Fluorescence Polarization Ratio Research Articles

Brush Printing Creates Polarized Green Fluorescence: 3D Orientation Mapping and Stochastic Analysis of Conductive Polymer Films.

Brush printing is a unique method used to obtain uniaxially oriented films, whereby a polymer solution is brushed onto a substrate. However, there have been only a few reports on the brush-printing method. Here, we report the preparation of a uniaxially oriented film of a green light-emitting conductive polymer, poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT). The fluorescence polarization ratio of the oriented F8BT films was as high as 11.3, and the average orientation factor reached 0.74 ± 0.06. The orientation factor and the torsion angle of F8BT were visualized by two mappings of fluorescence and Raman spectral measurements by confocal spectromicroscopy, respectively. These two x-y mapping data with many pixels (∼750 pixels) were evaluated by x-y-z mapping of the film thickness at a single position and were used to reveal the three-dimensional (3D) orientation mechanism from a stochastic approach. Polarized green fluorescence originates from polymer chains uniaxially oriented along the brush direction. The high orientation for a film thickness < 100 nm is established by shear stress, faster capillary flow, and flow-induced chain extension for a thin solution film on a substrate. The high orientation factor was also demonstrated by a high brushing speed, whereas an optimized brushing speed existed. We found that this optimization is attributed to the property of a non-Newtonian fluid. By applying this brush-printing method to the fabrication of an optoelectrical device, polarized green electroluminescence was preliminarily demonstrated by the OLED assembled from an oriented F8BT film.

Measuring fluorescence polarization with a dichrometer

A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer.

Fluorescence polarization-based method with bisulfite conversion-specific one-label extension for quantification of single CpG dinucleotide methylation.

To quantify the methylation at individual CpG dinucleotide sites in large biological or clinical samples, we developed a bisulfite conversion-specific one-label extension (BS-OLE) method using visualization by fluorescence polarization (FP) measurement of methylation at single CpG sites in small amounts of genomic DNA. Genomic DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil leaving 5-methylcytosine unaltered, and BS-PCR was used to generate DNA template containing target CpG sites. BS-OLE uses a BS-primer hybridized immediately upstream of the target CpG site being examined and then fluorescent dCTP or dUTP is incorporated into the methylated (CpG) or unmethylated (TpG) form of the target site through single-nucleotide chain extension, yielding an FP ratio between the fluorescent dCTP- and dUTP-incorporated products as a measure of methylation. This provides stable estimates of the methylation level of human genomic DNA and of a 250-bp plasmid DNA segment containing a single TCGA TaqI cleavage site, in accordance with the results of a combined bisulfite restriction analysis method. We used BS-OLE to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA methyltransferase inhibitor 5-aza-dC. BS-OLE is well suited to high-throughput multi-sample applications in biological and medical studies.

Intrinsic Fluorescence Polarization of Amniotic Fluid: II. Toward a Noninvasive Method for the Determination of Fetal Lung Maturity

The present study compares two methods for the determination of fetal lung maturity: the novel intrinsic fluorescence polarization ratio (IFPR) and the commercial TDx-FLMII. Amniotic fluid (AF) samples were collected from 69 women during the second and third trimesters of singleton pregnancies. Thirty-three samples were tested for IFPR only after centrifugation, and the rest were examined both before and after centrifugation. Of the latter 33 samples, 29 were assessed for lung maturity with the TDx-FLMII method as well. The results showed that IFPR values decreased with the advance in gestational age (r= 0.77, p < 0.05, n= 69). A significant correlation was found between IFPR of centrifuged and noncentrifuged samples (r= 0.94, p < 0.05, n= 36). A significant correlation was demonstrated between IFPR and TDx-FLMII values of centrifuged (r= 0.75, p < 0.05, n= 29) and noncentrifuged (r= 0.63, p < 0.05, n= 29) samples and moreover, samples considered mature by TDx-FLMII had low values of IFPR (n= 10). It can be concluded that the IFPR method can utilize noncentrifuged AF, thus suggested as a potential noninvasive method.

