[Elements of functional organic fluorometry. VII. Fluorometry of pyridine derivatives].
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Fluorometry↗
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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↗
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↗
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↗
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↗
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↗
The clinical use of laser-excited fluorometry.
Many areas of spectroscopy have benefited from the use of laser radiation sources. I present a discussion of the basic properties of the laser and how these properties can be advantageous when laser excitation is used in fluorometry. Although the laser has not yet been accepted as a routine instrument in the clinical laboratory, its unique properties have rendered it useful in several analytical methodologies that are based on fluorometry and used in the clinical or biological fields. Accordingly, I briefly review the practical aspects of some clinical applications of laser-excited fluorometry.
Biological Transport↗
Objective measurement of limb perfusion by dermal fluorometry. A criterion for healing of below-knee amputation.
Quantitative fluorometry has been recommended as an accurate adjunct to clinical judgment in the preoperative assessment of lower-extremity amputation level. In this prospective study of 56 patients who had below-knee amputation, clinical judgment was used as the sole criterion for site selection. Quantitative fluorometry was compared with clinical judgment in a prospective, blinded study. All patients were studied before amputation with administration of intravenous fluorescein. Fifteen minutes after injection, objective measurement of dye fluorescence was performed at multiple sites with a quantitative fluorometer, and a dye fluorescence index was derived. All limbs undergoing amputation were ischemic, manifested by rest pain, nonhealing ulcers, or gangrene. Five patients (8.7%) failed to heal at the below-knee level. The mean dye fluorescence index for the group that healed was 81 +/- 51 (range, 13 to 259) and for the group that failed to heal, 110 +/- 49 (range, 70 to 195). Objective measurement of fluorescein perfusion did not correlate with amputation healing at the below-knee level in our patient population.
Adult↗
Laser-Doppler versus fluorometry in the postoperative assessment of a cutaneous free flap.
To find the optimal means for monitoring the vascularity of a cutaneous free flap in the postoperative period, we have experimentally compared laser-Doppler velocimetry and fluorometry. Using the rat groin model, five groups were evaluated: 1. flap isolation without division of the pedicle vessels (island flap); 2) flap isolation, division, and repair of the pedicle artery and vein (free flap); 3) flap isolation, with ligation of the pedicle artery immediately or 1 hour later; 4) flap isolation, with ligation of the pedicle vein immediately or 1 hour later; 5) flap isolation, with ligation of the pedicle artery and vein immediately or 1 hour later. The laser-Doppler processes the signal by combination of the root mean square and differential amplification. The fluoroscan gives an index in relation to the fluorescence of a control area. The results obtained with both methods correlated well with findings in clinical situations. However, the laser-Doppler readings were more rapid and sensitive than those with fluorometry. We suggest that laser-Doppler velocimetry is a superior means of monitoring the vascular status of a free tissue transfer or digital replant.
Animals↗
Determination of spatial coordinates in ocular fluorometry.
A method has been developed for the determination of spatial coordinates of ocular fluorescence measurements made by a non-contact-lens type of fluorometer (Fluorotron, Coherent Inc., Palo Alto, Calif., USA). The method is based upon a mathematical model of the instrument and the eye. The model is adapted to the individual eye by the extraction of information from the fluorescence scan and the use of keratometry to determine the radius of curvature of the anterior corneal surface. The validity of the model was examined by comparison of the ocular axial length as measured by fluorometry vs ultrasonometry in 26 eyes of healthy human subjects. The results of fluorometry differed from those of ultrasonometry by less than +/- 2%.
Adult↗
Lens fluorometry: light-attenuation effects and estimation of total lens transmittance.
Front-face fluorometry of the axial fluorescence profile of the lens is influenced by attenuation of light along the optical pathways leading to and from the detection volume. The resulting distortion is evident when the curve is compared with the intrinsic profile that can be recorded by direct measurement on the transected lens. Assuming that attenuation of light by scatter and absorption is proportional to fluorescence, the intrinsic profile can be reconstructed from the non-invasive fluorescence profile. The calculations involve the deduction of total lens transmittance and fluorescence-related absorptivity, parameters that provide essential information about the optical quality of the lens. The method was applied to human lens fluorometry in vivo and in vitro (excitation 430-490 nm, fluorescence 530-630 nm). Even the most anterior lens fluorescence measurements were found to be markedly affected by attenuation of light in the lens. Lens transmittance estimates agreed within +/- 12% with direct measurements of lens transmittance. The differences were due in part to inter-individual variations in the shape of the intrinsic fluorescence profile of the lens.
Absorption↗
The use of quantitative perfusion fluorometry to measure relative tumor and liver blood flow after transient microembolization.
