Fluorescence-detected Linear Dichroism Research Articles

Advantages of a synchrotron light source for fluorescence-detected linear dichroism.

Fluorescence-detected linear dichroism (FD-LD) enables one to collect linear dichroism spectra for oriented fluorophores in the presence of other absorbing species and light scattering. The experiment proceeds by scanning the excitation wavelength and using a filter to collect only emitted photons from the fluorophore. Thus, it has the potential to give data with enhanced selectivity and quality. By using a synchrotron radiation light source and fluorescence-detection, we show data for a range of fluorophores in different orienting environments. Film and flow-oriented FD-LD spectra were collected down to 170 nm. Even for flow-oriented liposomes, we have data collected down to 210 nm. For strongly scattering samples, for example, liposomes, FD-LD has the clear advantage that scattering is absent for the longer wavelength fluorescence photons. The collimated and smaller beam size of the synchrotron radiation also gives rise to sharper and more well-defined features in the spectra.

Couette flow fluorescence detected linear dichroism for analytes in lipid bilayers.

Membranes are important sites of intermolecular interactions in biological systems. However, they present significant analytical challenges as they contain multiple analytes and are dynamic in nature. In this work, we show how a Jasco J-1500 circular dichroism spectropolarimeter can be used with a microvolume Couette flow cell and appropriate cut-off filters to measure excitation fluorescence detected linear dichroism (FDLD) of fluorophores embedded in liposomal membranes. The result is a spectrum that selectively probes the fluorophore(s) and eliminates the scattering that is apparent in the corresponding flow linear dichroism (LD) spectrum. The FDLD spectrum is opposite in sign from the LD spectrum with relative magnitudes modified by the quantum yields of the transitions. FDLD thus enables analyte orientations to be identified in a membrane. Data for a membrane peptide, gramicidin, and two aromatic analytes, anthracene and pyrene, are presented. Issues with the "leakage" of photons by the long pass filters used is also discussed.

Structure and principles of self-assembly of giant “sea urchin” type sulfonatophenyl porphine aggregates

Principles of molecular self-assembly into giant hierarchical structures of hundreds of micrometers in size are studied in aggregates of meso-tetra(4-sulfonatophenyl)porphine (TPPS4). The aggregates form a central tubular core, which is covered with radially protruding filamentous non-branching aggregates. The filaments cluster and orient at varying angles from the core surface and some filaments form bundles. Due to shape resemblance, the structures are termed giant sea urchin (GSU) aggregates. Spectrally resolved fluorescence microscopy reveals J- and H-bands of TPPS4 aggregates in both the central core and the filaments. The fluorescence of the core is quenched while filaments exhibit strong fluorescence. Upon drying, the filament fluorescence gets quenched while the core is less affected, showing stronger relative fluorescence. Fluorescence-detected linear dichroism (FDLD) microscopy reveals that absorption dipoles corresponding to J-bands are oriented along the filament axis. The comparison of FDLD with scanning electron microscopy (SEM) reveals the structure of central core comprised of multilayer ribbons, which wind around the core axis forming a tube. Polarimetric second-harmonic generation (SHG) and third-harmonic generation microscopy exhibits strong signal from the filaments with nonlinear dipoles oriented close to the filament axis, while central core displays very low SHG due to close to centrosymmetric organization. Large chiral nonlinear susceptibility points to helical arrangement of the filaments. The investigation shows that TPPS4 molecules form distinct aggregate types, including chiral nanotubes and nanogranular aggregates that associate into the hierarchical GSU structure, prototypical to complex biological structures. The chiral TPPS4 aggregates can serve as harmonophores for nonlinear microscopy.

Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools

Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.

Molecular Orientation Determination in Nanodiscs at the Single-Molecule Level.

The function of membrane-bound proteins often depends on their interactions with the lipid bilayer. Bulk absorption-based linear dichroism has been historically used to investigate molecular orientations in the phospholipid bilayer but cannot resolve the actual distribution of molecules embedded in the membrane and is often limited by a poor signal-to-noise ratio. Here, we present single-molecule orientation determination by fluorescence-detected linear dichroism visualization in Nanodisc grids or SOLVING, to determine the molecular orientation of molecules assembled into nanoscale lipid bilayers. We provide a proof-of-concept by using SOLVING to quantitate the orientation distribution of two commonly used fluorescent dyes, DiO and BODIPY, in 10 nm Nanodiscs. Besides confirming the mean orientation determined by bulk absorption measurement, SOLVING provides the actual distribution of orientations and promises to provide key molecular insights into the topology and interactions of multiprotein complexes, such as those observed in intracellular signal transduction.

Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment

Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples—without interfering with their ‘conventional’ fluorescence or transmission imaging (Steinbach et al. in Methods Appl Fluoresc 2:015005, 2014). The resolution and the quality of fluorescence imaging have been enhanced in the recently constructed Re-scan confocal microscopy (RCM) (De Luca et al. in Biomed Opt Express 4:2644–2656, 2013). In this work, we developed the RCM technique further, by adding a DP-attachment modulating the exciting laser beam via a liquid crystal (LC) retarder synchronized with the data acquisition system; by this means, and with the aid of a software, fluorescence-detected linear dichroism (FDLD), characteristic of the anisotropic molecular organization of the sample, could be recorded in parallel with the confocal fluorescence imaging. For demonstration, we show FDLD images of a plant cell wall (Ginkgo biloba) stained with Etzold’s staining solution.

Fluorescence detected linear dichroism of small molecules oriented on polyethylene film.

We have previously reported a novel method for measuring the spectral properties and orientation of fluorescent probes in solution, which we denoted fluorescence-detected linear dichroism (FDLD). In this work, we expand the scope of the technique by recording the FDLD spectra of the small organic fluorophores: anthracene, 4',6-diamidino-2-phenylindole (DAPI), 1,6-diphenylhexatriene (DPH), and 4-methylphenyl benzoate (MPB), oriented on stretched oxidised polyethylene film. Much like what was observed in solution, we found that the FDLD produced a large signal enhancement compared with absorbance LD, which lowers the sample requirement for measurement. We also found that FDLD has an increased sensitivity towards the immediate environment of the chromophore, revealing oligomeric structures on the film. We believe that FDLD has the potential to reveal important properties of molecules that are obscured or unattainable when using other methods.

Fluorescence-Detected Linear Dichroism of Wood Cell Walls in Juvenile Serbian Spruce: Estimation of Compression Wood Severity.

Fluorescence-detected linear dichroism (FDLD) microscopy provides observation of structural order in a microscopic sample and its expression in numerical terms, enabling both quantitative and qualitative comparison among different samples. We applied FDLD microscopy to compare the distribution and alignment of cellulose fibrils in cell walls of compression wood (CW) and normal wood (NW) on stem cross-sections of juvenile Picea omorika trees. Our data indicate a decrease in cellulose fibril order in CW compared with NW. Radial and tangential walls differ considerably in both NW and CW. In radial walls, cellulose fibril order shows a gradual decrease from NW to severe CW, in line with the increase in CW severity. This indicates that FDLD analysis of cellulose fibril order in radial cell walls is a valuable method for estimation of CW severity.

Studies of T4 Primosome DNA Unwinding by Single-Molecule Fluorescence-Detected Linear Dichroism

The replication machine (replisome) of the T4 bacteriophage is an important model system for studies of DNA synthesis in all higher organisms. The helicase-primase (primosome) complex is one of three major sub-assemblies of the replisome, whose function is to processively unwind double-stranded DNA to expose single-stranded daughter strands for template-directed DNA synthesis. The detailed mechanism by which the primosome complex assembles and performs duplex DNA unwinding is not understood. Recently, we developed and applied single-molecule fluorescence-detected linear dichroism (smFLD), which can detect fluctuations of the transition dipole orientation on microsecond-to-millisecond time scales. We have studied model DNA replication fork constructs with cyanine fluorophores rigidly incorporated at the fork junction to monitor 'breathing' of the backbone as it interacts with the weakly binding T4 hexameric helicase. Our recent results [1] indicate that the time scale and distribution of microsecond breathing fluctuations at the replication fork junction are heavily influenced by the presence of the weakly binding T4 helicase, and suggest that these motions are on the pathway to the formation of the fully assembled DNA-primosome complex. The possible significance of these findings for helicase mechanisms will be discussed.[1] Phelps, C.; Lee, W.; Jose, D.; von Hippel, P. H.; Marcus, A. H. “Single-Molecule FRET and Linear Dichroism Studies of DNA ‘Breathing’ and Helicase Binding at Replication Fork Junctions,” Proc. Nat. Acad. Sci. (in press, accepted July 26, 2013, doi/10.1073/ pnas.1314862110).