Intrinsic Fluorescence Polarization of Amniotic Fluid: II. Toward a Noninvasive Method for the Determination of Fetal Lung Maturity

The present study compares two methods for the determination of fetal lung maturity: the novel intrinsic fluorescence polarization ratio (IFPR) and the commercial TDx-FLMII. Amniotic fluid (AF) samples were collected from 69 women during the second and third trimesters of singleton pregnancies. Thirty-three samples were tested for IFPR only after centrifugation, and the rest were examined both before and after centrifugation. Of the latter 33 samples, 29 were assessed for lung maturity with the TDx-FLMII method as well. The results showed that IFPR values decreased with the advance in gestational age (r = 0.77, p < 0.05, n = 69). A significant correlation was found between IFPR of centrifuged and noncentrifuged samples (r = 0.94, p < 0.05, n = 36). A significant correlation was demonstrated between IFPR and TDx-FLMII values of centrifuged (r = 0.75, p < 0.05, n = 29) and noncentrifuged (r = 0.63, p < 0.05, n = 29) samples and moreover, samples considered mature by TDx-FLMII had low values of IFPR (n = 10). It can be concluded that the IFPR method can utilize noncentrifuged AF, thus suggested as a potential noninvasive method.

Intrinsic fluorescence polarization of amniotic fluid: evaluation of human fetal lung maturity.

This article reports a novel approach for the evaluation of fetal lung maturity based on fluorescence polarization (FP). The technique determines the intrinsic fluorescence polarization ratio (IFPR) of the amniotic fluid (AF). In vitro measurements of the IFPR indicate a clear dichotomy: high values for young pregnancies and low values for mature pregnancies. The new method has the potential to be a noninvasive procedure because the excitation of the AF and the collection of its fluorescence emission can be performed through the intact cervical amniotic membranes.

Fluorescence polarization of green fluorescence protein.

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60 degrees to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.

Fluorescence Polarization Transients from Rhodamine Isomers on the Myosin Regulatory Light Chain in Skeletal Muscle Fibers

Fluorescence polarization was used to examine orientation changes of two rhodamine probes bound to myosin heads in skeletal muscle fibers. Chicken gizzard myosin regulatory light chain (RLC) was labeled at Cys 108 with either the 5- or the 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLC (termed Cys 108-5 or Cys 108-6) was exchanged for the endogenous RLC in single, skinned fibers from rabbit psoas muscle. Three independent fluorescence polarization ratios were used to determine the static angular distribution of the probe dipoles with respect to the fiber axis and the extent of probe motions on the nanosecond time scale of the fluorescence lifetime. We used step changes in fiber length to partially synchronize the transitions between biochemical, structural, and mechanical states of the myosin cross-bridges. Releases during active contraction tilted the Cys 108-6 dipoles away from the fiber axis. This response saturated for releases beyond 3 nm/half-sarcomere (h.s.). Stretches in active contraction caused the dipoles to tilt toward the fiber axis, with no evidence of saturation for stretches up to 7 nm/h.s. These nonlinearities of the response to length changes are consistent with a partition of ∼90% of the probes that did not tilt when length changes were applied and 10% of the probes that tilted. The responding fraction tilted ∼30° for a 7.5 nm/h.s. release and traversed the plane perpendicular to the fiber axis for larger releases. Stretches in rigor tilted Cys 108-6 dipoles away from the fiber axis, which was the opposite of the response in active contraction. The transition from the rigor-type to the active-type response to stretch preceded the main force development when fibers were activated from rigor by photolysis of caged ATP in the presence of Ca 2+. Polarization ratios for Cys 108-6 in low ionic strength (20 mM) relaxing solution were compatible with a combination of the relaxed (200 mM ionic strength) and rigor intensities, but the response to length changes was of the active type. The nanosecond motions of the Cys 108-6 dipole were restricted to a cone of ∼20° half-angle, and those of Cys 108-5 dipole to a cone of ∼25° half-angle. These values changed little between relaxation, active contraction, and rigor. Cys 108-5 showed very small-amplitude tilting toward the fiber axis for both stretches and releases in active contraction, but much larger amplitude tilting in rigor. The marked differences in these responses to length steps between the two probe isomers and between active contraction and rigor suggest that the RLC undergoes a large angle change (∼60°) between these two states. This motion is likely to be a combination of tilting of the RLC relative to the fiber axis and twisting of the RLC about its own axis.

