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Schematics of the universal beam splitter and artificial molecule used with 2D POLIM. a) The universal beam splitter preserving fluorescence polarization. It can be used instead of a dichroic beam splitter. b) Artificial molecule (AM)—the test sample for measuring polarization artifacts. The AM possesses perfect dipolar absorption and dipolar emission due to its design. Further, by choosing an adequate luminescent solution, the emission is tuned to the exact spectral range used in the experiments. Therefore, when the AM is measured in an artifact free microscope it should possess Mex = Mem = 1 and equal modulation phases in excitation and emission corresponding to the angle θa (orientation of the AM in the sample plane). All these conditions should be true regardless of the orientation of the AM in the sample plane (angle θa). Deviations from the conditions mentioned above report on polarization artifacts of the microscope. Reproduced with permission.44 Copyright 2012, Elsevier.
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Fluorescence polarization is widely used to assess the orientation/rotation of molecules, and the excitation energy transfer between closely located chromophores. Emerging since the 1990s, single molecule fluorescence spectroscopy and imaging stimulate the application of light polarization for studying molecular organization and energy transfer bey...
Citations
... Fluorescent molecules exhibit dipole behavior and their orientation provides insights into the properties of the targeted molecular [1][2][3]. Numerous fluorescence polarization microscopes (FPM) [4][5][6][7] have been developed to measure dipole orientation, which helps to study the motion of molecular motors [8], conformation of DNA [9, 10], molecular arrangement of actin filaments [11][12][13], and lipid membranes [14-16], etc. To overcome the challenge of conventional FPM limited by optical diffraction, improved super-resolution FPM techniques have been proposed [17][18][19][20], such as single-molecule orientation-localization microscopy (SMOLM) [21][22][23], and 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.
... Fluorescent molecules exhibit dipole behavior and their orientation provides insights into the properties of the targeted molecular [1][2][3]. Numerous fluorescence polarization microscopes (FPM) [4][5][6][7] have been developed to measure dipole orientation, which helps to study the motion of molecular motors [8], conformation of DNA [9,10], molecular arrangement of actin filaments [11][12][13], and lipid membranes [14][15][16], etc. To overcome the challenge of conventional FPM limited by optical diffraction, improved super-resolution FPM techniques have been proposed [17][18][19][20], such as single-molecule orientation-localization microscopy (SMOLM) [21][22][23], and polarization modulation [24]. ...
... , , x y ρ coordinate is defined by the distance 2 R , which includes both the spatial distance and the spacing along the ρ -axis. Moreover, when considering the difference in polar and azimuthal angles of the dipoles, the resolution in ( ) , , , x y ρ η coordinate becomes the distance 3 R , ...
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 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.
... The self-aggregate and the co-assembly formed highly entangled networks which in principle, would assist in a long-range exciton migration. 83 This would involve a signicant homo-ET among the CS donors before the hetero-ET to the acceptor (NR) took place. Therefore, to determine the number of CS donors (n) taking part in ET per antenna, we employed a mathematical model combining both dynamic quenching (for homo-ET) and static quenching (hetero-ET) mechanisms. ...
Luminescent organic nanotubes derived from the co-assembly of cyanostilbene (CS) based cationic supramolecular polymers and bio-polyanion heparin, a known anticoagulant, have been utilized as highly efficient FRET (fluorescence resonance energy transfer) donors in aqueous media resulting in amplified acceptor emission in the orange-red and near-infrared (NIR). Energy transfer efficiencies higher than 80% and ultra-high antenna effect of 150 were achieved even at high donor/acceptor ratios (500:1-100:1) translating to emission quenching of several hundred donors by one acceptor. Utilizing the temperature responsiveness of the FRET process, these systems were employed as ratiometric emission thermometers in the temperature range 20-90⁰C. Moreover, the energy transfer was very effective in solid and polymer films. This allowed us to generate multi-color emissions ranging from blue to red including white light in the solution as well as in solid and polymer films.
... is widely used as an indicator of molecular size or diffusion in fluorescence spectroscopy and expresses the preferential excitation of molecules with transition dipoles oriented along a particular polarization direction [28]. LD (−1 ≤ LD ≤ 1) corresponds to the difference of two absorption spectra between two orthogonal linear polarizations and is linked to the macroscopic averaged orientation of the molecules [29]. ...
Diffuse reflectance spectroscopy (DRS) has proven to be a powerful, reliable, and non-invasive optical method for characterizing a specimen. Nevertheless, these methods are based on a rudimentary interpretation of the spectral response and can be irrelevant to understanding 3D structures. In this work, we proposed adding optical modalities into a customized handheld probe head in order to increase the number of parameters in DRS acquired from the light/matter interaction. It consists of (1) placing the sample in a reflectance manual rotation stage to collect spectral backscattered angularly resolved light and (2) illuminating it with two sequential linear polarization orientations. We demonstrate that this innovative approach leads to a compact instrument, capable of performing fast polarization-resolved spectroscopic analysis. Due to the significant amount of data available with this technique in a short time, we observe sensitive quantitative discrimination between two types of biological tissue provided by a raw rabbit leg. We believe that this technique can pave the way for rapid meat quality check or biomedical diagnosis of pathological tissues in situ at an early stage.
