Fluorescence Polarization Microscopy Research Articles

Pulling Forces Dampen Torsional Fluctuations of Actin Filaments and Reduce Cooperative Cofilin Binding.

A variety of potential biological roles of mechanical forces have been proposed in the field of cell biology. In particular, mechanical forces alter the mechanical conditions within cells and their environment, exerting a strong effect on the reorganization of the actin cytoskeleton. Single-molecule imaging studies have provided evidence that an actin filament may act as a mechanosensor. An increase in the tension within actin filaments causes changes in their conformation and affinity to their regulatory proteins. However, our current understanding of the molecular mechanisms of the tension sensing and the affinity change of regulatory proteins is still incomplete. In this study, we employed fluorescence polarization microscopy and magnetic tweezers to directly quantify the torsional fluctuations of single actin filaments and cofilin binding to the filament under several distinct mechanical conditions. When an actin filament was severed by scratching the filament with a pipette tip, the amplitude of twisting/torsional fluctuations and the rate of cooperative cofilin binding increased. On the other hand, when a piconewton force was applied to single actin filaments by magnetic tweezers, the amplitude of twisting fluctuations and the rate of cooperative cofilin binding decreased. This may be involved in the molecular mechanism behind the mechanical force-dependent severing of actin filaments in cells.

Boundaries and cross-linking densities modulate domain sizes of polydomain nematic elastomers.

When nematic liquid crystal elastomers (LCEs) crosslinked at their isotropic phase are quenched to the nematic phase, they show polydomain patterns, in which nematic microdomains with different orientations self-organize into a three-dimensional mosaic with characteristic correlation patterns. The orientational correlation length of the domain, which is usually in the micrometer range, is believed to emerge as a result of a competition between liquid crystalline ordering and frozen network inhomogeneity. Although polydomain patterns show potentials as the basic platform for optical, memory, and mechanical devices, no study exists regarding how they are modulated by experimentally accessible parameters. Here, using confocal polarized fluorescence microscopy, we study the effects of a solid-wall or open boundary on the domain size in conjunction with effects of cross-linking density. The LCE bounded by solid glass shows reduced domain size near the boundary. In contrast, increased domain size appears at the free surface. With increasing cross-linking density, the domain size decreases, also exhibiting the boundary effects. Guided by theoretical considerations, the results are explained by a picture that the effective strength of the inhomogeneity frozen in the polymer network, i.e., the effective disorder strength, varies depending on the cross-linking density and constrained states at boundaries. The results offer the first experimental approach to global and local modulation of the polydomain pattern in nematic LCEs.

Aptamer's Structure Optimization for Better Diagnosis and Treatment of Glial Tumors.

Background: Oncological diseases are a major focus in medicine, with millions diagnosed each year, leading researchers to seek new diagnostic and treatment methods. One promising avenue is the development of targeted therapies and rapid diagnostic tests using recognition molecules. The pharmaceutical industry is increasingly exploring nucleic acid-based therapeutics. However, producing long oligonucleotides, especially aptamers, poses significant production challenges. Objectives: This study aims to demonstrate the efficacy of using molecular modeling, supported by experimental procedures, for altering aptamer nucleotide sequences while maintaining their binding capabilities. The focus is on reducing production costs and enhancing binding dynamics by removing nonfunctional regions and minimizing nonspecific binding. Methods: A molecular modeling approach was employed to elucidate the structure of a DNA aptamer, Gli-55, facilitating the truncation of nonessential regions in the Gli-55 aptamer, which selectively binds to glioblastoma (GBM). This process aimed to produce a truncated aptamer, Gli-35, capable of forming similar structural elements to the original sequence with reduced nonspecific binding. The efficiency of the truncation was proved by flow cytometry, fluorescence polarization (FP), and confocal microscopy. Results: The molecular design indicated that the new truncated Gli-35 aptamer retained the structural integrity of Gli-55. In vitro studies showed that Gli-35 had a binding affinity comparable to the initial long aptamer while the selectivity increased. Gli-35 internalized inside the cell faster than Gli-55 and crossed the blood-brain barrier (BBB), as demonstrated in an in vitro model. Conclusions: The success of this truncation approach suggests its potential applicability in scenarios where molecular target information is limited. The study highlights a strategic and resource-efficient methodology for aptamer development. By employing molecular modeling and truncation, researchers can reduce production costs and avoid trial and error in sequence selection. This approach is promising for enhancing the efficiency of therapeutic agent development, particularly in cases lacking detailed molecular target insights.

