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PubMed1985

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↗
PubMed1990

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↗
PubMed1989

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↗
PubMed1997

Measuring cholesterol in macrophages: comparison of high-performance liquid chromatography and gas-liquid chromatography with enzymatic fluorometry.

Cholesterol and cholesteryl esters in human macrophages were analyzed by three different methods. Values obtained by high-performance liquid chromatography and by gas-liquid chromatography were compared with those obtained using enzymatic fluorometry. We also assessed fractional lipid recovery from these cells using radiolabeled cholesterol and cholesteryl ester. Enzymatic fluorometry substantially underestimated cellular cholesterol content. Two reasons for this were found. First, recovery into a variety of solvents was incomplete, particularly when extracted lipids were dried and redissolved in a second solvent. Second, the cells appeared to contain an intrinsic inhibitor of the enzymatic fluorometric method.

Cells, Cultured↗
PubMed1998

Direct fluorometry of phase-extracted tryptamine-based fast quantitative assay of L-tryptophan decarboxylase from Catharanthus roseus leaf.

An assay for the enzyme L-tryptophan decarboxylase (TDC; EC 4.1.1.28) is described. It is based on direct fluorometry of the enzymatic reaction product (tryptamine) selectively recovered in ethyl acetate from the reaction mixture. Catalytically formed tryptamine from tryptophan in the incubation mixture is selectively (free from tryptophan) physically separated as ethyl acetate solution under basic (pH > or = 11) conditions and subjected to direct fluorescence measurement in the organic solvent using a spectrofluorometer with excitation and emission wavelengths of 280 and 350 nm, respectively. Tryptamine production rate was quantitated from the luminescence response curve of tryptamine drawn under similar extraction and measurement conditions. Luminescence calibration curves were drawn for tryptamine in aqueous (water or buffer system) as well as in organic solvent as recovered from the varied aqueous solution conditions including those similar to the enzyme incubation mixture. The luminescence calibration graphs were linear for at least 0.5 to 10 microM tryptamine. The examination of interassay variations and the comparative magnitude of fluorescence response allowed to infer that a satisfactory and sufficient sample luminescence response was retained under the varied conditions including those akin to the enzymatic assay mixture, allowing adaptation of the fluorometry for the TDC activity quantitation. The assay was found to follow the proportionality principle of product formation with respect to catalytic reaction time as well as protein concentration in the assay mixture using Catharanthus roseus leaf crude homogenate as well as the enzyme preparation at different states of purity. The rate of tryptamine formation under the catalytic conditions was linear for at least 1 h at 30 degrees C. Though the assay has been demonstrated to use the C. roseus leaf as the enzyme source, it should be equally applicable to other plant and nonplant sources. The merits and precautions of the protocol have been discussed.

Alkalies↗
PubMed2000

Background suppression in frequency-domain fluorometry.

Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry. The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and instrumental conditions, the data can be analyzed as usual for frequency-domain data without further processing. We show by simulations that this method can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence.

Fluorescence↗
PubMed1999

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry.

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.

Actins↗
PubMed1991

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↗
PubMed1991

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↗
PubMed2005

Estimation of chlorophyll content and daily primary production of the major algal groups by means of multiwavelength-excitation PAM chlorophyll fluorometry: performance and methodological limits.

The performance and methodological limits of the Phyto-PAM chlorophyll fluorometer were investigated with laboratory grown algae cultures and natural phytoplankton from the rivers Saar and Saale. The Phyto-PAM is a 4-wavelength chlorophyll fluorometer with the functional combination of chlorophyll (Chl) estimation and assessment of photosynthetic activity, both differentiated into the main algal groups. The reliability of fluorescence-based Chl estimation strongly depends on the group specific calibration of the instrument and the resulting chlorophyll/fluorescence (Chl/F) ratios in reference algal cultures. A very high reliability of the Chl estimation was obtained in the case of constant Chl/F-ratios. Algae grown at different light intensities showed marked differences in Chl/F-ratios, reflecting differences in pigment composition and Chl a specific absorption (a*). When the Phyto-PAM was calibrated with laboratory grown diatoms, the Chl a in river grown diatoms was underestimated, due a lower content of accessory pigments and stronger pigment packaging. While this aspect presently limits the application of PAM fluorometry in limnology, this limitation may be overcome by future technical progress in the detection of dynamic changes in Chl/F-ratio via fluorescence-based measurements of the functional PS II absorption cross-section. Practically identical Chl/F-ratios were found for the diatom-dominated waters of the rivers Saar and Saale, suggesting that the same instrument calibration parameters may be applied for hydrographically similar surface waters. For this particular case, despite of the present methodological limitations, the potential of PAM fluorometry in limnology could be demonstrated. Light response curves were measured to estimate primary production with a spectrally resolved model in daily courses at two sampling sites. Fluorescence based primary production was closely correlated with measured oxygen evolution rates until midday. In the afternoon, at the water surface the fluorescence approach gave higher rates than the measured oxygen evolution. Possible explanations for the observed differences are discussed.