Confocal Fluorescence Detected Linear Dichroism Imaging of Isolated Human Amyloid Fibrils. Role of Supercoiling

Amyloids are highly organized insoluble protein aggregates that are associated with a large variety of degenerative diseases. In this work, we investigated the anisotropic architecture of isolated human amyloid samples stained with Congo Red. This was performed by fluorescence detected linear dichroism (FDLD) imaging in a laser scanning confocal microscope that was equipped with a differential polarization attachment using high frequency modulation of the polarization state of the laser beam and a demodulation circuit. Two- and three-dimensional FDLD images of amyloids provided information on the orientation of the electric transition dipoles of the intercalated Congo Red molecules with unprecedented precision and spatial resolution. We show that, in accordance with linear dichroism imaging (Jin et al. Proc Natl Acad Sci USA 100:15294, 2003), amyloids exhibit strong anisotropy with preferential orientation of the dye molecules along the fibrils; estimations on the orientation angle, of around 45°, are given using a model calculation which takes into account the helical organization of the filaments and fibrils. Our data also show that FDLD images display large inhomogeneities, high local values with alternating signs and, in some regions, well identifiable µm-sized periodicities. These features of the anisotropic architecture are accounted for by supercoiling of helically organized amyloid fibrils.

Imaging anisotropy using differential polarization laser scanning confocal microscopy

We have constructed differential polarization (DP) attachments to a laser scanning microscope (LSM) for imaging the main DP quantities of anisotropic microscopic objects. The DP-LSM operates with high-frequency modulation and subsequent demodulation and displays the main DP quantities pixel by pixel. These, for linearly polarized light, include: (i) linear birefringence (LB), which is exhibited by structurally and/or optically anisotropic material; (ii) linear dichroism (LD), which carries information on the anisotropic distribution of the molecules, i.e. of their absorbance transition dipole vectors, in the sample; (iii) fluorescence-detected LD (FDLD), which carries the same information for fluorescent dyes upon excitations with two orthogonally polarized light beams; (iv) anisotropy of the fluorescence emission (r), excited with non-polarized light, which is determined by the distribution of the emission transition dipole vectors in the sample and is analogous with LD and (v) the degree of polarization of the fluorescence emission (P), excited with polarized light, which depends on the depolarization of the emission e.g. due to the rotation of molecules during their excitation lifetimes. In fluorescence regimes, the DP images can be recorded in the confocal regime of the microscope, which thus warrants good spatial resolution and the possibility of mapping the anisotropy in three dimensions. In this paper, we outline the design and technical realization of our DP-LSM and give a few examples on DP imaging of different biological samples.

From molecular interactions to tissue organization—Diversity of evolving cytometric approaches from a Hungarian perspective

From molecular interactions to tissue organization—Diversity of evolving cytometric approaches from a Hungarian perspective

Imaging fluorescence detected linear dichroism of plant cell walls in laser scanning confocal microscope

Anisotropy carries important information on the molecular organization of biological samples. Its determination requires a combination of microscopy and polarization spectroscopy tools. The authors constructed differential polarization (DP) attachments to a laser scanning microscope in order to determine physical quantities related to the anisotropic distribution of molecules in microscopic samples; here the authors focus on fluorescence-detected linear dichroism (FDLD). By modulating the linear polarization of the laser beam between two orthogonally polarized states and by using a demodulation circuit, the authors determine the associated transmitted and fluorescence intensity-difference signals, which serve the basis for LD (linear dichroism) and FDLD, respectively. The authors demonstrate on sections of Convallaria majalis root tissue stained with Acridin Orange that while (nonconfocal) LD images remain smeared and weak, FDLD images recorded in confocal mode reveal strong anisotropy of the cell wall. FDLD imaging is suitable for mapping the anisotropic distribution of transition dipoles in 3 dimensions. A mathematical model is proposed to account for the fiber-laminate ultrastructure of the cell wall and for the intercalation of the dye molecules in complex, highly anisotropic architecture.

Structural analysis of RecA protein–DNA complexes by fluorescence-detected linear dichroism: Absence of structural change of filament for pairing of complementary DNA strands

We have developed a simple measuring system for fluorescence-detected linear dichroism and applied it to the structural analysis of the RecA–DNA complex filaments, which are intermediates of the homologous recombination reaction. Taking advantage of the selectivity of fluorescence signals, we distinguished the linear dichroism signals of ethidium bromide and tryptophan residues in the RecA–DNA–ethidium bromide complex, whereas the conventional (absorption-detected) linear dichroism measurement provides only the sum of the signals because signals overlap each other and that of DNA. We further observed that the tryptophan residue at position 290 of RecA in the RecA–DNA–adenosine-5′- O-(3-thiotriphosphate) complex was oriented parallel to the long axis of the filament, in good agreement with the previous site-specific linear dichroism analysis, and that this orientation was not significantly modified by the pairing of the complementary DNA strand. These results suggest that the pairing reaction occurs without a large structural change of the RecA filament.

