Orientation Of Single Molecules Research Articles

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.

Single-molecule orientation and position measurement assisted by deep learning and point spread function engineering

Single-molecule orientation and position measurement assisted by deep learning and point spread function engineering

Three-Dimensional Orientation and Localization Microscopy of Single Molecules Using Super-Resolution Imaging Technology (Invited)

Three-Dimensional Orientation and Localization Microscopy of Single Molecules Using Super-Resolution Imaging Technology (Invited)

Polarization microscopy: from ensemble structural imaging to single-molecule 3D orientation and localization microscopy

Optical contrasts in microscopy are sensitive to light polarization, whose interaction with molecular dipoles provides an important lever for probing molecular orientation. Polarization microscopy has evolved considerably during the last decade, integrating strategies ranging from traditional linear dichroism to single-molecule orientation and localization imaging. This review aims to provide a summary of concepts and techniques behind orientation and structural imaging at the molecular level, from ensemble microscopy in 2D to single-molecule super-resolution microscopy in 3D.

Revealing the Orientation Selectivity of Tetrapyridyl-Substituted Porphyrins Constrained in Molecular "Klotski Puzzles".

Understanding and controlling molecular orientations in self-assembled organic nanostructures are crucial to the development of advanced functional nanodevices. Scanning tunneling microscopy (STM) provides a powerful toolbox to recognize molecular orientations and to induce orientation changes on surfaces at the single-molecule level. Enormous effort has been devoted to directly controlling the molecular orientations of isolated single molecules in free space. However, revealing and further controlling molecular orientation selectivity in constrained environments remain elusive. In this study, by a combination of STM imaging/manipulations and density functional theory calculations, we report the orientation selectivity of tetrapyridyl-substituted porphyrins in response to various local molecular environments in artificially constructed molecular "Klotski puzzles" on Au(111). With the assistance of STM lateral manipulations, "sliding-block" molecules were able to enter predefined positions, and specific molecular orientations were adopted to fit the local molecular environments, in which the intermolecular interaction was revealed to be the key to achieving the eventual molecular orientation selectivity. Our results demonstrate the essential role of local molecular environments in directing single-molecule orientations, which would shed light on the design of molecular structures to control preferred orientations for further applications in molecular nanodevices.

Influence of the excitation polarization on single molecule 3D orientation imaging

Single Molecule Orientation and Localization Microscopy (SMOLM) is gaining an increasing interest in the community of localization microscopy, due to the capability to monitor orientational information in addition to spatial reconstruction. In many cases, molecule’s orientations are not random and their linker to a protein of interest is sufficiently rigid to be able to report orientation information from this protein. While several strategies exist to report single molecule orientation based on polarization splitting and point spread function engineering, the effect of the incident polarization has been often neglected by supposing its effect embedded in the retrieved angular parameters, or by supposing the excitation to be isotropic or the rotational diffusion to be very fast. In this work we quantify the amount of possible bias brought in SMOLM readout due to the incident polarization effect, using analytical derivations. We illustrate this effect experimentally on single molecules attached to a surface in presence of wobbling.

Regulating the orientation of a single coordinate bond by the synergistic action of mechanical forces and electric field.

The molecular binding orientation with respect to the electrode plays a pivotal role in determining the performance of molecular devices. However, accomplishing in situ modulation of single-molecule binding orientation remains a great challenge due to the lack of suitable testing systems and characterization approaches. To this end, by employing a developed STM-BJ technique, we demonstrate that the conductance of pyridine-anchored molecular junctions decreases as the applied voltage increases, which is determined by the repeated formation of thousands of gold-molecule-gold dynamic break junctions. In contrast, the static fixed molecular junctions (the distance between two electrodes is fixed) with identical molecules exhibit a reverse tendency as the bias voltage increases. Supported by flicker noise measurements and theoretical calculations, we provide compelling evidence that the orientation of nitrogen-gold bonds (a universal coordinate bond) in the pyridine-anchored molecular junctions can be manipulated to align with the electric field by the synergistic action of the mechanical stretching force and the electric fields, whereas either stimulus alone cannot achieve the same effect. Our study provides a framework for characterizing and regulating the orientation of a single coordinate bond, offering an approach to control electron transport through single molecular junctions.

