Polarized Fluorescence Emission Research Articles

Multifunctional Anthracene-Based Ni-MOF with Encapsulated Fullerenes: Polarized Fluorescence Emission and Selective Separation of C70 from C60.

We report an anthracene-based Ni-MOF [Ni(II) metal-organic framework, {[Ni(μ2-L)2Cl2]·x(C6H6)·y(MeOH)}n (1), L = anthracene-9,10-diylbis(methylene)diisonicotinate] whose crystal structure reveals the presence of hexagonal channels with a pore size of 1.4 nm that can accommodate guests such as C60 and C70. Both confocal fluorescence and Raman microscopy results are in agreement with a homogeneous distribution of fullerenes throughout the single crystals of 1. Efficient energy transfer from 1 to the fullerenes was observed, which emitted partially polarized fluorescence emission. Stronger binding between 1 and C70 versus C60 was confirmed from HPLC analysis of the dissolved material and provides a basis for the selective retention of C70 in liquid chromatography columns packed with 1.

One Nanometer Wide Functional Patterns with a Sub-10 Nanometer Pitch Transferred to an Amorphous Elastomeric Material

Decades of work in surface science have established the ability to functionalize clean inorganic surfaces with sub-nm precision, but for many applications, it would be useful to provide similar control over the surface chemistry of amorphous materials such as elastomers. Here, we show that striped monolayers of diyne amphiphiles, assembled on graphite and photopolymerized, can be covalently transferred to polydimethylsiloxane (PDMS), an elastomer common in applications including microfluidics, soft robotics, wearable electronics, and cell culture. This process creates precision polymer films <1 nm thick, with 1 nm wide functional patterns, which control interfacial wetting and reactivity, and template adsorption of flexible, ultranarrow Au nanowires. The polydiacetylenes exhibit polarized fluorescence emission, revealing polymer location, orientation, and environment, and resist engulfment, a common problem in PDMS functionalization. These findings illustrate a route for patterning surface chemistry below the length scale of heterogeneity in an amorphous material.

Methods toward simplification of time resolved fluorescence anisotropy in proteins labeled with NBD (4-chloro-7-nitrobenzofurazan) adducts

The analysis of time resolved fluorescence anisotropy for NBD tagged proteins is difficult when multiple exponential components arise from heterogeneous amino acid fluorescent adducts. Two approaches were taken toward simplification. First, N terminal selective labeling of tear lipocalin with NBD-Cl was attempted at pH 7.0. While lysines were predominantly labeled at pH 8.0, selective N terminal labeling was attained at neutral pH. Second, fluorescence anisotropic decay analysis was simplified to recover only the rotational correlation time of the protein not the side chain. The boundaries for analysis of anisotropic decays were limited to the longer lifetimes. A modified tail fit enabled fitting the anisotropic decay to a single exponential. The correlation time for tear lipocalin matched published values. Additionally, a method for normalization of acquisition times of vertically (VV) and horizontally (VH) polarized fluorescence emission decays is presented for time-resolved anisotropy. Here it is applied to Picoharp software (Picoquant, Berlin). Picoharp software is programmed with an automatic stop at unequal acquisition times if the fluorescent counts exceeds a default. The method adjusts the intensity decays to the same acquisition time and is applicable to all time-resolved anisotropic decay data collected with time-correlated photon counting.•NBD labeling at pH 7.0 was not selective for N terminus of LCN1.•Constraints for range simplifies fittings of anisotropic decays.•Different acquisition times for decays can be normalized to facilitate fitting in data obtained by Picoharp.

8.1: Invited Paper: Alignment of Quantum Rods

Electrospinning and a fine‐pitch groove structure are used to align semiconductor quantum rods (QRs). Embedding the QRs in an electrospun polymer nanofiber sheet yields polarized fluorescence emission. The fine‐pitch groove structure results in QRs aligned parallel to the grooves.

Polarized fluorescent emission from aligned electrospun nanofiber sheets containing semiconductor nanorods

We fabricated sheets consisting of aligned electrospun polymer nanofibers embedded with semiconductor nanorods (NRs) and confirmed the production of polarized fluorescence emission. Electrospun nanofibers with embedded semiconductor NRs were rolled around a drum to form a sheet in which each nanofiber was aligned parallel to the rotation direction. The thickness and polarization ratio of the sheet were 1.5 μm and 0.6, respectively. We also examined how electrospinning parameters affected the polarization ratio of the sheet.

Influence of the glass transition on rotational dynamics of dyes in thin polymer films: single-molecule and ensemble experiments.