Illuminating cellular architecture and dynamics with fluorescence polarization microscopy.

Ever since Robert Hooke's 17th century discovery of the cell using a humble compound microscope, light-matter interactions have continuously redefined our understanding of cell biology. Fluorescence microscopy has been particularly transformative and remains an indispensable tool for many cell biologists. The subcellular localization of biomolecules is now routinely visualized simply by manipulating the wavelength of light. Fluorescence polarization microscopy (FPM) extends these capabilities by exploiting another optical property - polarization - allowing researchers to measure not only the location of molecules, but also their organization or alignment within larger cellular structures. With only minor modifications to an existing fluorescence microscope, FPM can reveal the nanoscale architecture, orientational dynamics, conformational changes and interactions of fluorescently labeled molecules in their native cellular environments. Importantly, FPM excels at imaging systems that are challenging to study through traditional structural approaches, such as membranes, membrane proteins, cytoskeletal networks and large macromolecular complexes. In this Review, we discuss key discoveries enabled by FPM, compare and contrast the most common optical setups for FPM, and provide a theoretical and practical framework for researchers to apply this technique to their own research questions.

Formation of subwavelength tunable flat-top focus with double foci characteristic by tightly focused cylindrical vector beams

We report a numerical investigation on the generation of a subwavelength flat-top focus by tuning the polarization angle of the incident beam under tight focusing conditions. Our approach uses a binary phase mask containing two regions of opposite phases, with a variable diameter of the inner region. Properly tuning the diameter of the inner region of the mask, the flat-top profile of variable sizes (above and below the subwavelength region) is generated in a controlled manner with the various incident polarization angles of the beam and with the different numerical aperture (NA) value of the lens. Our study reveals that a particular cut-off value of polarization angle, above which the subwavelength flat-top is generated, corresponds to a specific NA that increases with the increase in the NA value. In addition, the emergence of the dual foci structure is observed in the longitudinal direction with increasing NA of the lens. The flat-top's polarization degrees-of-freedom and dual foci properties open up promising routes for potential applications in fluorescence polarization microscopy and multiplane particle trapping.

OOPS: Object-Oriented Polarization Software for analysis of fluorescence polarization microscopy images.

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.

Three-dimensional dipole orientation mapping with high temporal-spatial resolution using polarization modulation

Fluorescence polarization microscopy is widely used in biology for molecular orientation properties. However, due to the limited temporal resolution of single-molecule orientation localization microscopy and the limited orientation dimension of polarization modulation techniques, achieving simultaneous high temporal-spatial resolution mapping of the three-dimensional (3D) orientation of fluorescent dipoles remains an outstanding problem. Here, we present a super-resolution 3D orientation mapping (3DOM) microscope that resolves 3D orientation by extracting phase information of the six polarization modulation components in reciprocal space. 3DOM achieves an azimuthal precision of 2° and a polar precision of 3° with spatial resolution of up to 128 nm in the experiments. We validate that 3DOM not only reveals the heterogeneity of the milk fat globule membrane, but also elucidates the 3D structure of biological filaments, including the 3D spatial conformation of λ-DNA and the structural disorder of actin filaments. Furthermore, 3DOM images the dipole dynamics of microtubules labeled with green fluorescent protein in live U2OS cells, reporting dynamic 3D orientation variations. Given its easy integration into existing wide-field microscopes, we expect the 3DOM microscope to provide a multi-view versatile strategy for investigating molecular structure and dynamics in biological macromolecules across multiple spatial and temporal scales.

Dsg2 ectodomain organization increases throughout desmosome assembly

ABSTRACT Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Polarized Fluorescence Microscopy and Recent Progress (Invited)

Polarized Fluorescence Microscopy and Recent Progress (Invited)

Mechanical Stress Decreases the Amplitude of Twisting and Bending Fluctuations of Actin Filaments

A variety of biological roles of mechanical forces have been proposed in cell biology, such as cell signaling pathways for survival, development, growth, and differentiation. Mechanical forces alter the mechanical conditions within cells and their environment, which strongly influences the reorganization of the actin cytoskeleton. Single-molecule imaging studies of actin filaments have led to the hypothesis that the actin filament acts as a mechanosensor; e.g., increases in actin filament tension alter their conformation and affinity for regulatory proteins. However, our understanding of the molecular mechanisms underlying how tension modulates the mechanical behavior of a single actin filament is still incomplete. In this study, a direct measurement of the twisting and bending of a fluorescently labeled single actin filament under different tension levels by force application (0.8–3.4 pN) was performed using single-molecule fluorescence polarization (SMFP) microscopy. The results showed that the amplitude of twisting and bending fluctuations of a single actin filament decreased with increasing tension. Electron micrograph analysis of tensed filaments also revealed that the fluctuations in the crossover length of actin filaments decreased with increasing filament tension. Possible molecular mechanisms underlying these results involving the binding of actin-binding proteins, such as cofilin, to the filament are discussed.