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching.

Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (ϕ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5′s orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, ϕ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins.

(Digital Presentation) Monitoring and Controlling Tautomerization in Phthalocyanines, Porphyrines and Porphycenes By Optical Single-Molecule Imaging and Spectroscopy

Tautomerization is an intramolecular chemical reaction defined as structural interconversion of a molecule between two isomers separated by a low energy barrier. Tautomerization in free base phthalocyanines, porphyrins or porphycenes has fascinated and attracted chemists and physicists alike over the last decades for its fundamental importance in various chemical and biological systems and many applications in technology [1,2]. Particularly interesting is the reaction rate which can range over orders of magnitude depending on the temperature and the local environment. In solution, this reaction in porphycenes occurs usually in several picoseconds or even faster, as determined via anisotropy studies by ultrafast transient absorption spectroscopy [3] and in porphyrins at cryogenic temperatures it can last for hours or days as determined form the live time of narrow spectral holes photochemically burned in the inhomogeneously broadened absorption band with a narrow band laser [4]. However, a spectacular and unexpected observation has been made by optical single molecule imaging and spectroscopy [5]. Examining a large number of single molecules embedded at low concentration in various polymer matrices at room temperature has revealed unquestionably that the rate in these molecules can vary dramatically over time such that tautomerization can be frozen for seconds and longer under ambient conditions[6]. The interconversion of the inner protons of phthalocyanine changes the orientation of the transition dipole moment by 90o degrees with respect to the molecular structure. Hence, if these molecules are embedded with a fixed orientation in a transparent solid polymer film at high dilution and tautomerization is frozen, the orientation of their transition dipole moments can be determined by polarization spectroscopy.Here we report on our recent experimental and theoretical studies of monitoring tautomerization by fluorescence imaging of single molecules of phthalocyanines, porphyrins, or porphycenes excited in the tightly focused azimuthally and radially polarized laser beam with a sample scanning confocal optical microscope equipped with single photon counting electronics and a fluorescence spectrometer. This technique has been used to determine the transition dipole moment orientation of single molecules, the polarizability of plasmonic nanoparticles or the dimensionality of single quantum dots. Different diffraction limited fluorescence intensity patterns are observed for molecules that undergo tautomerization and for those that do not. The latter corresponds to a molecule with a fixed transition dipole moment direction, whereas the tautomerizing molecule can be treated as the superposition of two emitters with different transition dipole moment orientations.Previous studies have shown that tautomerization in porphyrins or phthalocyanines can occur via ground state tunneling and is frozen at low temperatures, particularly at cryogenic temperatures. The structural heterogeneity in a polymer matrix leads to a distribution of asymmetrically distorted ground state double well potential and hence can lead to long residence times in the deeper energy minimum. However, even at helium temperatures tautomerization can be induced by electronic excitation to the first excited state, making photochemical spectral hole-burning possible. Recently we could demonstrate in a single-molecule study that the tautomerization rate of phthalocyanine tetrasulfonate embedded in a thin PVA-film at room temperature could be controlled in a tunable λ/2 Fabry Perot resonator by weak coupling between the electronically excited state and the vacuum electromagnetic field of a resonant cavity mode.

Simultaneous super-resolution estimation of single-molecule position and orientation with minimal photon fluxes.

The orientation of a single molecule provides valuable information on fundamental biological processes. We report a technique for the simultaneous estimation of single-molecule 2D position and 2D orientation with ultra-high localization precision (∼2-nm precision with ∼500 photons under a typical 100-nm diameter of excitation beam pattern), which is also compatible with tracking in living cells. In the proposed method, the theoretical precision limits are calculated, and the localization and orientation performance along with potential applications are explored using numerical simulations. Compared to other camera-based orientation measurement methods, it is confirmed that the proposed method can obtain reasonable estimates even under very weak signals (∼15 photons). Moreover, the maximum likelihood estimator (MLE) is found to converge to the theoretical limit when the total number of photons is less than 100.