Optimal chemotherapy delivery to the tumor depends on regional drug concentration, tumor perfusion, tissue drug uptake, and metabolism. Modulation of tumor blood flow has been used to improve tumor response to treatment. Transient microembolization is one method to alter regional blood flow, but its effects on relative changes in tumor and liver blood flow have not been previously measured. This study used quantitative perfusion fluorometry (QPF) to evaluate blood flow distribution in liver and tumor before and after hepatic arterial infusion of degradable starch microspheres (DSMs) in 10 New Zealand white rabbits. QPF was compared with radioactive xenon-133 washout, an established method for measuring blood flow. Xenon-133 was injected intraparenchymally and the clearance rate was measured allowing calculation of relative blood flow. QPF was then used to measure liver and tumor blood flow in a hepatic VX-2 tumor model after hepatic artery injection of DSMs. Initial tumor blood flow was 55% of liver flow. DSMs produced a significant and transient decrease in hepatic blood flow that was decreased to 40% of baseline after 25 min. Changes in relative hepatic blood flow after DSMs as measured by QPF correlated strongly with results obtained by xenon-133 washout (R = 0.97, P less than 0.01). Fluorometry's simplicity and reliability may be clinically useful to evaluate tumor blood flow characteristics.
Animals↗
Automation of the resazurin reduction test using fluorometry of microtitration trays.
Microtitration plate technique and -fluorometry was applied to automate the resazurin reduction test for monitoring bacterial numbers in broth cultures and milk. The effect of resazurin and resorufin concentration, bacterial species, growth medium, pH, and redox potential on the fluorescence response was studied. The timing of the appearance of maximum fluorescence was directly related to the logarithm of the number of colony-forming units (log CFU). Fresh milk and heat-treated milk contain interfering redox systems. The technique based on microtitration plate fluorometry, when fully automated, seems to provide a high-capacity system for analyzing bacterial numbers in foodstuffs and other media.
Animals↗
Estimation of 5-aminosalicylic acid and its metabolite in human serum by front-face fluorometry: a simple and sensitive method.
Salicylazosulfapyridine (SASP), commonly used in the treatment of inflammatory bowel disease, breaks down in the colon into sulfapyridine and 5-aminosalicylic acid (5-ASA), the active moiety of SASP. We report a sensitive method to measure 5-ASA and its known major metabolite acetyl 5-ASA (Ac-5-ASA) directly from the serum without any extraction procedure. Using front-face fluorometry, 5-ASA and Ac-5-ASA were detected at the excitation wavelength of 310 nm with emission maxima at 475 nm and 440 nm, respectively. Standard curves were obtained by adding known amounts of 5-ASA and Ac-5-ASA to several individual and pooled human sera. Presence of sulfapyridine (0 to 20 micrograms/ml) and SASP (0 to 15 micrograms/ml) in the serum did not interfere with the assays. Five microliters of acetic anhydride was added to the serum to convert all 5-ASA to Ac-5-ASA. The difference in the spectrum before and after addition of acetic anhydride represented the concentration of free 5-ASA. The values thus estimated were within 1% of the expected readings from the standard curves. This assay was compared with the organic extraction method for the determination of free and acetylated 5-ASA in sera of patients given olsalazine (azodisalicylate). The results demonstrate that direct analysis of the sera by front-face fluorometry enables us to measure 5-ASA and Ac-5-ASA at levels as low as 0.1 micrograms/ml in serum, making this method at least 10-fold more sensitive than the current available extraction methods.
Aminosalicylic Acids↗
Correlation of redox fluorometry and analytical measurements of pyridine nucleotide.
Pyridine nucleotide levels in the corneal epithelium were measured using redox fluorometry, a noninvasive method for monitoring the metabolic status of corneal tissue, and a sensitive bioluminescent assay and an improved extraction procedure that allows the simultaneous extraction and measurement of both NADH and NAD. The same corneas were measured using each of the two methods to enable comparison of the results. The NADH/(NADH + NAD) fraction in the normal epithelium measured by the bioluminescent assay was 0.14 +/- 0.06. Incubation of corneas in 1 mM potassium cyanide (KCN) to mimic the anoxic state increased the NADH/(NADH + NAD) fraction significantly to 0.24 +/- 0.03 (P less than 0.001). The autofluorescence from reduced pyridine nucleotide measured by redox fluorometry also increased with KCN from 2840 +/- 605 to 5147 +/- 738 (P less than 0.0001). A plot of the fluorescence and analytical data for each cornea showed a positive correlation between the two methods, with a correlation coefficient (r value) of 0.80. The correlation was improved but was not dependent on the high values of the KCN treated corneas; an r value of 0.73 was obtained for the non-KCN treated corneas alone. Additional measurements of the temperature dependence of the fluorescence intensity of an NADH solution and the cornea gave a decrease in intensity of 17% from 25 degrees C to 35 degrees C for the NADH solution and 11% (P = 0.0004) for the reduced pyridine nucleotide fluorescence in the cornea over the same temperature range.
Animals↗