A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein. 相似文献
The ability of eight soil microfungal species, Alternaria alternata, Clonostachys rosea f. rosea, Exophiala cf. salmonis, Fusarium cf. coeruleum, Fusarium redolens, Paecilomyces lilacinus, Penicillium canescens and Phoma sp., and two known basidiomycete humic acid (HA) degraders, Trametes versicolor and Phanerochaete chrysosporium, to modify fluorescence properties of fulvic acids (FA) and/or HAs was determined. Effects of minerals and/or glucose on the modifications were examined. FA purified on polyvinyl-polypyrrolidone (PVPP) chromatography column was used. Purification of FA on PVPP column removed the low-molar-mass FA-structural components and excess of extractant (NaOH) used during FA preparation. Excitation spectra of FA entering the purification, purified FA and the removal solution indicate that organic compounds rich in carboxylic groups dominate in the removal solution and higher content of phenolic groups is a characteristic of purified FA. Many microfungal species shifted the emission maximum (measured at 470 and 468 nm of excitation wavelength) of FA, and also HA to longer wavelengths. The opposite effect (shift of the HA emission maximum to shorter wavelengths) of microfungi was observed for HA complemented by glucose. Depending on the presence of glucose in the medium, most microfungi changed also the shape of the emission spectra of HA and FA and the excitation spectra of FA. HA excitation spectrum measured at 590 nm of emission wavelength was significantly affected by the presence of glucose. Mineral ions caused a minor shift in the position of excitation maximum (measured at 590 nm of emission wavelength) toward longer wavelengths. 相似文献
The degeneration of articular cartilage is the main cause of osteoarthritis (OA), a common cause of disability among elderly patients. The aim of this study is to understand the correlation between intrinsic fluorescence of articular cartilage and its biomechanical properties in patients with osteoarthritis. Cylindrical samples of articular cartilage 6 mm in diameter were extracted via biopsy punch from the femoral condyles of 6 patients with advanced OA undergoing knee replacement surgery. The mechanical stiffness and fluorescence of each cartilage plug were measured by indentation test and spectrofluorometry. Maps of fluorescence intensity, at excitation/emission wavelengths of 240–520/290‐530 nm, were used to identify wavelengths of interest. The mechanical stiffness and fluorescence intensity were correlated using a Spearman analysis. The excitation/emission maps demonstrated three fluorescence peaks at excitation/emission wavelength pairs 330/390, 350/430 and 370/460 nm. The best correlation between the fluorescence intensity and stiffness of cartilage was obtained for the 330 nm excitation band [R=0.82, p=0.04]. The intrinsic fluorescence of articular cartilage may have application in optically assessing the state of cartilage in patients with osteoarthritis.
Plants dissipate excess excitation energy as heat by non‐photochemical quenching (NPQ). NPQ has been thought to resemble in vitro aggregation quenching of the major antenna complex, light harvesting complex of photosystem II (LHC‐II). Both processes are widely believed to involve a conformational change that creates a quenching centre of two neighbouring pigments within the complex. Using recombinant LHC‐II lacking the pigments implicated in quenching, we show that they have no particular role. Single crystals of LHC‐II emit strong, orientation‐dependent fluorescence with an emission maximum at 680 nm. The average lifetime of the main 680 nm crystal emission at 100 K is 1.31 ns, but only 0.39 ns for LHC‐II aggregates under identical conditions. The strong emission and comparatively long fluorescence lifetimes of single LHC‐II crystals indicate that the complex is unquenched, and that therefore the crystal structure shows the active, energy‐transmitting state of LHC‐II. We conclude that quenching of excitation energy in the light‐harvesting antenna is due to the molecular interaction with external pigments in vitro or other pigment–protein complexes such as PsbS in vivo, and does not require a conformational change within the complex. 相似文献
Protochlorophyll was found in roots of dark-grown plants of seven species investigated. It was identified by absorbance and fluorescence spectra of acetone and ether extracts. Chlorophyll was also found in roots of one pea species. The concentration of protochlorophyll was usually highest in young root tips and decreased upwards along the roots. The maxima of the in vivo absorbance spectra of the species studied varied between 634 and 638 nm. Low temperature in vivo fluorescence emission spectra had two maxima, one at ca 633 and the other at ca 642 nm, when the wavelengths of the excitation light were 440 and 460 nm, respectively. In vivo fluorescence excitation spectra displayed a shift of the excitation maximum from 438 to 445 nm, when emission varied from 620 to 647.5 nm. Deconvolution of these three types of spectra into Gaussian components made it possible to identify two spectral forms of protochlorophyll: protochlorophyll629–633 and protochlorophyll638–642. 相似文献
Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5‐aminolevulinic‐acid‐induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50‐fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well‐defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose‐gel phantoms. Even in direct contact, blood layers of 20–40 µm between probe and tissue must be expected, obscuring 405‐nm‐excited PpIX fluorescence almost completely, but reducing the 633‐nm‐excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX‐assisted detection of high‐grade gliomas in stereotactic biopsy.
PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation. 相似文献
Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling. 相似文献
The physicochemical mechanism for merocyanine 540 (M540) binding to unilamellar phosphatidylcholine (PC) vesicles was examined by steady-state and dynamic fluorescence and fluorescence stopped-flow methods. At 530-nm excitation, aqueous M540 has an emission peak at 565 nm, which red shifts to 580 nm with formation of membrane-bound monomers (M); bound dimers (D) are nonfluorescent. Equilibrium fluorescence titrations show that 50% of total M540 partitions into the membrane to form D at [M540]/[PC] (Rm/p)_approximately 0.6. M and D concentrations are equal at Rm/p approximately 0.05. For Rm/p less than 0.1, M540 has a single fluorescence lifetime (tau), which decreases with Rm/p [tau-1 (ns-1) = 0.48 + 3.3Rm/p], indicating a rapid collisional rate between M to form D. Dynamic depolarization studies show that hindered rotation of M (r infinity = 0.13 at Rm/p = 0.006) becomes more rapid (rotational rate 0.2-1.9 ns-1) with increasing Rm/p (0.006-0.075). The efficiencies of energy transfer between n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12, 16) and bound M540 suggest that M is oriented parallel to the phospholipids near the membrane surface; studies of efficiencies of n-AF quenching by D are consistent with an orientation of D perpendicular to the phospholipids. In stopped-flow fluorescence measurements in which M540 is mixed with PC vesicles, there is a rapid (1 ms) followed by a slower (10-50 ms) concentration-dependent fluorescence increase.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
A new highly sensitive high‐performance liquid chromatographic method with fluorescence detection (HPLC–FLD) in zero‐order emission mode was developed for the first time for the simultaneous determination of piroxicam (PRX) and norfloxacin (NRF) in biological fluids. The fluorescence detector wavelengths were set at 278 nm for excitation and zero‐order mode for emission. The zero‐order emission mode produced greater sensitivity for the measurement of both drugs than a fixed emission wavelength (446 nm). The new developed method was validated according to International Conference of Harmonization (ICH) guidelines. Linearity was found to be over concentration ranges 0.001–20 μg/ml and 0.00003–0.035 μg/ml for PRX and NRF, respectively. The limits of detection were 4.87 × 10?4 and 1.32 × 10?5 μg/ml for PRX and NRF, and the limits of quantitation were 1.47 × 10?3 and 4.01 × 10?5 μg/ml, respectively. The current fluorescence method was found to be more sensitive than most commonly used analytical methods and was successfully applied for simultaneous determination of PRX and NRF in biological fluids (serum and urine) with recoveries ranging from 91.67% to 100.36% for PRX and from 96.00% to 101.43% for NRF. 相似文献
Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358 +/- 0.134 X 10(4) at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells. 相似文献
Sensitive and selective spectrophotometric and spectrofluorimetric methods have been developed for the estimation of two anti-migraine drugs, namely sumatriptan succinate (SUM) and zolmitriptan (ZOL). These methods depend on producing a yellow-coloured product after the reaction of the two drugs with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). The reaction products exhibited maximum absorbance at 481 nm in borate buffer of pH 9 and fluorescence emission peak at 540 nm after excitation at 470 nm for the two drugs. The linear ranges were 5–60 μg/ml for SUM and 5–50 μg/ml for ZOL in the spectrophotometric method (Method I), whereas this was 0.4–4 μg/ml for SUM and 0.5–5 μg/ml for ZOL in the spectrofluorimetric method (Method II). The method validity was assessed according to International Council for Harmonisation (ICH) guidelines. Statistical analysis of the results obtained from the proposed and comparison methods confirmed that the proposed methods were highly accurate and precise. The suggested methods could be used for the determination of the mentioned drugs in both pure form and in tablets. 相似文献
Summary Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358±0.134×104 at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.Supported by NIH grants EY00012, EY07035 and EY01583 相似文献
Plants are one of the most important parts of the ecological system and demand a reliable method for accurate classification. In this study, the first‐derivative fluorescence spectral curves (FDFSCs) based on laser‐induced fluorescence technology were proposed for the characterization of plant species. The measurement system is mainly composed of a spectrometer, an excitation light source (the two excitation wavelengths are 460 and 556 nm, respectively), and an intensified charge‐coupled device camera. FDFSCs were calculated from the deviation between the fluorescence values at each wavelength, plus and minus one band, divided by the wavelength range. Principal component analysis was utilized to analyze the FDFSCs by extracting the main attributes and reducing the dimensionality of variables. A support vector machine was used to evaluate FDFSC performance for the identification of plant species. Plant species that are difficult to distinguished by the naked eye, can be identified effectively using the proposed FDFSCs. For the 556 nm and 460 nm excitation wavelengths, the overall identification rates of the six plant species evaluated were 93.3% and 91.7%, respectively. Experimental results demonstrated that the combination of the FDFSCs with multivariate analysis could provide a simple and reliable method for the characterization of plant species. 相似文献
The first step in the Visual Cycle, the series of reactions that regenerate the vertebrate visual pigment rhodopsin, is the reduction of all-trans retinal to all-trans retinol, a reaction that requires NADPH. We have used the fluorescence of all-trans retinol to study this reduction in living rod photoreceptors. After the bleaching of rhodopsin, fluorescence (excitation, 360 nm; emission, 457 or 540 nm) appears in frog and wild-type mouse rod outer segments reaching a maximum in 30-60 min at room temperature. With this excitation and emission, the mitochondrial-rich ellipsoid region of the cells shows strong fluorescence as well. Fluorescence measurements at different emission wavelengths establish that the outer segment and ellipsoid signals originate from all-trans retinol and reduced pyridine nucleotides, respectively. Using outer segment fluorescence as a measure of all-trans retinol formation, we find that in frog rod photoreceptors the NADPH necessary for the reduction of all-trans retinal can be supplied by both cytoplasmic and mitochondrial metabolic pathways. Inhibition of the reduction reaction, either by retinoic acid or through suppression of metabolic activity, reduced the formation of retinol. Finally, there are no significant fluorescence changes after bleaching in the rod outer segments of Rpe65(-/-) mice, which lack 11-cis retinal. 相似文献