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At least 73 records · Page 4Linked to original sources
PubMed2004

Development of an efficient fluorescence-based microneutralization assay using recombinant human cytomegalovirus strains expressing green fluorescent protein.

MedImmune Vaccines has created four, live, attenuated human cytomegalovirus (HCMV) vaccine candidates, each derived from defined portions of the parental strains, Towne and Toledo. To determine each candidate's ability to induce HCMV specific immunity, a fluorescence-based microneutralization assay was developed using recombinants of Toledo and Towne which express enhanced green fluorescent protein (EGFP). Replication of the EGFP recombinants in cell culture was the same as the respective parental strains. Using the EGFP recombinants, this fluorescence-based microneutralization assay was compared with the traditional plaque reduction assay. Serum samples were analyzed by both the fluorescence microneutralization and plaque reduction assays and regression analysis showed a correlation of R2 > or = 0.90 between the two assays. As an alternative to measuring fluorescence, infected cells were examined microscopically and the number of green fluorescent cells was counted automatically. Regression lines between fluorescent cell counting and fluorescence in the well also showed a high correlation (R2 > or = 0.92). An excellent linear concordance in titers was observed between the two assays. Using the plaque reduction assay, serum samples were identified that preferentially neutralized the Toledo strain compared to the Towne strain. The same preferences were observed with the fluorescence-based microneutralization assay. This new assay is adaptable to rapid, automated collection of neutralization data and would therefore be suitable for the examination of large numbers of clinical serum samples.

Cell Line↗
PubMed2006

Selective determination of native fluorescent bioamines through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

In this paper, we introduce a novel approach for the highly selective and sensitive analysis of native fluorescent bioamines (indoleamines and catecholamines). This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the bioamines' amino groups. In this detection process, we monitored the FRET from the native fluorescent moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 15 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated the FRET most effectively. The OPA derivatives of the native fluorescent bioamines emitted OPA fluorescence (445 nm) through an intermolecular FRET process when they were excited at the excitation maximum wavelengths of the native fluorescent bioamines (280 nm). The generation of FRET was confirmed through comparison with the analysis of a nonfluorescent amine (isoleucine) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the indoleamines and catecholamines when performing LC on an ODS column. The detection limits (signal-to-noise ratio, 3) for the indoleamines and catecholamines, at a 20-muL injection volume, were 17-120 and 28-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than those of systems that take advantage of both native fluorescence detection (i.e., without derivatization) and the conventional detection of OPA derivatives. Furthermore, this method provides enough selectivity and sensitivity for the determination of the indoleamines present in the urine of healthy humans.

Biogenic Amines↗
PubMedDate not supplied

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin.

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 </= n </= 4). The results showed an astounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even at high Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3 conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the recommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderate fluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at high ligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanisms are responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surface causes an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs with Cy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much more pronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of the labels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at 600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and to some extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into two groups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensive quenching occurs in antibodies labeled with Cy5 and Cy7.

Animals↗
PubMed2005

Enhancement of the fluorescence of the blue fluorescent proteins by high pressure or low temperature.

Green fluorescent proteins bearing the Y66H mutation exhibit strongly blue-shifted fluorescence excitation and emission spectra. However, these blue fluorescent proteins (BFPs) have lower quantum yields of fluorescence (Phi(f) approximately 0.20), which is believed to stem from the increased conformational freedom of the smaller chromophore. We demonstrate that suppression of chromophore mobility by increasing hydrostatic pressure or by decreasing temperature can enhance the fluorescence quantum yield of these proteins without significantly affecting their absorption properties or the shape of the fluorescence spectra. Analysis of the fluorescence lifetimes in the picosecond and nanosecond regimes reveals that the enhancement of the fluorescence quantum yield is due to the inhibition of fast quenching processes. Temperature-dependent fluorescence measurements reveal two barriers ( approximately 19 and 3 kJ/mol, respectively) for the transition into nonfluorescing states. These steps are probably linked with dissociation of the hydrogen bond between the chromophore and His148 or an intervening water molecule and to the barrier for chromophore twisting in the excited state, respectively. The chromophore's hydrogen-bond equilibrium at room temperature is dominated by entropic effects, while below approximately 200 K the balance is enthalpy-driven.

Green Fluorescent Proteins↗
PubMed1990

Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence.

Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.