We performed polarized fluorescence emission studies of Nile Red (NR) in poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate) (PEMA), and poly(butyl methacrylate) (PBMA) at the single molecule (SM) and at the ensemble level to study the in cage movements of the ground-state molecule in polymer films of nanometric thickness at room temperature. Experiments were performed with wide field irradiation. At the ensemble level, the linearly polarized irradiation was used to induce a photoselection by bleaching, which is compensated by rotational diffusion. Both results show an appreciable difference in mobility of NR in the films that is correlated with the different glass-transition temperatures of the films, particularly in PEMA, which displays a clearly distinct behavior between the 200 nm films, representing a rigid environment, and the 25 nm ones, showing much higher mobility. We developed a model of broad application for polarized photobleaching that allows obtaining rotational diffusion coefficients and photobleaching quantum yields in an easy way from ensemble experiments. The parameters obtained from ensemble measurements correlate well with the results from SM experiments.

Photophysics, RET Probes and Imaging Applications for Fluorescent BSA Au Nanoclusters

Protein protected fluorescent metal nanoclusters are attracting great deal of attention from fluorescence community. This work explores photo-physics, fluorescence polarization properties; resonance energy transfer (RET) probes, and cellular imaging applications of novel fluorescent probe bovine serum albumin protected gold (BSA Au) nanoclusters. Important findings were long fluorescence lifetime (> 1µs) and polarized fluorescence emission studied in solvents of different viscosity. The BSA Au cluster, due to long lifetime and high polarization, can potentially be used in developing fluorescence polarization immunoassays. Moreover, we describe an approach by developing RET of BSA Au clusters with the near infra-red (NIR) dye Dylight 750 (Dy750), where BSA Au cluster act as a donor to Dy750, showing 46% energy transfer efficiency to the NIR dye Dy750 with long lifetime component in acceptor decay through RET. Such RET probe with narrow emission spectrum and long lifetime component can be explored in microscopy imaging applications. Furthermore, we describe the use of these long lived BSA Au clusters in cellular and tissue imaging applications. We have shown using multiple pulse excitation (MPE) scheme that one can increase the intensity of long lived BSA Au cluster in cells without increasing the short lived auto-fluorescence background thereby increasing signal to background ratio at least by 15 times. Moreover, by applying time gated detection strategy to MPE imaging experiment we increased the signal to noise ratio to 30, a dramatic improvement in contrast for low fluorescence quantum yield dye. In summary, BSA Au clusters can be used as a fluorescent cellular stain advancing the bioimaging technology via their long fluorescence lifetime.

Controlling the Emission Polarization from Single Crystals Using Light: Towards Photopolic Materials

Turning light: A crystalline fullerene material based on H-bonding interactions exhibits highly polarized fluorescence emission, whose angle of polarization can be turned using light (see picture). This process can be used to write optical information onto the material, which can be read through the polarization component of the luminescence emitted by the material.

Sensing ligand binding to a clinically relevant lectin by tryptophan fluorescence anisotropy

Increasing insights into the involvement of endogenous lectins in disease processes fuel the interest to develop potent inhibitors. As a consequence, robust assay procedures are required. Due to their activity as adhesion/growth-regulatory effectors this study focussed on galectins. The human proto-type galectin-1 was selected as representative of this family with conserved presence of a tryptophan moiety in the binding site. This structural feature was taken advantage of to establish its use as reporter for ligand contact measuring polarized fluorescence emission. The experimentally determined anisotropy r(0) was about 0.2, altered by about 5% in the presence of the cognate disaccharide lactose. This parameter change enabled calculating the equilibrium binding constant and kinetic rate constants. The detailed analysis of the depolarization process further indicated fast conformational dynamics within the binding site. Since an inherent property of the protein was exploited, no labeling is needed. Owing to tryptophan's presence in carbohydrate-binding sites, also in other classes of lectins as well as in carbohydrate-binding modules and glycoenzymes (glycosyltransferases, glycosidases), this assay procedure can have relevance beyond galectins.

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.

Highly directional fluorescence emission from dye molecules embedded in a dielectric layer adjacent to a silver film

We report measurements on the angular radiation patterns from dye molecules embedded in a polymethyl methacrylate thin film spin-coated on a thin silver film. We systematically studied the influence of the thickness of the silver films and the thickness of the dielectric layers on the radiation pattern. We present the detailed radiation patterns over a large angular range showing highly polarized fluorescence emission coupled into the surface plasmon modes or waveguide modes. We also studied the influence of the polarization of the excitation beam on the radiation patterns. The experimental data are compared with numerical simulations using an asymptotic approach based on the Lorentz reciprocity theorem.