Orientation changes in myosin regulatory light chains following photorelease of ATP in skinned muscle fibers

The orientation of the light-chain region of myosin heads in muscle fibers was followed by polarized fluorescence from an extrinsic probe during tension transients elicited by photolysis of caged ATP. Regulatory light chain from chicken gizzard myosin was covalently modified with iodoacetamidotetramethylrhodamine and exchanged into skinned fibers from rabbit psoas muscle without significant effect of the tension transients. Fluorescence polarization ratios Q parallel = (parallel I parallel-perpendicular I parallel)/ (parallel I parallel+perpendicular I parallel) and Q perpendicular = perpendicular I perpendicular - parallel I perpendicular)/ (perpendicular I perpendicular + parallel I perpendicular), where mIn denote fluorescence intensities for excitation (pre-subscript) and emission (post-subscript) parallel or perpendicular to the fiber axis, were simultaneously measured at 0.5 ms time resolution. Q perpendicular decreased and Q parallel increased promptly after ATP release in the presence or absence of CA2+, indicating changes in orientation of the light-chain region associated with ATP binding or cross-bridge detachment. Little further change in the Q signals accompanied either active tension development (+Ca2+) or the final relaxation (-Ca2+). The Q and tension transients slowed when liberated ATP concentration was reduced. Assuming that ATP is released at 118 s-1 (20 degrees C), the apparent second-order rate constants were 3-10 x 10(5) M-1 s-1 for Q parallel, 1-5 x 10(5) M-1 s-1 for Q perpendicular, and 0.5-2 x 10(5) M-1 s-1 for the convergence of tension traces starting from different rigor values. Fitting of model orientation distributions to the Q signals indicated that the angular disorder increases after ATP binding. This orientation change is specific to ATP because photo release of ADP caused much smaller changes in the Q signals.

Steady-state polarization from cylindrically symmetric fluorophores undergoing rapid restricted motion

Steady-state fluorescence polarization measurements provide a relatively simple method for investigating the orientation of molecular components in ordered biological systems. However, the observed fluorescence polarization ratios also depend on any mobility of the fluorophores on the time scale of the fluorescence lifetime. Such mobility commonly arises from incomplete immobilization of a fluorescent probe on the macromolecule of interest. A theoretical formalism is presented for the interpretation of steady-state fluorescence polarization ratios in the presence of such rapid fluorophore motion. It is assumed that the fluorophores move freely within a cone between absorption and emission of a photon. Only one new parameter is introduced to describe fluorophore motion, the semi-angle of the cone, and this can be measured in separate experiments on an isotropic system. The fluorophore orientations are assumed to have cylindrical symmetry, but the symmetry axis need not be in the same plane as the center axes of the absorption and emission cones. This geometry applies to muscle and other biological fibers.

Organization and structure of actin filament bundles in Listeria-infected cells.

During its motion inside host cells, Listeria monocytogenes promotes the formation of a column of actin filaments that extends outward from the distal end of the moving bacterium. The column is constructed of short actin filaments that polymerize at the bacteria-column interface. To get a measure of filament organization in the column, Listeria grown in cultured PtK2 cells were studied with steady state fluorescence polarization, confocal microscopy, and whole cell intermediate voltage electron microscopy. Although actin filament ordering was higher in nearby stress fibers than in the Listeria-associated actin, four distinct areas of ordering could be observed in fluorescence polarization ratio images of bacteria: 1) the surface of the bacteria, 2) the cytoplasm next to the bacteria, 3) the outer shell of the actin column, and 4) the core of the column. Filaments were preferentially oriented parallel to the long axis of the column with highest ordering along the long axis of the bacterial surface and in the shell of the tail. The lowest ordering was in the core (where filaments are possibly also shorter with respect to the cup and the shell), whereas in the adjacent cytoplasm, filaments were oriented perpendicular to the column. A mutant of Listeria that can polymerize actin around itself but cannot move intracellularly does not have its actin organized along the bacterial surface. Thus the alignment of the actin filaments along the bacterial surfaces may be important for the intracellular movement. These conclusions are also supported by confocal microscopy and whole mount electron microscopic data that also reveal that actin filaments can be deposited asymmetrically around the long axis of the bacteria, a distribution that may affect the direction of motility of Listeria monocytogenes inside infected cells.

Lipid order in hepatocyte plasma membrane blebs during ATP depletion measured by digitized video fluorescence polarization microscopy

Low-light digitized video fluorescence polarization microscopy was used to measure lipid order parameters in plasma membrane blebs of single, cultured rat hepatocytes during ATP depletion with the metabolic inhibitors cyanide and iodoacetic acid. Hepatocytes were labeled on the microscope stage with the plasma membrane probe trimethylammoniumdiphenylhexatriene at successive stages of cell injury. A pair of fluorescence polarization ratio images was obtained from a series of four fluorescence images recorded with a polarizer in the emission path oriented first parallel and then perpendicular to each of two orthogonal excitation light polarization directions. From the polarization ratio images, the lipid order parameter S was determined in individual plasma membrane blebs. Results indicate that the plasma membrane becomes uniformly rigid within a few minutes of the addition of metabolic inhibitors when small surface blebs have formed and ATP levels have fallen by greater than 95%. The measured order parameter of S approximately 0.95 in plasma membrane blebs, compared with S approximately 0.75 in normoxic cell plasma membranes, remained unchanged throughout the course of bleb development and ultimate cell death. These findings demonstrate that significant alteration in hepatocyte plasma membrane structure occurs early in hypoxic cell injury.