Cell Adhesion↗
PubMed1985

A cytoplasmic component of pyridine nucleotide fluorescence in rat diaphragm: evidence from comparisons with flavoprotein fluorescence.

Pyridine nucleotide (PN) and flavoprotein (Fp) fluorescence were monitored in the isolated intact rat diaphragm. A substantial increase in PN fluorescence occurred when N2 replaced O2 in glucose medium. This response was much reduced in pyruvate medium and/or by pretreatment with iodoacetic acid (IAA). The anaerobic levels of Fp fluorescence were less affected by substrate and IAA. Substitution of glucose by pyruvate did not alter the PN fluorescence of the resting aerobic tissue, but increased Fp fluorescence. After a tetanus with glucose present the PN of the anaerobic muscle, but not the Fp underwent a substantial transient oxidation. This oxidation was absent in pyruvate medium. It is concluded that a cytoplasmic component of the PN fluorescence is present in skeletal muscle. The levels of Fp fluorescence in the resting and contracting aerobic tissue supplied with pyruvate suggest that the resting tissue respiration was ADP limited. On this basis the level of PN fluorescence in the aerobic resting state was less than expected; the source of the PN fluorescence was both mitochondrial and cytoplasmic.

Aerobiosis↗
PubMed2004

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles.

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.

Artifacts↗
PubMed2003

A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling.

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.

Amino Acid Sequence↗
PubMed2002

Unique fluorescence properties of a cyanobacterium Gloeobacter violaceus PCC 7421: reasons for absence of the long-wavelength PSI Chl a fluorescence at -196 degrees C.

We investigated the reason for the absence of the long-wavelength PSI Chl a fluorescence at -196 degrees C in the cyanobacterium Gloeobacter violaceus using two methods: p-nitrothiophenol (p-NTP) treatment and time-resolved fluorescence spectra. The p-NTP treatment showed that PSII Chl a fluorescence was specifically affected in a manner similar to that for Synechocystis sp. PCC 6803 and spinach chloroplasts, although there were no components modified by the p-NTP treatment, indicating an absence of the long-wavelength PSI Chl a fluorescence. The time-resolved fluorescence spectra with a time resolution of 1.3 ps and spectral resolution of 1.0 nm gave no indication of the presence of the long-wavelength PSI fluorescence in the wavelength region between 700 nm and 760 nm, indicating that a very fast energy transfer among Chl a molecules could not account for the absence of the long-wavelength PSI fluorescence. From these data, it seems that the absence of the long-wavelength PSI fluorescence is due to a lack of the formation of a component responsible for the fluorescence at -196 degrees C, which may originate from a difference in the amino acid sequence. We discuss the significance of this phenomenon and interpret our findings in terms of the evolution of cyanobacteria.

Chlorophyll↗
PubMed2003

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.

Algorithms↗
PubMedDate not supplied

[The lipophilic fluorescent probe 4-dimethylaminochalcone: factors responsible for the fluorescence yield].

Factors responsible for fluorescence quenching of the lipophylic fluorescent probe 4-dimethylaminochalcone in nonpolar and polar media were studied. The femtosecond dynamics of 4-dimethylaminochalcone excited state was measured using the absorption method of "excitation probing". In nonpolar hexane where the fluorescence quantum yield is very low (0.001), all excited 4-dimethylaminochalcone molecules go to the triplet state with a rate constant of 2.10(11) s(-1). At the same time, the radiation rate constant is 1000 times lower; therefore, such a fast transition to triplet is the major cause of the very small fluorescence yield. In polar acetone, the fluorescence yield is 220 times higher than in hexane. In acetone, no transitions to triplet state were detected. At the same time, a radiationless conversion to the ground state took place with a rate constant of 10(9) s(-1), which decreased the fluorescence yield to 0.22. The activation energy of the quenching processes is polarity dependent and decreases from 6 in nonpolar to 3 kcal/mol in polar media. The yield of 4-dimethylaminochalcone fluorescence varies hundreds times in media of different polarity but is a linear function of the Lippert's polarity parameter f(epsilon,n) where epsilon is the dielectric constant at low frequencies. It is supposed that polar media stabilize the "flat" conformation of the 4-dimethylaminochalcone molecule prior to its excitation, and this conformation is optimal for fluorescence. In this case, stabilization is determined only by medium polarity.

