of ischemia. Fluorometry, furthermore , suggested that flaps constructed 24 hours previously had an improved hemodynamic status with a significantly reduced period of poor reflow following clamp release. The improved hemodynamics were associated with increased viability, confirming the increased tolerance of 24-hour-old flaps to warm ischemia.
Animals↗
Detection of adenovirus in clinical specimens by polymerase chain reaction and liquid-phase hybridization quantitated by time-resolved fluorometry.
In addition to tests for the group-specific hexon antigen of adenoviruses, adenoviruses can be detected in clinical specimens by hybridization assays utilizing the widely shared base sequences of the region of the hexon gene that codes for the group-reactive determinants. We have developed a liquid-phase hybridization system with biotin- and europium-labeled probes which are reacted after DNA amplification of a 161-bp region of the hexon gene and which are quantitated by time-resolved (TR) fluorometry in streptavidin-coated microtiter wells. Polymerase chain reaction (PCR)-TR fluorometry is not a rapid test in the usual sense, but it is highly useful for specimens with inherent toxicity or with low virus yield, such as organ minces and specimens obtained late in the course of an illness. In a survey of 103 specimens tested by this method, including urine, stool, and tissue suspensions, the agreement with the hexon-specific TR fluoroimmunoassay antigen test for positive specimens was 100% and the sensitivity compared with that of virus culture was 91%. The PCR-TR fluorometry system was also shown to be advantageous as a quantitative measure of PCR products.
Adenoviruses, Human↗
Noninvasive redox fluorometry: how light can be used to monitor alterations of corneal mitochondrial function.
Ultraviolet light can result in corneal, lenticular and retinal damage; however it can also be used (at much lower intensities) to measure the light induced alteration of cellular respiration and function. Mitochondrial function can be measured by noninvasive redox fluorometry which measures the intrinsic mitochondrial fluorescence of the reduced pyridine nucleotides (NADH + NADPH) and of the oxidized flavoproteins. Impaired mitochondrial respiration results in an increase in the reduced pyridine nucleotide fluorescence signal (366 nm excitation and 450 nm emission) and in a decrease in the oxidized flavoprotein fluorescence signal (450 nm excitation and 550 nm emission). These redox signals are sensitive to the cellular supply and utilization of oxygen and glucose as well as the mitochondrial work load. The effects of a reduced oxygen supply to the corneal epithelial surface can be measured. While redox fluorometry has been applied to the study of corneal hypoxia, it may also be used to monitor the effects of light induced damage to the lens and the retina. Noninvasive redox fluorometry is a sensitive technique to measure the effects of light on mitochondrial function in ocular tissue.
Animals↗
Fluorometry of poultry semen: its application in the determination of viability, enzyme leakage, and fertility.
The accuracy of fluorometry for estimating percentages of dead chicken spermatozoa was investigated by comparing this technique with the eosin-nigrosin differential staining procedure and with glutamic oxaloacetic transaminase (GOT) concentration in seminal plasma. The relationship between percent dead sperm measured by fluorometry and fertility was also examined. The correlation coefficient of percentage of dead spermatozoa determined by fluorometry with eosin-nigrosin counts was highly significant (r = .99; P less than .001). Similarly, the correlation coefficient of GOT activity with percentage of dead spermatozoa was .99 (P less than .001). Percent fertility, fertile egg production, and duration of fertility were negatively correlated with percent dead spermatozoa; 4 = -.55, -.51, and -.44 (P less than .001), respectively.
Animals↗
Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).
The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.
Animals↗