Molecular orientation and optimization of membrane dyes based on conjugated oligoelectrolytes

Conjugated oligoelectrolytes (COEs) are amphiphilic, fluorogenic molecules that spontaneously associate with lipid bilayer membranes and are gaining attention as molecular reporters, particularly for exosome detection by flow cytometry. Questions nonetheless remain on how to best design COEs for optimal performance and on the geometry of lipid bilayer intercalation. In response, we designed a series of oligo-phenylenevinylene COEs with varying lengths and numbers of charged groups to address these uncertainties. Examination of the organization within lipid bilayers through polarized fluorescence microscopy shows that the optical transition moments are perpendicular to the bilayer plane, with the conjugated segment flanked by hydrophobic phospholipid tails. COEs initially form a disorganized layer on the vesicle periphery, reflecting electrostatic association before intercalation. Uptake experiments show that longer dimensions and increased numbers of charges allow for a higher degree of cellular association. Both shorter core length and increased number of charges accelerate the rate needed to achieve emission saturation.

Molecular Mechanisms of Deregulation of Muscle Contractility Caused by the R168H Mutation in TPM3 and Its Attenuation by Therapeutic Agents

The substitution for Arg168His (R168H) in γ-tropomyosin (TPM3 gene, Tpm3.12 isoform) is associated with congenital muscle fiber type disproportion (CFTD) and muscle weakness. It is still unclear what molecular mechanisms underlie the muscle dysfunction seen in CFTD. The aim of this work was to study the effect of the R168H mutation in Tpm3.12 on the critical conformational changes that myosin, actin, troponin, and tropomyosin undergo during the ATPase cycle. We used polarized fluorescence microscopy and ghost muscle fibers containing regulated thin filaments and myosin heads (myosin subfragment-1) modified with the 1,5-IAEDANS fluorescent probe. Analysis of the data obtained revealed that a sequential interdependent conformational-functional rearrangement of tropomyosin, actin and myosin heads takes place when modeling the ATPase cycle in the presence of wild-type tropomyosin. A multistep shift of the tropomyosin strands from the outer to the inner domain of actin occurs during the transition from weak to strong binding of myosin to actin. Each tropomyosin position determines the corresponding balance between switched-on and switched-off actin monomers and between the strongly and weakly bound myosin heads. At low Ca2+, the R168H mutation was shown to switch some extra actin monomers on and increase the persistence length of tropomyosin, demonstrating the freezing of the R168HTpm strands close to the open position and disruption of the regulatory function of troponin. Instead of reducing the formation of strong bonds between myosin heads and F-actin, troponin activated it. However, at high Ca2+, troponin decreased the amount of strongly bound myosin heads instead of promoting their formation. Abnormally high sensitivity of thin filaments to Ca2+, inhibition of muscle fiber relaxation due to the appearance of the myosin heads strongly associated with F-actin, and distinct activation of the contractile system at submaximal concentrations of Ca2+ can lead to muscle inefficiency and weakness. Modulators of troponin (tirasemtiv and epigallocatechin-3-gallate) and myosin (omecamtiv mecarbil and 2,3-butanedione monoxime) have been shown to more or less attenuate the negative effects of the tropomyosin R168H mutant. Tirasemtiv and epigallocatechin-3-gallate may be used to prevent muscle dysfunction.

Fluorescence based HTS-compatible ligand binding assays for dopamine D3 receptors in baculovirus preparations and live cells.

Dopamine receptors are G-protein-coupled receptors that are connected to severe neurological disorders. The development of new ligands targeting these receptors enables gaining a deeper insight into the receptor functioning, including binding mechanisms, kinetics and oligomerization. Novel fluorescent probes allow the development of more efficient, cheaper, reliable and scalable high-throughput screening systems, which speeds up the drug development process. In this study, we used a novel Cy3B labelled commercially available fluorescent ligand CELT-419 for developing dopamine D3 receptor-ligand binding assays with fluorescence polarization and quantitative live cell epifluorescence microscopy. The fluorescence anisotropy assay using 384-well plates achieved Z' value of 0.71, which is suitable for high-throughput screening of ligand binding. The assay can also be used to determine the kinetics of both the fluorescent ligand as well as some reference unlabeled ligands. Furthermore, CELT-419 was also used with live HEK293-D3Rcells in epifluorescence microscopy imaging for deep-learning-based ligand binding quantification. This makes CELT-419 quite a universal fluorescence probe which has the potential to be also used in more advanced microscopy techniques resulting in more comparable studies.