... If a = 1, there is a well-defined in-plane preferential direction with no dipoles oriented perpendicular to it. One can show that this is the most general distribution that can be resolved with linear polarizers, 45 implying that the directions ϕ 0 and ϕ 0 + π cannot be distinguished. It is possible to lift this ambiguity by using, for example, a dual-view design with detection under different angles. ...
The transition dipole orientations of dye assemblies in heterostructures have a crucial impact on the efficiency of novel optoelectronic devices such as organic thin-film transistors and light-emitting diodes. These devices are frequently based on heterojunctions and tandem structures featuring multiple optical transitions. Precise knowledge of preferred orientations, spatial order, and spatial variations is highly relevant. We present a fast and universal large-area screening method to determine the transition dipole orientations in dye assemblies with diffraction-limited spatial resolution. Moreover, our hyperspectral imaging approach disentangles the orientations of different chromophores. As a demonstration, we apply our technique to dye monolayers with two optical transitions sandwiched between two ultrathin silicate nanosheets. A comprehensive model for dipole orientation distributions in monolayers reveals a long-range orientational order and a strong correlation between the two transitions.
... 6 Natural materials for polarization modulation exhibit many limitations, such as a large volume, large loss, low efficiency, strict operating conditions, and narrow working bandwidth. [7][8][9] In recent years, the application of metamaterials to regulate polarization has attracted considerable attention. [10][11][12] Specifically, metamaterials are artificial materials with unit cells that are manually designed and arranged periodically. ...
In this study, we describe the design, fabrication, and characterization of a cross-shaped structure reflective broadband THz polarization converter. The operating bandwidth of the proposed polarization converter was in the range of 0.8–1.6 THz. The polarization conversion rate (PCR) exceeded 85% in the frequency range of 1.07–1.35 THz and was as high as 91% at 1.35 THz. The cross PCR value exceeded 85% in the frequency range of 1.05–1.35 THz and reached a maximum value of 91%. The performance characteristics (bandwidth and PCR) of the proposed polarization converter were compared with those of similar previously reported devices, and the results indicated that the proposed polarization converter exhibits better performance. The proposed THz polarization converter is suitable for a wide range of applications in communication and polarization manipulation devices.
... This means that FA measurements are not directly applicable at the single-molecule level because individual molecules and particles usually exhibit anisotropic absorption. 5,[21][22][23] To overcome this problem and extract maximum quantitative information from fluorescence polarization measurements, an advanced polarization technique called two-dimensional polarization imaging (2D POLIM) was introduced several years ago. 23,24 2D here indicates that fluorescence intensity (I) is measured as a function of two variables: the orientation angle of the linearly polarized excitation light (φex) and the orientation of the transmission axis of the polarization analyzer (φem) placed in front of the detector. ...
... 24 This parameter is calculated within the so-called single funnel approximation (SFA). 24 2D POLIM and the analysis in the SFA framework have been successfully used to quantitatively assess energy transfer efficiency in various systems, including single molecules, [21][22][23] conjugated polymer films, 26,27 and natural light-harvesting system, 5,25 and to detect protein aggregation in brain tissue. 8 Estimation of the energy transfer efficiency using the SFA has the following advantages: 23 (i) It is insensitive to the preferential alignment of the sample, so it is applicable to multichromophoric systems with linear dichroism. ...
... 24 2D POLIM and the analysis in the SFA framework have been successfully used to quantitatively assess energy transfer efficiency in various systems, including single molecules, [21][22][23] conjugated polymer films, 26,27 and natural light-harvesting system, 5,25 and to detect protein aggregation in brain tissue. 8 Estimation of the energy transfer efficiency using the SFA has the following advantages: 23 (i) It is insensitive to the preferential alignment of the sample, so it is applicable to multichromophoric systems with linear dichroism. (ii) It does not require any prior knowledge of the chromophores' organization. ...
Two-dimensional polarization imaging (2D POLIM) is an experimental method where correlations between fluorescence excitation- and fluorescence emission-polarization properties are measured. One way to analyze 2D POLIM data is to apply a so-called single funnel approximation (SFA). The SFA allows for quantitative assessment of energy transfer between chromophores with identical spectra [homo-FRET (Förster resonance energy transfer)]. In this paper, we run a series of computer experiments to investigate the applicability of the analysis based on the SFA to various systems ranging from single multichromophoric systems to isotropic ensembles. By setting various scenarios of energy transfer between individual chromophores within a single object, we were able to define the borders of the practical application of SFA. It allowed us to reach a more comprehensive interpretation of the experimental data in terms of uncovering the internal arrangement of chromophores in the system and energy transfer between them. We also found that the SFA can always formally explain the data for isotropic ensembles and derived a formula connecting the energy funneling efficiency parameter and traditional fluorescence anisotropy.
