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At least 19 recordsLinked to original sources

PubMed1985

[Hydrophobic acridine dyes for fluorescent staining of mitochondria in living cells. 3. Specific accumulation of the fluorescent dye NAO on the mitochondrial membranes in HeLa cells by hydrophobic interaction. Depression of respiratory activity, changes in the ultrastructure of mitochondria due to NAO. Increase of fluorescence in vital stained mitochondria in situ by irradiation].

The hydrophobic fluorescence dye 10-n-nonyl-acridinium-orange-chloride, NAO, stains specifically the mitochondria of living HeLa-cells. A dye concentration of 1 X 10(-8) M is sufficient for vital staining and at 5 X 10(-7) M an incubation time less than 1 min is enough to generate the bright green fluorescence of the mitochondria. The retention of NAO by the mitochondria is longer than 7 days. The dye accumulation is not affected by the ionophores valinomycin, nigericin, gramicidin, the uncoupling agents DNP, CCCP or by ouabain. In contrast to Rh 123 the trans-membrane potential is not the driving force of the NAO accumulation. We assume that NAO is bound to the hydrophobic lipids and proteins in the mitochondrial membranes by hydrophobic interaction. With valinomycin, 500 ng/ml, 10 min, the mitochondria in HeLa-cells swell. Now it is possible to observe some details in the enlarged mitochondria by light microscopy. After vital staining with NAO, 5 X 10(-7) M, 10 min, the periphery of the swollen mitochondria shows an intense green fluorescence, the inner part is dark. Obviously the dye is bound to the membranes. By electron microscopy it can be shown that the valinomycin treated and NAO stained mitochondria have outer and inner membranes and cristae. They differ from untreated mitochondria mainly in the size. After incubation of the HeLa-cells with relatively high NAO concentrations, 5 X 10(-6) M, 10 min, the mitochondria show a weak orange fluorescence. It is generated by the dimers D of NAO. Therefore the dye concentration in the mitochondrial membranes is locally very high and causes dye dimerisation. The weak orange fluorescence is instable and disappears within a few seconds. Instead we observe a green fluorescence with growing intensity that is generated by the monomers M of NAO. The intensity has its maximum value after a few seconds. Using low NAO concentrations for incubation, 1 X 10(-7) M, 10 min, we observe only the green fluorescence with increasing intensity. In this case the orange fluorescence is too weak for observation (concentration quenching). It can be shown by experiments and quantum mechanics that the orange fluorescence is assigned to an optical forbidden, the green fluorescence to an allowed electronic transition of D or M respectively. Our results indicate a dissoziation of D in 2 M by irradiation of the mitochondria under the fluorescence microscope.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoacridines↗

PubMed1999

Porphyrin-like fluorescence in oral cancer: In vivo fluorescence spectral characterization of lesions by use of a near-ultraviolet excited autofluorescence diagnosis system and separation of fluorescent extracts by capillary electrophoresis.

BACKGROUND: Red fluorescence from malignant tumors was observed in experimentally induced rat sarcoma by Policard (1924) and in ulcerated human oral carcinoma by Harris et al. (1987) by examination with ultraviolet (UV) irradiation. The objective of the current study was twofold: to examine in vivo the spectral characteristics of red fluorescence emitted from oral carcinomas and to separate the red fluorescent compounds in these lesions by the capillary electrophoretic (CE) method. METHODS: In vivo fluorescence spectral characteristics of oral carcinoma were examined by a near-UV excited autofluorescence diagnosis (NEAD) system developed by the authors. Fluorescence spectra of the extract from carcinomas were measured using a spectrofluorometer. CE was used to separate fluorescent compounds from the oral carcinomas. RESULTS: Of the 78 oral carcinomas examined using the NEAD system, 66 carcinomas (85%), including 2 adenoid cystic carcinomas (ACCs) and 14 recurrent squamous cell carcinomas (SCCs), showed porphyrin-like fluorescence spectra. The CE study was performed on three oral SCCs, two of which contained fluorescent compounds other than protoporphyrin IX and zinc protoporphyrin IX, whereas the other SCCs contained the compounds with the same migration time as protoporphyrin IX. CONCLUSIONS: Seventy-eight oral carcinomas, including ACCs and recurrent SCCs, were examined using the NEAD system. When exposed to UV light at a wavelength of 410 nm, 85% of the carcinomas showed porphyrin-like fluorescence spectra, whereas the normal mucosa in the oral cavity did not. Porphyrin-like fluorescent compounds were extracted from oral carcinomas and separated by a CE system equipped with a fluorescence detector. The CE data clearly show that compounds vary in each individual carcinoma.

Aged↗

PubMed2003

A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants.