Fluorescent leukotriene B4: potential applications

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Surface-Mediated Photoalignment of Discotic Liquid Crystals on Azobenzene Polymer Films

This paper describes a simple strategy for the formation of photoaligned and micropatterned discotic liquid crystal (DLC) film on the surface of photoirradiated azobenzene-containing polymer thin film. The key material for the surface-mediated photoalignment of the DLCs was poly[4-(4-cyanophenylazo)phenyl methacrylate] (pMAzCN). Optical anisotropy was generated in a pMAzCN film by oblique exposure to nonpolarized light which resulted in angle-selective photoisomerization and reorientation of the azobenzenes. Subsequent annealing of the film at 240 degrees C enhanced the photoaligned state of the p-cyanoazobenzenes due to strong intermolecular dipole-dipole interaction and semicrystalline nature of the pMAzCN. This combination of photoirradiation and subsequent annealing of the pMAzCN film made it possible to realize the surface-assisted orientation control of a DLC molecule, which displays both columnar (Col) and discotic nematic (N(D)) phases over 152 degrees C. When the pMAzCN film was exposed to linearly polarized light from the surface normal, the DLC molecules showed homeotropic orientation with the director perpendicular to the substrate surface. In the contrast, oblique irradiation of the pMAzCN film with nonpolarized light gave rise to tilted DLC orientation with well-ordered optical birefringence at the N(D) phase. Rapid cooling from the N(D) phase produced a well-aligned glassy N(D) state at room temperature, which was adequately stable for 10 months even though no covalent cross-linking among the DLCs was performed. The spatial orientation of photoaligned DLCs in both their bulk film and in their interface region was characterized by means of optical birefringence, X-ray diffraction, and fluorescence measurements. At the N(D) phase, the DLC molecules were aligned in a hybrid manner such that their tilt angles varied throughout the thickness of DLC film. The direction of tilted DLCs was opposite to the propagation of the actinic nonpolarized light. The photoaligned DLC films exhibited polarized fluorescence emission with an s-polarized/p-polarized intensity ratio of 4.1, despite the nonpolarized excitation of only DLC at outmost surface. These results indicate that the three-dimensionally aligned azobenzene moieties of the pMAzCN thin film were transferred to the tilted DLC molecules at air/DLC interface. Finally, we demonstrated micrometer-scale photopatterned orientation of DLC molecules on the pMAzCN surface by oblique nonpolarized irradiation of the film through a photomask.

Polarization Measurements on Single Pigment-Protein Complexes

Individual antenna complexes from different photosynthetic units have been investigated by single molecule spectroscopy. In such energy transfer systems the polarization of the fluorescence emission gives valuable information about the nature of the emitting state, which is not readily available with other methods like fluorescence excitation or emission spectroscopy. The peripheral antenna light harvesting complex from purple bacterium Rhodopseudomonas Acidophila shows predominantly linear polarized fluorescence emission at low temperature, whereas at room temperature the fluorescence is randomly polarized. This is attributed to the fact, that at low temperature in the fluorescence emitting state the excitation energy is localized mainly on 4-5 chromophores. Analysis of the fluorescence emission of single peripheral antenna complexes of green plants indicate that for trimers of this species more than one Chlorophyll is responsible for the final fluorescence emission, which points towards a weak intermonomer coupling in the complex.

Manganese deficiency alters high-density lipoprotein subclass structure in the sprague-dawley rat

Dietary manganese affects lipid and lipoprotein metabolism. In the Sprague-Dawley rat, manganese deficiency affects high-density lipoprotein composition and may alter lipoprotein structural stability. This study assessed the effects of manganese on high-density lipoprotein subclass structure, dynamics, and mobility, using fluorescence quenching and fluorescence polarization techniques. Male Sprague-Dawley rats were fed diets deficient or adequate in manganese. High-density lipoprotein 1 (HDL 1, density 1.05 to 1.077 kilograms/liter) and high-density lipoprotein 2 (HDL 2, density 1.077 to 1.21 kilograms/liter) were separated by density gradient ultracentrifugation. Iodide quenching of intrinsic (tryptophan) fluorescence showed that there were at least two types of fluorophores in both lipoprotein subclasses. Differences in dynamics and accessibility were noted in HDL 1 (deficient, K sv = 16.9 (mol/L) −1, f a = 20%; adequate, K sv = 9.8 (mol/L) −1, f a = 27%) and in HDL 2 (deficient, K sv = 0.89 (mol/L) −1, f a = 100%; adequate, K sv= 4.71 (mol/L) −1, f a = 36%). Differences in polarized fluorescence emission were seen in HDL 2 but not in HDL 1 (deficient, P = 0.193; adequate, P = 0.213). There were no differences in dynamics, accessibility, or polarized fluorescence in apolipoproteins from either subclass. The data suggest structural changes in HDL 1 and changes in the rotational motion of the proteins in HDL 2. These changes may affect HDL metabolism in the rat.