Chalcone↗
PubMed2005

How to improve quality assurance in fluorometry: fluorescence-inherent sources of error and suited fluorescence standards.

The scope of this paper is to illustrate the need for an improved quality assurance in fluorometry. For this purpose, instrumental sources of error and their influences on the reliability and comparability of fluorescence data are highlighted for frequently used photoluminescence techniques ranging from conventional macro- and microfluorometry over fluorescence microscopy and flow cytometry to microarray technology as well as in vivo fluorescence imaging. Particularly, the need for and requirements on fluorescence standards for the characterization and performance validation of fluorescence instruments, to enhance the comparability of fluorescence data, and to enable quantitative fluorescence analysis are discussed. Special emphasis is dedicated to spectral fluorescence standards and fluorescence intensity standards.

Journal Article↗
PubMed2006

Extreme fluorescence sensitivity of some aniline derivatives to aqueous and nonaqueous environments: mechanistic study and its implication as a fluorescent probe.

Effects of solvent water on the photophysical properties of a series of meta- and para-substituted anilines have been investigated by means of time-resolved fluorescence, transient absorption, and photoacoustic measurements. Some aniline derivatives exhibit extremely short fluorescence lifetime (tau(f)) and small quantum yield (Phi(f)) in water (e.g., tau(f) = 45 ps and Phi(f) = 0.0019 for m-cyanoaniline (m-ANCN) in H(2)O), which is in marked contrast with their much larger values in nonaqueous solvents (tau(f) = 7.3 ns and Phi(f) = 0.14 for m-ANCN in acetonitrile). Photoacoustic and transient absorption measurements show that the remarkable fluorescence quenching of m-ANCN in water is attributed almost exclusively to fast internal conversion. The lifetime measurements of m-ANCN in H(2)O/acetonitrile binary solvent mixtures reveal that the quenching is related to variation of hydrogen-bonding interactions between the amino group and water molecules and the conformational change of the amino group upon electronic excitation. Similar fluorescence quenching due to solvent water is also found for N-alkylated m-ANCNs. The drastic differences in the fluorescence intensity and lifetime of m-ANCNs under hydrophobic and hydrophilic environments and also the large solvent polarity dependence of the fluorescence band position suggest the possibility that they can be utilized as fluorescent probes for investigating the microenvironment of biological systems. In suspensions of human serum albumin (HSA) in water, remarkable enhancement of the fluorescence intensity and lifetime is observed for m-ANCN and its N-alkylated derivatives, demonstrating that m-ANCNs can be a candidate for novel fluorescent probe with small molecular size.

Journal Article↗
PubMed1984

Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity.

The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities.

Journal Article↗
PubMed2001

Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable to high-throughput screening technologies.

BACKGROUND: Reliable assessment of cell death is now pivotal to many research programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Such approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening (HTS) technologies. We describe a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP). METHODS: Cell death was induced by a variety of apoptotic stimuli in various EGFP-expressing mammalian cell lines, including those routinely used in anti-cancer drug screening. Decrease in fluorescence was assessed either by flow cytometry (and compared with other apoptotic markers) or by a fluorescence microplate reader. RESULTS: Cells expressing EGFP exhibited a decrease in fluorescence when treated by various agents, such as chemotherapeutic drugs, UV irradiation, or caspase-independent cell death inducers. Kinetics and sensitivity of this EGFP-based assay were comparable to those of traditional apoptosis markers such as annexin-V binding, propidium iodide incorporation, or reactive oxygen species production. We also show that the decrease in EGFP fluorescence is directly quantifiable in a fluorescence-based microplate assay. Furthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradation of the protein. CONCLUSIONS: This novel GFP-based microplate assay combines sensitivity and rapidity, is easily amenable to HTS setups, making it an assay of choice for cytotoxicity evaluation.

3T3 Cells↗
PubMed1993

Measurement of restricted rotational diffusion of fluorescent lipids in supported planar phospholipid monolayers using angle-dependent polarized fluorescence photobleaching recovery.