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu(222)-->Gly (E222G) substitution, which removed the evolutionarily invariant Glu(222). Re-introduction of Glu(222) into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of 'humanized' aceGFP and beta-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

Animals↗

PubMed1981

Fluorescence titration and fluorescence stopped-flow studies on skeletal troponin C labeled with fluorescent maleimide reagent or dansylaziridine.

Skeletal muscle troponin C (TN-C) labeled with N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) shows about 5% fluorescence increase and 82% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites (sites III and IV) of TN-C, respectively. TN-C labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) shows about 26% fluorescence decrease and 22% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites, respectively. These findings indicate that environmental change around Cys-98, where the maleimide reagent bind, induced by Ca2+ binding to the high affinity Ca2+-binding sites is very different from that induced by Mg2+ binding to the same sites. These dye-protein conjugates do not show fluorescence intensity change upon Ca2+ binding to the low affinity Ca2+-binding sites (sites I and II). Dansylaziridine (DANZ)-labeled TN-C shows more than 100% fluorescence increase upon Ca2+ binding to the low affinity Ca2+-binding sites of TN-C. Hence, we can observe the kinetic processes which TN-C undergoes upon Ca2+ binding to or removal from the high affinity Ca2+-binding sites by following the fluorescence intensity change of ANM-labeled TN-C, respectively. THe kinetic processes of the fluorescence intensity change associated with the Ca2+ binding and removal reactions with the high affinty Ca2+-binding sites (rate constants, 3.7-157 s-1) are slower than the kinetic processes associated with the low affinity Ca2+-binding sites (rate constants, equal to or higher than 230 s-1) in the absence of MgCl2. In the presence of 2 mM MgCl2, a new slow phase (rate constant, 10-16 s-1) appears in the kinetic processes associated with the low affinity Ca2+-binding sites, in addition to the rapid phase which is already observed in the absence of MgCl2. Kinetic properties associated with the high affinity Ca2+-binding sites are not essentially altered by addition of 2 mM MgCl2 to the system, but ANM-labeled TN-C shows lower rate constants (0.67-26 s-1).

Animals↗

PubMed2003

Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm.

BACKGROUND: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser. METHODS: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564-606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters. Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached. Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry. RESULTS: Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd. Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564-606 nm. A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564-606 nm, consistent with the conclusion that FRET was detected comparably by both methods. A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR --> YFP-FBPcd. FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached. The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry. CONCLUSION: These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser. The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique.

Argon↗

PubMed1998

Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy.

We have investigated the pH dependence of the dynamics of conformational fluctuations of green fluorescent protein mutants EGFP (F64L/S65T) and GFP-S65T in small ensembles of molecules in solution by using fluorescence correlation spectroscopy (FCS). FCS utilizes time-resolved measurements of fluctuations in the molecular fluorescence emission for determination of the intrinsic dynamics and thermodynamics of all processes that affect the fluorescence. Fluorescence excitation of a bulk solution of EGFP decreases to zero at low pH (pKa = 5.8) paralleled by a decrease of the absorption at 488 nm and an increase at 400 nm. Protonation of the hydroxyl group of Tyr-66, which is part of the chromophore, induces these changes. When FCS is used the fluctuations in the protonation state of the chromophore are time resolved. The autocorrelation function of fluorescence emission shows contributions from two chemical relaxation processes as well as diffusional concentration fluctuations. The time constant of the fast, pH-dependent chemical process decreases with pH from 300 microseconds at pH 7 to 45 microseconds at pH 5, while the time-average fraction of molecules in a nonfluorescent state increases to 80% in the same range. A second, pH-independent, process with a time constant of 340 microseconds and an associated fraction of 13% nonfluorescent molecules is observed between pH 8 and 11, possibly representing an internal proton transfer process and associated conformational rearrangements. The FCS data provide direct measures of the dynamics and the equilibrium properties of the protonation processes. Thus FCS is a convenient, intrinsically calibrated method for pH measurements in subfemtoliter volumes with nanomolar concentrations of EGFP.

Fluorescence↗

PubMed1997

Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators.

We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the calmodulin-binding domain from smooth muscle myosin light chain kinase. When (Ca2+)4-calmodulin is bound to the indicator (Kd = 0.4 nM), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases 6-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio-temporal changes in the fluorescence of the indicator. Changes in indicator fluorescence in these cells are coupled with no discernible lag (<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to approximately 1 microM. This observation suggests that the activity of a calmodulin target with a typical 1 nM affinity for (Ca2+)4-calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than calmodulin.