Polarized fluorescence emission measurements in mixtures of 2-butoxyethanol, cetyltrimethylammonium bromide, and water

Polarized fluorescence emission and fluorescence lifetime measurements are reported for two fluorescent probes, diphenylbutadiene (DPB) and 2-(p-toluidino) naphthalene-6-sulfonate, potassium salt (2,6-TNS), dissolved in binary water-surfactant systems for several different surfactants. As well, data for the probe DPB in ternary solutions of 2-butoxyethanol (BE)-cetyltrimethylammonium bromide (CTAB)-water are presented

Orientation of the pigments in Photosystem II: A low-temperature linear dichroism and polarized fluorescence emission study of chlorophyll-protein complexes isolated from Chlamydomonas reinhardtii

The orientation of the various absorbing and fluorescing dipoles in Photosystem II have been investigated by linearly polarized light spectroscopy at 5 K, performed on macroscopically oriented PS II complexes derived from Chlamydomonas reinhardtii. Linear dichroism and absorption spectra show that the Q Y transitions of the chlorophyll molecules are mostly tilted at less than 35° from the plane of largest cross-section of the particle (which in vivo coincides with the plane of the thylakoid membrane). The chlorophyll forms absorbing at 676 and 683 nm are oriented closer to the membrane than the forms absorbing at 665 and 670 nm which are tilted at approximately 35° from the plane. A dip observed around 680 nm in the LD/absorption spectra indicates a component tilted at a larger angle away from the membrane plane than the 676 nm- and 683 nm-absorbing species. A component weakly absorbing around 693 nm and exhibiting a negative LD (tilt larger than 35°) is clearly resolved. The amplitude of the LD at 693 nm relative to that observed at the maximum (676 nm) varies from sample to sample. In the blue spectral region, two populations of carotenoids are observed; one absorbs around 460 and 490 nm, while the other absorbs around 510 nm. They are oriented out of and near to the thylakoid plane, respectively. Comparison of polarized absorption and fluorescence spectra from the same oriented samples allows the assignment of the 695 nm fluorescence emission to the dipoles responsible for the LD signal at 693 nm.

Orientation of photosystem-I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission

Using absorption and fluorescence experiments at low temperature with polarized light on oriented samples, the orientation of PS-I-related pigments, both in green plants and in Chlamydomonas reinhardtii, has been investigated on isolated pigment-protein complexes and intact thylakoids. The following observations have been made. (i) The isolation procedure of PS I 110, PS I 65, LHC I and CP0) particles from pea and C. reinhardtii do not alter significantly the intrinsic orientation of the pigments inside the complexes; (ii) Chl b is a structural component of PS I, linked to the peripheral antenna, with an orientation with respect to the thylakoid plane different from that observed in the main light-harvesting complex (iii) PS I 65 (i.e., ‘core’ PS I) of pea and C. reinhardtii contains identical chromophores having the same orientation with respect to the geometrical longest axis (axes) of the complexes. (iv) LHC I and CP0 (i.e., PS I ‘peripheral antenna’) of pea and C. reinhardtii have identical oriented chromophores, except that a long-wavelength component with a high anisotropy is only present in green plants. This set of pigments, which absorbs at 705–725 nm, has the same orientation as the dipoles emitting F 735 and also as the Q Y transition of P-700. (v) All the long-wavelength fluorescence properties of the various studied membranes are explained by these data on isolated PS I complexes: wild-type C. reinhardtii and Chl- b-less barely fluoresce from the core pigments, while a CP1 deficient mutant of C. reinhardtii and wild-type barley fluoresce from the antenna pigments.

Effect of rotational relaxation on the intensity and polarization of fluorescence emission caused by sequential two-photo excitation

Abstract General formulas are derived for the intensity and the degree of polarization of the S m -fluorescence emission ( m ⩾ 2) of a sample excited by the sequential two-photon excitation process (S n ← S 1 ← S 0 , n ⩾ 2) with plane-polarized pulsed light. They show how the S m -fluorescence intensity and anisotropy depend on the relative orientation of the relevant transition dipoles within a molecule and on the degree of rotational relaxation of molecules in the intermediate state (S 1 ) and in the S m state (for the case m = n ), or in the S n → S m process ( m ≠ n ).

Analysis of linearly polarized fluorescence of chloroplasts oriented in polyacrylamide gel

Linearly polarized fluorescence was measured in mesophyll chloroplasts of maize aligned in polyacrylamide gel. The dependence of fluorescence polarization on the squeezing parameter of the gel sample was studied at room temperature. Polarized fluorescence emission and polarization ratio spectra were recorded at low temperature (− 140°C). A theoretical model is proposed for a quantitative description of the polarized fluorescence emission and its dependence on the squeezing parameter. It is shown that chloroplasts are aligned as disc-shaped particles with no detectable deformation of their ultrastructure. The fluorescence emission and the polarization ratio spectra as well as the calculated orientation angles between the transition dipoles and the membrane plane were similar to those obtained with magnetically oriented chloroplasts.