Mapping the chemical composition and nanoscale structure of lipid membranes using polarized fluorescence from single molecules

Single-molecule orientation localization microscopy (SMOLM) provides insight into biological activities that cannot be resolved using standard superresolution techniques. Here, we present a radially and azimuthally polarized (raPol) microscope for tracking the 2D positions and 3D orientations of fluorescent molecules. This technique is simply implemented by adding a commercially available vortex half waveplate and polarizing beamsplitter to an epifluorescence microscope. Compared to other SMOLM techniques, the raPol microscope exhibits superior detectability and excellent measurement precision, which enables us to resolve the chemical composition and nanoscale structure of lipid membranes.

Probing Multidimensional Structural Information of Single Molecules Transporting through a Sub-10 nm Conical Plasmonic Nanopore by SERS.

Probing the orientation and oxygenation state of single molecules (SMs) is of great importance for understanding the advanced structure of individual molecules. Here, we manipulate molecules transporting through the hot spot of a sub-10 nm conical gold nanopore and acquire the multidimensional structural information of the SMs by surface enhanced Raman scattering (SERS) detection. The sub-10 nm size and conical shape of the plasmonic nanopore guarantee its high detection sensitivity. SERS spectra show a high correlation with the orientations of small-sized single rhodamine 6G (R6G) during transport. Meanwhile, SERS spectra of a single hemoglobin (Hb) reveal both the vertical/parallel orientations of the porphyrin ring and oxygenated/deoxygenated states of Hb. The present study provides a new strategy for bridging the primary sequence and the advanced structure of SMs.

Effects of interfaces on structure and dynamics of water droplets on a graphene surface: A molecular dynamics study

The structure and dynamics of water droplets on a bilayer graphene surface are investigated using molecular dynamics simulations. The effects of solid/water and air/water interfaces on the local structure of water droplets are analyzed in terms of the hydrogen bond distribution and tetrahedral order parameter. It is found that the local structure in the core region of a water droplet is similar to that in liquid water. On the other hand, the local structure of water molecules at the solid/water and air/water interfaces, referred to as the interface and surface regions, respectively, consists mainly of three-coordinated molecules that are greatly distorted from a tetrahedral structure. This study reveals that the dynamics in different regions of the water droplets affects the intermolecular vibrational density of states: It is found that in the surface and interface regions, the intensity of vibrational density of states at ∼50 cm-1 is enhanced, whereas those at ∼200 and ∼500 cm-1 are weakened and redshifted. These changes are attributed to the increase in the number of molecules having fewer hydrogen bonds in the interface and surface regions. Both single-molecule and collective orientation relaxations are also examined. Single-molecule orientation relaxation is found to be marginally slower than that in liquid water. On the other hand, the collective orientation relaxation of water droplets is found to be significantly faster than that of liquid water because of the destructive correlation of dipole moments in the droplets. The negative correlation between distinct dipole moments also yields a blueshifted libration peak in the absorption spectrum. It is also found that the water-graphene interaction affects the structure and dynamics of the water droplets, such as the local water structure, collective orientation relaxation, and the correlation between dipole moments. This study reveals that the water/solid and water/air interfaces strongly affect the structure and intermolecular dynamics of water droplets and suggests that the intermolecular dynamics, such as energy relaxation dynamics, in other systems with interfaces are different from those in liquid water.

Molecular Mechanism of Acceleration and Retardation of Collective Orientation Relaxation of Water Molecules in Aqueous Solutions.

The collective orientation relaxation (COR) of water molecules in aqueous solutions is faster or slower with an increase in the concentration of the solutions than that in pure water; for example, acceleration (deceleration) of the COR is observed in a solution of sodium chloride (tetramethylammonium chloride) with increasing concentration. However, the molecular mechanism of the solution and concentration dependence of the relaxation time of the COR has not yet been clarified. We theoretically investigate the concentration dependence of the COR of water molecules in solutions of tetramethylammonium chloride (TMACl), guanidinium chloride (GdmCl), and sodium chloride (NaCl). Based on the Mori-Zwanzig equation, we identify two opposing factors that determine the COR of water molecules in any aqueous solution: the correlation of dipole moments and the single-molecule orientation relaxation. We reveal the molecular mechanism of the concentration dependence of the relaxation time of the COR in the TMACl, GdmCl, and NaCl solutions in terms of these two factors.