A theory describing the shapes of polarized fluorescence photobleaching recovery (PFPR) curves for a population of fluorophores undergoing restricted rotational diffusion in two-dimensional systems such as planar membranes has been developed. In this model, restricted rotational diffusion of the fluorophores is described by using reflective boundary conditions, in which the fluorophores are assumed to diffuse freely but only within an angular space of width 2 omega. The magnitude and apparent rate of the PFPR postbleach fluorescence curves are a function of both omega and the angle between the bleaching and observation beam polarizations psi. It is shown that estimates of the degree of rotational restriction omega may be obtained from changes in the psi-dependent postbleach fluorescence intensities. Using angle-dependent PFPR, slow rotational reorientations of the fluorescent lipid analogue 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine in distearoylphosphatidylcholine Langmuir-Blodgett monolayers deposited on octadecyltrichlorosilane-treated fused quartz were measured. As theoretically predicted for a rotationally restricted fluorophore population, both the initial F psi (0) and final F psi (infinity) postbleach fluorescence intensities varied as a function of psi, and no measurable change in the postbleach fluorescence intensities was observed for psi = 45 degrees. Using the theory for restricted rotational motion, the psi-dependent variations of the final fluorescence intensities F psi (infinity) obtained at two bleaching intensities gave an average apparent omega approximately 52 degrees. However, to adequately fit the F psi (0) data, inclusion of the theoretical effects of rapid (faster than the duration of the photobleaching pulse) fluorophore dynamics was also required. Best fits of the F psi (0) and F psi (infinity) data were obtained when the fluorophores were assumed to rapidly wobble within a cone of semiangle delta approximately 30 degrees-50 degrees while slowly rotating within an angular space defined by semiangle omega approximately 35 degrees-60 degrees. Subsequent analysis of the time- and psi-dependent changes in the post-bleach fluorescence curves F psi (t) gave apparent diffusion coefficients ranging from D approximately 10(-3) s-1 to 4 x 10(-2) s-1.

Carbocyanines↗
PubMed1996

Fluorescence polarization study on the dynamics and location of peroxidized fluorescent phospholipids in liposomes.

Motional properties of fluorescent substances produced by lipid peroxidation by a time-resolved fluorescence polarization technique were studied. When liposomes containing phosphatidylethanolamine (PE) and linoleic hydrocarbon chain were incubated at 37 degrees C, fluorophores absorbing maximally at 360 nm and emitting near 430 nm were produced. Their fluorescence anisotropy decay measured at 23 degrees C was fitted well with a sum of a fast relaxation and a time-independent residual term. With the increase of oxidation degree, the time constant of the relaxation term increased. This may be explained by alteration in the membrane structure or by modification of the fluorescent products themselves. Information on the location of the fluorescent products was obtained when their motional property was compared with those of various extrinsic probes that were incorporated at different positions of the lipid bilayer. It was found that the motional property of the fluorescent oxidation products is similar to that of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, a rod-shaped hydrophobic probe with a charged terminal. Other probes sensing the polar region or the hydrophobic region of the membrane were characterized by a lower order parameter. It is suggested that the fluorescent oxidation products have a polar moiety located at the membrane surface and attached to the amino group of PE while the tail part being buried in the hydrophobic region of the membrane. This picture is supported by fluorescence quenching experiments with the aqueous quencher Co2+. On the other hand, fluorophores produced by the reaction of malondialdehyde and PE suggested to have a chemical structure in which the angle between the absorption and emission dipole moments is very large. On the basis of these observations, the production pathway of fluorophores in oxidized membranes is discussed.

Fluorescence Polarization↗
PubMed2003

Spatial and temporal patterns of green fluorescent protein (GFP) fluorescence during leaf canopy development in transgenic oilseed rape, Brassica napus L.

The green fluorescent protein (GFP) holds promise as a field-level transgene marker. One obstacle to the use of GFP is fluorescence variability observed within leaf canopies. In growth chamber and field experiments, GFP fluorescence in transgenic oilseed rape ( Brassica napus) was shown to be variable at each leaf position over time and among different leaves on the same plant. A leaf had its highest GFP fluorescence after emergence and, subsequently, its fluorescence intensity decreased. GFP fluorescence intensity was directly correlated with the concentration of soluble protein. The concentration of the genetically linked recombinant Bacillus thuringiensis (Bt) cry1Ac endotoxin protein also was examined, and GFP fluorescence was positively correlated with Bt throughout development. The results show that GFP can be used as an accurate transgene marker but that aspects of plant developmental should be taken into account when interpreting fluorescence measurements.

Bacillus thuringiensis Toxins↗