Site-selective laser spectroscopy of deuterated SrF2:Er3+

Laser selective excitation and optical absorption of deuterated SrF2:0.05% Er3+ crystals have revealed eight Er3+ ion centers involving D− ion charge compensation, nine new Er3+–F− centers not associated with D− ions and several approximately cubic symmetry Er3+ centers. In contrast to the trigonal symmetry of the principal F− center, the dominant arrangements in the D− ion charge compensation case are derived from a tetragonal symmetry center. Detailed spectroscopic results for the new centers are presented. Fluorescence polarization ratios are listed for three centers ( J, B, and G1) present in either SrF2:Er3+ or CaF2:Er3+ and crystal-field analyses of these, based on assumed trigonal symmetry, are also reported.

Low-lying singlet states in the two-photon excitation spectrum of chrysene

The two-photon fluorescence excitation spectrum and polarization ratio of chrysene have been measured in the spectral region 370-230 nm. Totally symmetric electronic transitions have been observed which provide direct evidence of A g ππ * states in the near-UV. Comparison with MO-CI calculations offers strong support to our experimental assignment.

Electronic Spectroscopy of Mixed Cyclophanes: [2.2](9,10)Anthraceno(1,4)naphthalenophane

The absorption and fluorescence spectra of [2.21(9,10) anthraceno (1,4) naphthalenophane are reported. The anisotropy of the absorption bands was measured by two methods, ( i ) from fluorescence polarization ratios in a rigid glass and (ii) from single crystal absorption spectra at 23 K. The two states arising from the 1La states of the two chromophores were identified together with two states arising from other chromophore 1B2u states. Two states arising from the 1La states of the two chromophores were also identified. Whereas a coupled chromophore model accounts reasonably well for the latter two states, the energies and intensities of the bands arising from 1La chromophore states cannot be reconciled with this approach. Long axis polarized absorption intensity lying under the 1La, bands appears to be vibronically induced and not due to the 1Lb states. The absorption spectrum, fluorescence spectrum and fluorescence polarization ratios of a stable dimer were also observed. Its structure is similar to that of the stable dimer of anthracene in which the molecular long axes are parallel but the short axes make an angle of about 70� with each other.

Changes in chemical structure and function in Escherichia coli cell membranes caused by freeze-thawing. I. Change of lipid state in bilayer vesicles and in the original membrane fragments depending on rate of freezing

The effect of different rates of freezing on the character of lipids in unilamellar lipid bilayer vesicles and in the original membrane fragments of Escherichia coli B cells was investigated by measuring the temperature-dependent fluorescence polarization ratio changes of cis- and trans-parinaric acids. In lipid bilayer vesicles, both slow and rapid freezing brought about significant alterations in fluorescence polarization ratios in the specimens derived from both logarithmic and stationary-phase cells. In the original membrane fragments derived from logarithmic-phase cells, slow freezing gave rise to a similar alteration in fluorescence polarization ratio change, but no such alteration was found in the case of rapid freezing. Logarithmic-phase cells suffered from a membrane permeability change during slow freezing, which subsequently resulted in low cell viability. The cells suffered only slight impairment in membrane function during rapid freezing, and maintained higher viability. These results suggest that the primary site of damage due to freezing of the cells is the cellular membranes, and this destruction is due to a lipid state change in the membranes brought about by freezing.

The light-harvesting chlorophyll a/b protein acts as a torque aligning chloroplasts in a magnetic field

Displacement of particles from the purified light-harvesting chlorophyll a/b protein aggregate (LHC) was studied in magnetic fields of various strengths (0 to 1.6 T) by polarized fluorescence measurements. Macromolecular aggregates of LHC have a considerable magnetic susceptibility which enables the particles to rotate and align with their nematic axes parallel with H. As LHC is embedded in a transmembrane direction thylakoids should align perpendicular to H, the mode of alignment experimentally observed in thylakoids. The value of the magnetic susceptibility could be estimated by relating it to the integral susceptibility of the chlorophyll molecules in LHC. The fitting of this value with the field strength dependency of the fluorescence polarization ratio (FP) revealed a relationship between the LHC content of various photosynthetic membranes and their capacity for alignment, which suggested that LHC might be the torque ordering chloroplasts in a magnetic field.