Amino Acid Sequence↗

PubMed1990

Time-resolved fluorescence studies of flavodoxin. Fluorescence decay and fluorescence anisotropy decay of tryptophan in Desulfovibrio flavodoxins.

The time-resolved fluorescence characteristics of tryptophan in flavodoxin isolated from the sulfate-reducing bacteria Desulfovibrio vulgaris and Desulfovibrio gigas have been examined. By comparing the results of protein preparations of normal and FMN-depleted flavodoxin, radiationless energy transfer from tryptophan to FMN has been demonstrated. Since the crystal structure of the D. vulgaris flavodoxin is known, transfer rate constants from the two excited states 1La and 1Lb can be calculated for both tryptophan residues (Trp 60 and Trp 140). Residue Trp 60, which is very close to the flavin, transfers energy very rapidly to FMN, whereas the rate of energy transfer from the remote Trp 140 to FMN is much smaller. Both tryptophan residues have the indole rings oriented in such a way that transfer will preferentially take place from the 1La excited state. The fluorescence decay of all protein preparations turned out to be complex, the parameter values being dependent on the emission wavelength. Several decay curves were analyzed globally using a model in which tryptophan is involved in some nanosecond relaxation process. A relaxation time of about 2 ns was found for both D. gigas apo- and holo-flavodoxin. The fluorescence anisotropy decay of both Desulfovibrio FMN-depleted flavodoxins is exponential, whereas that of the two holoproteins is clearly non-exponential. The anisotropy decay was analyzed using the same model as that applied for fluorescence decay. The tryptophan residues turned out to be immobilized in the protein. A time constant of a few nanoseconds results from energy transfer from tryptophan to flavin, at least for D. gigas flavodoxin. The single tryptophan residue in D. gigas flavodoxin occupies a position in the polypeptide chain remote from the flavin prosthetic group. Because of the close resemblance of steady-state and time-resolved fluorescence properties of tryptophan in both flavodoxins, the center to center distance between tryptophan and FMN in D. gigas flavodoxin is probably very similar to the distance between Trp 140 and FMN in D. vulgaris flavodoxin (i.e. 20 A).

Desulfovibrio↗

PubMed1986

Enhancement of fluorescence development of end products by use of a fluorescence developer solution in a rapid and sensitive fluorescent spot test for specific detection of microbial beta-lactamases.

A fluorescent spot test method for specific detection of microbial beta-lactamases as previously published (K. C. S. Chen, J. S. Knapp, and K. K. Holmes, J. Clin. Microbiol. 19:818-825, 1984) was improved by the use of a fluorescence developer solution. The fluorescence developer solution used in this study consisted of 0.78 M sodium tartrate buffer containing 12% formaldehyde at a final pH of 4.5. An addition of 1 volume of fluorescence developer solution to 5 volumes of ampicillin or cephalex substrate solution incubated with beta-lactamase-producing organisms, followed by heating the mixture at 45 degrees C for 10 min resulted in enhancement of fluorescence of the end products of beta-lactamase activity. This provides a more sensitive assay for microbial beta-lactamases and offers the potential for direct detection of beta-lactamases in clinical specimens.

Amidohydrolases↗

PubMed1994

Comparisons of the relative effects of polyhydroxyl compounds on local versus long-range motions in the mitochondrial inner membrane. Fluorescence recovery after photobleaching, fluorescence lifetime, and fluorescence anisotropy studies.

This laboratory has been interested in understanding the relationship between molecular motion and electron transport rates in the mitochondrial inner membrane. We have previously noted a sucrose-induced decrease in both multicomponent electron transport rates and lateral diffusion of redox components. The decreases in lateral diffusion and the related mobile fraction of redox components were greater than expected from hydrodynamic theory. In this report we sought to understand how the presence of increasing aqueous concentrations of polyhydroxyl agents affect short-range motions in different regions of the inner membrane bilayer, frequently expressed in terms of 'viscosity' and order, compared to lateral diffusion. Fluorescence recovery after photobleaching was used to monitor long-range phospholipid and integral protein diffusion. Multifrequency fluorescence lifetime and steady-state fluorescence anisotropy techniques were used to monitor local dynamics of diphenylhexatriene (DPH) and trimethylaminodiphenylhexatriene (TMA-DPH). Light scattering corrections were found to be essential for inner membrane measurements by the latter two techniques. DPH and TMA-DPH each exhibited two-lifetime components. Generally, increasing the aqueous concentration of polyhydroxyl agents decreased the average DPH lifetime and increased the average TMA-DPH lifetime. In general, under the same conditions fluorescence anisotropies increased. Our results indicated that changes in the rotational diffusion coefficient, microviscosity and order were being induced at both the phospholipid headgroup and in the acyl chain regions of the membrane bilayer. Our results suggest that these changes may be due in part to induced changes in the interaction and distribution of water with membranes. Long-range lateral diffusion was found to be significantly retarded by increasing concentrations of polyhydroxyl agents. We conclude that the discrepancies between bulk viscosity predicted decreases in long-range diffusion may result, in part, from the aforementioned membrane/water interactions. We also note an apparent qualitative relationship between long-range lateral diffusion reported diffusion coefficient with local TMA-DPH reported rotational diffusion coefficient and apparent microviscosities.