Total Internal Reflection Fluorescence and Electrocapillary Investigations of Adsorption at the Water−Dichloroethane Electrochemical Interface. 2. Fluorescence-Detected Linear Dichroism Investigation of Adsorption-Driven Reorientation of Di-N-butylaminonaphthylethenylpyridiniumpropylsulfonate

The potential-dependent adsorption and orientation of the zwitterionic amphiphile, di-N-butylaminonaphthylethenylpyridiniumpropylsulfonate, I, in the presence of dilauroylphosphatidylcholine (DLPC) at the H2O−1,2-dichloroethane (DCE) interface was investigated using a combination of steady-state fluorescence, fluorescence-detected linear dichroism, and electrocapillary measurements. From electrocapillary measurements, DLPC was found to dominate the interfacial composition at all potentials, when DLPC and I were present in bulk DCE and H2O at ca. 2 and 1 μM concentrations, respectively. At potentials Ew − Eo > 0.32 V, the affinity of DLPC for the interface is diminished, and I becomes a more effective competitor for interfacial sites. Over the potential range 0.32 V ≤ Ew − Eo ≤ 0.47 V, the total interfacial excess of species is reduced, but the ratio / is enhanced. The DC fluorescence signal increased in response to the enhanced interfacial population of the unprotonated monomeric I at positive potentials....

Function of spindle microtubules in directing cortical movement and actin filament organization in dividing cultured cells.

The mitotic spindle has long been recognized to play an essential role in determining the position of the cleavage furrow during cell division, however little is known about the mechanisms involved in this process. One attractive hypothesis is that signals from the spindle may function to induce reorganization of cortical structures and transport of actin filaments to the equator during cytokinesis. While an important idea, few experiments have directly tested this model. In the present study, we have used a variety of experimental approaches to identify microtubule-dependent effects on key cortical events during normal cell cleavage, including cortical flow, reorientation of actin filaments, and formation of the contractile apparatus. Single-particle tracking experiments showed that the microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following anaphase onset, while the microtubule stabilizing drug taxol causes profound changes in the overall pattern of receptor movements. These effects were accompanied by a related set of changes in the organization of the actin cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less ordered than in controls. Measurements with fluorescence-detected linear dichroism indicated a decrease in the alignment of filaments along the spindle axis. In contrast, actin filaments in taxol-treated cells showed an increased alignment along the equator on both the ventral and dorsal cortical surfaces, mirroring the redistribution pattern of surface receptors. Together, these experiments show that spindle microtubules are involved in directing bipolar flow of surface receptors and reorganization of actin filaments during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the contractile apparatus of dividing tissue culture cells.

Myosin S1 changes the orientation of caldesmon on actin.

Changes in the orientation of caldesmon bound to actin in skeletal ghost myofibrils caused by the binding of myosin subfragment 1 (S1) were measured by fluorescence-detected linear dichroism using fluorescence microscopy. Gizzard caldesmon, labeled with acrylodan at its two Cys residues (CaD*), bound to actin with a probe angle that was unaffected by actin-bound tropomyosin. Irrigation of fibrils with myosin S1 dissociated most of the bound CaD*, but reintroduction of CaD* resulted in its rebinding to actin, without dissociation of S1, with a 7 degrees difference in probe angle. A similar change in probe angle was also observed when a 27-kDa actin-binding C-terminal fragment of caldesmon, labeled with acrylodan at its single Cys 580 (CaD-27*), was used. Introducing MgADP, which bound to S1 in the CaD*-actin-S1 ternary complex in the fibril, reversed the bound CaD* dichroism. These results indicate that (i) myosin heads and caldesmon compete for a common actin binding site; (ii) a ternary complex of CaD*-actin-S1 can be formed with an orientation of CaD* different from that in the CaD*-actin binary complex, and (iii) MgADP, which binds to and reorients myosin S1, affects the orientation of CaD* in the ternary complex. These results are consistent with a two-state binding model of caldesmon for actin in which state 1 involves a site that is competitive with S1 binding and state 2 involves a site that is formed in the presence of bound S1.

DNA orientation during electrophoresis in a viscoelastic solution

The objective of the present study was to examine the possibility of using a viscoelastic (free) solution to separate DNA molecules. The electrophoretic orientation of λ DNA in an uncross-linked polyacrylamide solution was measured by a fluorescence-detected linear dichroism of bound ethidium bromide. The λ DNA molecules oriented almost parallel to the applied electric field direction during electrophoresis in a relatively low concentration of the polyacrylamide solution for a moderate electric field strength of 90 V/cm, and the extent of orientation increased with the field strength. The analogy between the DNA molecule and a thin cylindrical body in the polyacrylamide solution suggests that when one perfects the separation device, very large DNA molecules can be separated using a viscoelastic solution.

Orientation and three-dimensional organization of actin filaments in dividing cultured cells.

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.

Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils

Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.