Fluorescence polarization spectra of granal and stromal membranes treated with linolenic acid. Orientation of the Photosystem I core complex within the membrane

Polarized fluorescence spectra and fluorescence polarization ratios were compared in aligned isolated intact thylakoids and in granal and stromal membranes, without and after linolenic acid treatment at liquid N 2 temperature in squeezed polyacrylamide gel. Separation of granal membranes from stromal membranes allowed an improved alignment of the membranes as compared to isolated intact thylakoids. As a result, a higher anisotropy of fluorescence was measured with fragments than with chloroplasts. Incorporation of linolenic acid into the membranes affected the energy migration between the complexes, and induced changes in the orientation of the complexes within the membranes, as shown by a reduced fluorescence intensity and decreasing values (but still larger than 1) of fluorescence polarization ratios at longer wavelengths. In order to interpret these changes in the fluorescence polarization ratios, model calculations were carried out, the following parameters being taken into account: the direction of the absorption and emission dipoles in the complex, the orientation of the complex in the membrane, and the fluctuation of the orientation. Calculated values of the fluorescence polarization ratio changed in a similar manner as those observed experimentally. The character of the changes of the fluorescence polarization ratio suggests a picture of the orientation of the complexes within the membranes.

The effect of exogenous glycerophospholipids on the fluorescence polarization ratios of Escherichia coli cells labelled with diphenylhexatriene

Saturated, unsaturated, and short acyl chain analogues of phosphatidylcholine and phosphatidylcholine and phosphatidylethanolamine were incorporated into a deep heptoseless mutant of Escherichia coli, strain D21F2, and into the parent wild-type strain, K12. Normal and lipid-treated cells or lipid extracts from such cells were labelled with diphenylhexatriene and their fluorescence polarization ratios were measured as a function of temperature. Incorporations of dipalmitoyl analogues of phosphatidylethanolamine and/or phosphatidylcholine in the presence of Ca 2+ caused an increase in polarization ratios over a wide temperature range and the appearance of new phase transitions at 25–30°C as measured in whole D21F2 cells. Incorporation into D21F2 of the dioleoyl analogues of these glycerophospholipids under similar conditions had the opposite effect on the polarization ratios and, in the case of dioleoylphosphatidylethanolamine, caused the occurrence of a new phase transition at 20°C. Incorporation of these same lipids in K12 cells, in the presence of Ca 2+, caused changes in the polarization ratios similar to those recorded for D21F2 cells when measurements were made on whole cells. Furthermore incorporation of didecanoyl-phosphatidylcholine in wild-type cells, in the presence of Ca 2+, substantially decreased the polarization ratio and broadened the phase transition as could be measured with cell preparations. Since Ca 2+ stimulates incorporation of lipid, the changes in polarization ratio were always greater when cells had been exposed to exogenous lipid in the presence of this cation. However, even in cells not treated with lipid, Ca 2+ caused increases in the polarization ratio and affected the thermotropic structural transitions. The polarization ratios of extracted lipids were always reduced when compared to whole cells. Generally there was an attenuation of any differences in polarization ratio between normal and glycerophospholipid-treated samples. Extracted lipids also displayed broadened phase transitions. The results as a whole indicated that E. coli cells respond to the uptake of lipid and to the presence of Ca 2+ by changes in their thermotropic mesomorphic behaviour. These changes reflect to a large extent the fluidity of the incorporated lipid and are exerted on a structural system the phase transitions of which are strongly influenced by the presence of non lipid components in the membrane.

GENETIC VARIATIONS AND LINOLENIC ACID INDUCED CHANGES IN THE ORIENTATION PATTERN OF CHLOROPHYLL a IN THYLAKOID MEMBRANES

Abstract Orientation pattern of the Qy absorption and emission dipoles of chlorophyll a were studied in wild type Scenedesmus obliquus and in mutants deficient in chlorophyll b and carotenoids. Fluorescence polarization ratio at –140°C and linear dichroism at 25°C were measured in whole cells and thylakoids aligned in polyacrylamide gel. Unlike normal thylakoids, mutants displayed fluorescence polarization ratios significantly lower than 1.0 and showed a negative LD signal around 672 nm, indicating the tendency of the Qy dipoles to tilt out from the membrane plane. Such an orientation pattern can also be artificially induced by treating normal thylakoids with linolenic acid.