Diffusion↗

PubMed1980

A new lysozyme assay based on fluorescence polarization or fluorescence intensity utilizing a fluorescent peptidoglycan substrate.

A new sensitive, rapid and simple method for lysozyme assay is described which is based on either fluorescence polarization or fluorescence intensity using fluorescein-labeled peptidoglycan as a substrate. The peptidoglycan was obtained from Micrococcus lysodeikticus after extensive digestion with Pronase and washing with Triton X-100 followed by various solvents. Subsequently, it was labeled with fluorescein isothiocyanate (FITC) at the amino group of the peptide. When the FITC-labeled substrate was subjected to lysozyme digestion, an increase of fluorescence intensity or a decrease of fluorescence polarization value (P value) was apparent in five minutes at a lysozyme concentrations as low as 0.1 or 0.01 micrograms/ml, respectively. The effect of other hydrolytic enzymes including alpha-mannosidase, proteases and RNase on the P value was found to be negligible. The measured values represented the specificity and dose of lysozyme added. Apparent Vmax and Km values for two different lysozymes, chicken egg white and human, could be determined by this method.

Amino Acids↗

PubMed2001

Diagnostic potential of fluorescence of formalin-fixed paraffin-embedded malignant melanoma and pigmented skin lesions: quantitative study of fluorescence intensity using fluorescence microscope and digital imaging.

The background for this study was reports in the literature of stronger fluorescence observed visually for melanomas compared with benign naevi in formalin-fixed paraffin-embedded sections. Our objective was to carry out a quantitative study of the phenomenon and to investigate if such an approach could be used in the detection of melanomas. Microscopic digital imaging was used to measure quantitatively the fluorescence intensity in specimens from 50 malignant melanomas, four basal cell carcinomas and 58 benign lesions. The mean fluorescence intensity of the melanomas was considerably higher than of the other lesions. For melanomas, the intensity depended both on the distance from the skin surface and the distance from the centre of the lesion. A simple algorithm based on the intensity threshold correctly classified the melanomas with a sensitivity of 74% and a specificity of 59%. Quantitative measurements of the fluorescence of the pigmented skin lesions fixed with formalin and embedded in paraffin can be a useful auxiliary tool for differentiating melanoma from other pigmented lesions histopathologically.

Basal Cell Carcinoma↗

PubMed1988

Comparison of fluorescence characteristics of products of peroxidation of membrane phospholipids with those of products derived from reaction of malonaldehyde with glycine as a model of lipofuscin fluorescent substances.

The fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to product (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorescent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.

Animals↗

PubMed1997

Analysis of the intracellular processing of proteins: application of fluorescence polarization and a novel fluorescent probe.

Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in fluorescence polarization was due to movement of the probe through a pH gradient within the cell, suggestive of transport through the endocytic system. By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processing of proteins within macrophages.

Animals↗

PubMed1961

Studies on fluorescent antibody staining. I. Non-specific fluorescence with fluorescein-coupled sheep anti-rabbit globulins.

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules.

Animals↗

PubMed2000

The effect of decreasing temperature up to chilling values on the in vivo F685/F735 chlorophyll fluorescence ratio in Phaseolus vulgaris and Pisum sativum: the role of the photosystem I contribution to the 735 nm fluorescence band.

The effect of leaf temperature (T), between 23 and 4 degrees C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 microE m-2 s-1) and low (30-35 microE m-2 s-1) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1-state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence life-time measurements support this interpretation. At a critical temperature (about 6 degrees C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spill-over from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F0), maximal (Fm) and steady-state (Fs) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

Chlorophyll↗

PubMed1998

Enhanced detection sensitivity of "fluorescence reduction" by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio.

Nonfluorescing protein bands can be detected by the fluorescence optics of the commercial gel electrophoresis apparatus with automated scanning of the migration path (HPGE-1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins ("fluorescence reduction"). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of "fluorescence reduction" equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by "fluorescence reduction" provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 microg/lane of the gel tray. A theory of "fluorescence reduction" is presented which accounts for the existence of a linear relationship between band area and load.

Animals↗

PubMed2003

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease, and 1 phosphatase.

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.

Biological Assay↗