... To study variations between chlorosomes, the application of single-molecule spectroscopy is indispensable. [44][45][46][47] Since chlorosomes contain many thousands of molecules, we rather refer to the application of this type of spectroscopy to chlorosomes as singleobject spectroscopy. Prior studies have applied single-object linear polarization (SOLP) resolved fluorescence excitation spectroscopy to individual chlorosomes, in which fluorescence excitation spectra for different linear polarization directions of the exciting light are recorded; hereafter, we refer to this as SOLP spectroscopy. ...
We theoretically investigate the possibility to use single-object spectroscopy to probe size variations of the bacteriochlorophyll aggregates inside chlorosomes. Chlorosomes are the light-harvesting organelles of green sulfur and non-sulfur bacteria. They are known to be the most efficient light-harvesting systems in nature. Key to this efficiency is the organization of bacteriochlorophyll molecules in large self-assembled aggregates that define the secondary structure inside the chlorosomes. Many studies have been reported to elucidate the morphology of these aggregates and the molecular packing inside them. It is widely believed that tubular aggregates play an important role. Because the size (radius and length) of these aggregates affects the optical and excitation energy transport properties, it is of interest to be able to probe these quantities inside chlorosomes. We show that a combination of single-chlorosome linear polarization resolved spectroscopy and single-chlorosome circular dichroism spectroscopy may be used to access the typical size of the tubular aggregates within a chlorosome and, thus, probe possible variations between individual chlorosomes that may result, for instance, from different stages in growth or different growth conditions.
... However, most fluorophores are dipoles, whose absorption efficiency of light is related to the dipole orientation and the polarization. [15][16][17] This anisotropic polarization response exists in most specimens labeled by fluorescent proteins or molecular probes, especially in the specimen of cytoskeletal filaments, lipid membranes, and biocomplex. [18][19][20] Since another benefit of SIM is its compatibility with standard fluorescence samples, the investigation on how this inherent polarization modulation will affect the SIM imaging results can reinforce the SIM theory and further broaden the application of SIM. ...
... Therefore, the intensity of the Fourier components of every direction is the same. However, most fluorophores are dipoles, which means that its absorption and emission property is strongly polarization-dependent. [15] Under linear polarization excitation, the absorption efficiency of the dipole E is determined by the angle between its natural orientation α and the polarization direction θ, reaching the maximum when parallel and the minimum when perpendicular. The quantitative relationship is described as Eq. ...
Structured illumination microscopy (SIM) requires polarization control to guarantee the high-contrast illumination pattern. However, this modulated polarization will induce artifacts in SIM when imaging fluorescent dipoles. Here we proposed the polarization weighted recombination of frequency components to reconstruct SIM data with suppressed artifacts and better resolving power. Both the simulation results and experimental data demonstrate that our algorithm can obtain isotropic resolution on dipoles and resolve a clearer structure in high-density sections compared to the conventional algorithm. Our work reinforces the SIM theory and paves the avenue for the application of SIM on a polarized specimen.
... Some additional drawbacks of FPs can severely hamper microscopic analysis: low quantum efficiency, blinking behaviour, a high photobleaching rate during long-term observations (Reck-Petersen et al., 2006), photoswitching (Morisaki and McNally, 2014) as well as the tendency of FPs to form oligomers (Miyawaki et al., 2003). These drawbacks are especially unfavourable when using advanced fluorescence microscopy methods such as two-photon, high content, single molecule, subdiffractional polarisation or super-resolution imaging that typically require fluorescence tags of high stability and brightness (Moerner and Kador, 1989;Orrit and Bernard, 1990;Bode et al., 2008;Holleboom et al., 2014;Liao et al., 2010Liao et al., /2011Godin et al., 2014;Loison et al., 2018;Camacho et al., 2019). ...
... Many subcellular structures are smaller which hampers their detailed observation (Huang et al., 2009). To circumvent these restrictions, advanced fluorescence imaging methods such as single molecule detection, subdiffractional polarisation imaging or super-resolution microscopy (SRM) techniques have been developed to improve the resolution and to allow studying molecular processes more detailed (Moerner and Kador 1989;Orrit and Bernard, 1990;Bode et al., 2008;Holleboom et al., 2014;Liao et al., 2010Liao et al., /2011Godin et al., 2014;Loison et al., 2018;Camacho et al., 2019). However, stable fluorescence molecules are needed for such advanced imaging techniques which is hard to realise by standard fluorescence proteins. ...
An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP based protein-protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualisation of protein complex localisation. Due to its simple protocols, it has become one of the most frequently used methods. However, standard fluorescent proteins entail several drawbacks for sophisticated microscopy.
With the HaloTag ® system, these drawbacks can be overcome as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we established the Split-HaloTag ® imaging assay in plants which is based on the reconstitution of a functional HaloTag ® protein upon protein-protein interaction and subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using well-characterised interactions as an example for protein-protein interaction at cellular structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Additionally, a specific interaction was visualised with subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging.
Split-GFP and Split-HaloTag ® can complement one another as Split-HaloTag ® represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag ® imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interaction with specific microscopic techniques such as 3D-imaging, single molecule tracking and super-resolution microscopy.