OOPS: A GUI-based software package for object-oriented analysis of fluorescence polarization microscopy data.

Excitation-resolved fluorescence polarization microscopy (FPM) is a powerful technique that enables the study of orientational order and dynamics in biological systems. Compared to methods like X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy, FPM offers key advantages, particularly when applied to lipid membranes, membrane proteins, or membrane-associated protein assemblies. Unlike traditional structural methods, FPM can be configured on standard optical microscopes by simply adding a polarization modulator to the excitation path. However, these experimental advantages are countered by the extensive image processing and analysis required to interpret the polarization response data. Consequently, the use of FPM remains limited to a small subset of laboratories, typically relying on custom-built software designed for a particular biological system of interest. Here, we describe OOPS (Object-Oriented Polarization Software), a software package written in MATLAB that offers a convenient graphical user interface, designed to make FPM analysis accessible to researchers at all skill levels. We demonstrate the power and flexibility of our approach by analyzing FPM data acquired of both punctate and filamentous structures. First, we apply OOPS to desmosomes, cell adhesion complexes that are incredibly difficult to study using traditional methods. Our analyses reveal novel, domain-specific differences in orientational order in the transmembrane components of desmosomes, the desmosomal cadherins. We further show how FPM data can be used to reveal the underlying geometry of large, membrane-associated protein assemblies. Finally, to illustrate the flexibility of OOPS, we also collected and analyzed FPM data of F-actin. We demonstrate how our software can quickly and reliably retrieve pixel-by-pixel F-actin filament orientations under a variety of experimental conditions. Importantly, OOPS does not rely on a specific labeling method, meaning the approaches detailed here could easily be extended to a wide array of biological systems.

Regulation of troponin and myosin conformation in thin- and thick-filament domains in the sarcomere of skeletal muscle myofibrils.

The contractility of striated muscle is controlled by a dual-filament regulatory mechanism which couples calcium-dependent structural changes in thin filaments with those in thick filaments triggering the release of folded-helical myosin motors from the filament surface. The folded-helical conformation of motors in relaxed muscle may be stabilized in the region of the thick filament containing myosin-binding protein-C (C-zone). Moreover, in active muscle actin-bound myosin motors might locally alter the activation level of the thin filament. To test these hypotheses we developed a novel fluorescence-based approach to measure troponin and myosin orientation in subdomains of the sarcomere in isolated myofibrils. Bifunctional rhodamine probes labelling troponin-C (TnC) and the myosin regulatory light chain (RLC) were exchanged into myofibrils from rabbit psoas muscle, and the spatial distribution of probe orientations in the sarcomere was measured by fluorescence polarization microscopy. In the absence of ATP, the RLCs of actin-attached myosin motors were perpendicular to the filament axis and the orientation of the TnC probes in the region of overlap with thick filaments was markedly different from that in the rest of the sarcomere, indicating that myosin binding to actin alters the conformation of troponin in the thin filament. In relaxing conditions all TnC probes had the same conformation which was insensitive to temperature. In contrast, the RLC probes became more parallel to the filament axis as temperature was increased above 12°C, and at 30°C in the presence of 4% Dextran their orientation was more parallel in the C-zone, indicating enrichment of folded myosin motors in this filament domain. These results show directly that troponin and myosin have distinct conformations in different sarcomere subdomains in situ (supported by the Wellcome Trust and the Royal Society, UK).

Liquid crystal-like active layer for high-performance organic field-effect transistors

High carrier mobility and uniform device performance are of crucial importance for organic field-effect transistor (OFET)-based device and integrated circuit applications. However, strategies for achieving high device performance with small variations from batch to batch are still desired. Here, we report a thin liquid crystal-like film of 2,8-difluoro-5,11-bis(triethylsilylethynyl) anthradithiophene (dif-TES-ADT) grown on a N,N′-ditridecylperylene-3,4,9,10-tetra-carboxylic diimide (PTCDI-C13) template, confirmed by atomic force microscopy and polarized fluorescence microscopy. The liquid crystal-like films with large crystalline domains are further employed as carrier transport channels for OFETs. As a result, we achieved high-performance OFETs with a saturation carrier mobility of 1.62 ± 0.26 cm2 V−1 s−1 and a small variation of 16% among three batches. This finding provides a new strategy to design materials and device structures to simultaneously achieve high carrier mobility and device uniformity.