Chlorophyll↗
PubMed1988

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↗
PubMed1998

Two-round enzymatic amplification combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity in DNA hybridization assays.

Microtiter well-based DNA hybridization assays are developed in which two rounds of enzymatic amplification are combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity. The target DNA is immobilized on the wells (through digoxigenin/anti-digoxigenin interaction) and then hybridized with a biotinylated oligonucleotide probe. The hybrids are reacted with a streptavidin-horseradish peroxidase conjugate. Peroxidase catalyzes the oxidation of biotinylated tyramine by hydrogen peroxide, resulting in the attachment of multiple biotin moieties to the solid phase. Alkaline phosphatase-labeled streptavidin is then allowed to bind to the immobilized biotins. The activity of alkaline phosphatase is measured by using the phosphate ester of 5'-fluorosalicylate as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb3+, which is measured by time-resolved fluorometry. We observed a 30-fold improvement of the signal and a 10-times enhancement of the signal-to-background ratio compared to the assay that uses a single round of enzymatic amplification (only alkaline phosphatase). The CV was in the range of 11.2-14.4%.

Chelating Agents↗
PubMedDate not supplied

Application of scanning fluorometry for monitoring of a fermentation process.

Conventional observation of a fermentation process by using NAD(P)H-dependent fluorescence provides a mean for cell monitoring, but its practical application has been very limited due to many factors that affect culture fluorescence. In this work we studied correlation of scanning fluorometry signals with various process variables and with cell metabolic states and showed that scanning fluorometry is more informative than conventional NAD(P)H-dependent fluorescence. A stepwise multiple-regression procedure has been used to select meaningful signals from the whole spectrum, and we found that with three or four components of the excitation-emission plane the process variables can be portrayed with a rather good accuracy. The approach is demonstrated on the examples of wild-type or recombinant Escherichia coli and Saccharomyces cerevisiae fermentations.

Anti-Bacterial Agents↗
PubMed2001

Interaction of beta-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: nonlinear quenching effects related to protein--protein association.

Although a thorough characterization of binding parameters is essential for application of beta-lactoglobulin as a carrier for a variety of small hydrophobic ligands, the binding parameters derived in various studies using various techniques are inconsistent. The bindings of several small ligands as detected by fluorometry and equilibrium dialysis were compared. Fluorescence spectroscopy showed that beta-ionone, retinol, and fatty acid lactones all bound in the vicinity of a tryptophan residue. Retinol and fatty acid lactone competed for the same binding site. Exclusively for ligands that quench the beta-lactoglobulin fluorescence through a resonance energy transfer mechanism, fluorometry yielded a systematically higher binding affinity than equilibrium dialysis. The binding overestimation in fluorometric measurements can be explained by oligomer formation of protein, together with an underestimation of the limiting quenching level at saturating ligand concentrations due to the use of a limited set of data points.

Dialysis↗
PubMed1991

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↗
PubMed1999

[Examining interaction of phages with microorganisms by fluorometry and electro-orientation spectroscopy].

Bacterial sensitivity to different various phages was examined by electro-orientation spectroscopy, fluorometry, and electron microscopy. The strains of Pseudomonas aeruginosa, Staphylococcus aureus, Yersinia pestis, Mycobacterium smegmatis, and Xanthomonas campestris were used. The fluorescence intensity of a membranotropic agent in the ANS-cell-phage system was shown to depend on the interaction of a bacterial virus and a microorganism. Fluorometric data correlated with electro-orientation spectroscopic findings. An analysis of the low-frequency site makes it possible to determine phage adsorption on the bacterial surface. The changes in electro-orientation effects at high frequencies suggest that there are barrier dysfunctions in the external membranes and that there is cellular phage reproductions. Whether fluorometry and electro-orientation spectroscopy can be further used for rapid identification of microorganisms by using phages is discussed.

Bacteriophages↗
PubMed1990

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↗
PubMed1989

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↗