100th Anniversary of Macromolecular Science Viewpoint: Enabling Advances in Fluorescence Microscopy Techniques.

In the past few decades there has been a revolution in the field of optical microscopy with emerging capabilities such as super-resolution and single-molecule fluorescence techniques. Combined with the classical advantages of fluorescence imaging, such as chemical labeling specificity, and noninvasive sample preparation and imaging, these methods have enabled significant advances in our polymer community. This Viewpoint discusses several of these capabilities and how they can uniquely offer information where other characterization techniques are limited. Several examples are highlighted that demonstrate the ability of fluorescence microscopy to understand key questions in polymer science such as single-molecule diffusion and orientation, 3D nanostructural morphology, and interfacial and multicomponent dynamics. Finally, we briefly discuss opportunities for further advances in techniques that may allow them to make an even greater contribution in polymer science.

Single-Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes.

In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm-2 ) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single-molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus "wobble") of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme-induced compositional heterogeneity within membranes, where NR within liquid-ordered vs. liquid-disordered domains shows a ≈4° difference in polar angle and a ≈0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid-lipid, lipid-protein, and lipid-dye interactions with single-molecule, nanoscale resolution.

Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils.

Simultaneous measurements of single-molecule positions and orientations provide critical insight into a variety of biological and chemical processes. Various engineered point spread functions (PSFs) have been introduced for measuring the orientation and rotational diffusion of dipole-like emitters, but the widely used Cramér-Rao bound (CRB) only evaluates performance for one specific orientation at a time. Here, we report a performance metric, termed variance upper bound (VUB), that yields a global maximum CRB for all possible molecular orientations, thereby enabling the measurement performance of any PSF to be computed efficiently (~1000× faster than calculating average CRB). Our VUB reveals that the simple polarized standard PSF provides robust and precise orientation measurements if emitters are near a refractive index interface. Using this PSF, we measure the orientations and positions of Nile red (NR) molecules transiently bound to amyloid aggregates. Our super-resolved images reveal the main binding mode of NR on amyloid fiber surfaces, as well as structural heterogeneities along amyloid fibrillar networks, that cannot be resolved by single-molecule localization alone.

A device for exploring the full angular excitation space – Can more angular projections improve determination of a molecules 3D-orientation in the presence of noise?

Using rotation of linearly polarized illumination from different directions enables reconstructing the full 3D orientation of single molecules. Theoretically, already two projections suffice for a full 3D orientation determination if the intensities are properly calibrated and in the absence of noise. The question arises if a limited set of projections that cannot excite all molecules equally contains already the maximum information on the 3D orientation when also considering photon and experimental noise, or if detecting more projections or even the full angular projection space provides more information. To answer this question, we constructed a device exploring the full angular excitation space available with a distinct microscope objective and tested several different polarization modulation schemes using simulated as well as experimental single molecule data. For noisy experimental data, we found three projections ideal and no significant improvement in angular precision for larger projection numbers or when detecting the full projection space.

The effects of ligand charge, orientation and size on the binding of potential inhibitors for aldehyde dehydrogenase

l-DOPA, used as a therapy for patients with Parkinson’s disease, is transformed into needed dopamine in the brain. This dopamine can then be deactivated via metabolism by a series of enzymes, including aldehyde dehydrogenase (ALDH). The targeted inhibition of the ALDH enzyme may help to prolong l-DOPA therapy. A series of potential inhibitors has been studied viaab initiomodels using a crystal-structure of the ALDH enzyme with an inhibitor bound in its active site (PDB ID: 4WP7). The positions of novel dopaminergic derivatives were optimized in the active site using M062X/6-31G with implicit solvation and relaxed amino-acid side-chains. This work examines different single molecule orientations, as well as double molecule configurations. Various sizes of ligands were also studied. Interaction energies between the ligands and the protein were calculated using M062X with the 6–311 + G* basis set. Some potential inhibitors show promising results such as the LP and CAM series.

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.