Intra-annual fluctuation in morphology and microfibril angle of tracheids revealed by novel microscopy-based imaging.

Woody cells, such as tracheids, fibers, vessels, rays etc., have unique structural characteristics such as nano-scale ultrastructure represented by multilayers, microfibril angle (MFA), micro-scale anatomical properties and spatial arrangement. Simultaneous evaluation of the above indices is very important for their adequate quantification and extracting the effects of external stimuli from them. However, it is difficult in general to achieve the above only by traditional methodologies. To overcome the above point, a new methodological framework combining polarization optical microscopy, fluorescence microscopy, and image segmentation is proposed. The framework was tested to a model softwood species, Chamaecyparis obtusa for characterizing intra-annual transition of MFA and tracheid morphology in a radial file unit. According our result, this framework successfully traced the both characteristics tracheid by tracheid and revealed the high correlation (|r| > 0.5) between S2 microfibril angles and tracheidal morphology (lumen radial diameter, tangential wall thickness and cell wall occupancy). In addition, radial file based evaluation firstly revealed their complex transitional behavior in transition and latewood. The proposed framework has great potential as one of the unique tools to provide detailed insights into heterogeneity of intra and inter-cells in the wide field of view through the simultaneous evaluation of cells' ultrastructure and morphological properties.

Defining domain-specific orientational order in the desmosomal cadherins

Desmosomes are large, macromolecular protein assemblies that mechanically couple the intermediate filament cytoskeleton to sites of cadherin-mediated cell adhesion, thereby providing structural integrity to tissues that routinely experience large forces. Proper desmosomal adhesion is necessary for the normal development and maintenance of vertebrate tissues, such as epithelia and cardiac muscle, while dysfunction can lead to severe disease of the heart and skin. Therefore, it is important to understand the relationship between desmosomal adhesion and the architecture of the molecules that form the adhesive interface, the desmosomal cadherins (DCs). However, desmosomes are embedded in two plasma membranes and are linked to the cytoskeletal networks of two cells, imposing extreme difficulty on traditional structural studies of DC architecture, which have yielded conflicting results. Consequently, the relationship between DC architecture and adhesive function remains unclear. To overcome these challenges, we utilized excitation-resolved fluorescence polarization microscopy to quantify the orientational order of the extracellular and intracellular domains of three DC isoforms: desmoglein 2, desmocollin 2, and desmoglein 3. We found that DC ectodomains were significantly more ordered than their cytoplasmic counterparts, indicating a drastic difference in DC architecture between opposing sides of the plasma membrane. This difference was conserved among all DCs tested, suggesting that it may be an important feature of desmosomal architecture. Moreover, our findings suggest that the organization of DC ectodomains is predominantly the result of extracellular adhesive interactions. We employed azimuthal orientation mapping to show that DC ectodomains are arranged with rotational symmetry about the membrane normal. Finally, we performed a series of mathematical simulations to test the feasibility of a recently proposed antiparallel arrangement of DC ectodomains, finding that it is supported by our experimental data. Importantly, the strategies employed here have the potential to elucidate molecular mechanisms for diseases that result from defective desmosome architecture.

Polarization-fluorescence Microscopy in the Study of Aggregates and Concrete

Concrete structures may develop deleterious damage, which significantly reduces service life, structural integrity, and safety, posing serious issues in large or otherwise critical infrastructure. Routine petrographic assessments, including microstructure, texture, and fabric, of concrete and its (gravel and sand) aggregate and binder constituents in thin section using polarization-fluorescence microscopy (PFM) enables the unequivocal identification of features that would otherwise remain hidden in conventional petrography. Rigorous preparation procedures preserve original microstructural details, make preparation artefacts recognizable, and ensure that the fluorescent emission can be quantified. This contribution outlines the preparation of fluorescence-impregnated thin sections and elaborates on the application of PFM to damaged concrete, with further examples from selected rock types commonly used for concrete aggregate.

Expanded repertoire of polaris, a versatile fluorescent probe for molecular orientation

Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, we reported a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest) (PNAS 2021).