Fluorescence Resonance Research Articles

Gold-iron oxide nanoparticle: A unique multimodal theranostic approach for thrombosis

Thrombosis is a major concern worldwide as thrombosis-related deaths are attributed to one in five deaths globally. Establishing a clot-targeting theranostic particle for diagnosis and treatment of thrombosis related diseases is crucial to provide rapid life-saving intervention. Herein, we developed activated platelet targeted gold-iron oxide nanoparticles (AuIONP) as a robust and powerful probe facilitating triple imaging modalities, including magnetic resonance imaging (MRI), photoacoustic imaging (PAI), and fluorescence imaging (FLI). Additionally, the antibody tagged AuIONP was utilised to facilitate photothermal thrombolysis, stimulated by the exposure to near-infrared (NIR) laser at 808 nm. In vivo observation of the targeted AuIONP showed a significant increase in MRI T2*-weighted contrast and up to 3-fold more prominent photoacoustic signal and fluorescence intensity compared to the non-targeted group. Upon exposure to NIR laser at 1.5 W/cm2, the localised temperature increase at the clot was observed in the targeted AuIONP treated group, facilitating a significantly higher reduction of thrombus area compared to the non-targeted treated group. Our work offers an innovative theranostic probe with excellent triple modality imaging and thrombolysis.

Nucleotide dependent conformational changes of MutL.

MutL is one of the enzymes required for mismatch repair (MMR). Mutations in MutL are linked to Lynch Syndrome, which predisposes individuals to colorectal and other cancers. In MMR, MutL is known to nick the error-containing daughter strand of mismatch DNA as well as form complexes with other MMR proteins such as MutS. MutL accomplishes this by undergoing a series of conformational changes which include extended, one-arm, semi-condensed, and condensed states in the presence of ATP. These dynamic conformational changes are needed to coordinate MMR, but the exact sequence of events and the impact of disease-associated mutations are unknown. We used site-directed mutagenesis to fluorescently label sites in the different domains of Thermus Aquaticus MutL, including the N-terminal, linker arms, and C-terminal domains. We expressed MutL variants with wild-type and ATPase-deficient activity and monitored their dynamic conformational changes under differing nucleotide conditions with pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) in solutions of freely diffusing molecules. Using the same technique, we also monitored the interactions of MutL with MutS, the enzyme that initially recognizes a mismatch, to reveal the specific interactions and conformational changes that are necessary to initiate mismatch repair.

Investigating the impact of ATPase mutation on MutL function.

DNA mismatch repair (MMR) is an enzymatic pathway for fixing incorrect base pairing that may arise during DNA replication. Mutations in the enzyme MutL have been linked to Lynch syndrome, which is the leading cause of hereditary colorectal cancer. MutL is an important part of the MMR pathway and is thought to nick the error containing DNA strand for repair as well as perform other critical functions. To accomplish this, MutL has several large conformational changes that occur in the presence of ATP. While we believe these conformational changes are important for MMR, the exact order of the process and the direct effect of disease-related mutations are unknown. To better understand this process, we used Thermus Aquaticus MutL as a model system for human MutLalpha and mutated the ATPase domain by changing the glutamic acid in residue 28 to an alanine (E28A, aligned to E34A in human MLH1, a common mutation in Lynch syndrome patients). We then conducted an ATPase assay to compare wild-type and E28A activity and fluorescently labeled each MutL variant for single molecule fluorescence studies. We monitored MutL conformational dynamics under varying nucleotide conditions using pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) to reveal the impact of the E28A mutation on the dynamic function of MutL.

Dual functions of the SARS-CoV-2 nucleocapsid domains mediate RNA binding and phase separation.

The primary purpose of the nucleocapsid (N) protein of the Sars-CoV-2 coronavirus is to interact with viral RNA and drive the formation of the viral capsid, mediated by multivalent interaction of both the N- and C-terminal domains (NTD and CTD) of N with RNA. N additionally forms liquid-liquid phase separated droplets with RNA, both in vitro and in infected cells, in a process believed to be important to viral transcription and replication. The degree to which N phase separates with RNA is dependent on the specific RNA being tested, suggesting that the secondary structure of the RNA plays a role in the propensity for phase separation. The ∼32 kb CoV-2 genomic RNA contains a heterogeneous mix of paired and unpaired bases, and while studies suggest that paired RNA is the primary driver of phase separation, little is known at the structural level about how different RNA structures interact with the nucleocapsid. Here we present investigation of binding between N and short designer oligos built to be homogenously single-stranded or double-stranded (ssRNA or dsRNA). Interestingly, we find that our 14-mer dsRNA phase separates readily with the nucleocapsid, while the ssRNA does not, indicating underlying structural differences between the two interactions. Further, we demonstrate by nuclear magnetic resonance (NMR) spectroscopy, fluorescence anisotropy and gel shift assays, that the NTD binds preferentially to ssRNA, and the CTD to dsRNA. We show that the NTD interacts nonspecifically with dsRNA, and that mutations in the NTD that disrupt RNA binding promote this nonspecific interaction. Lastly, we demonstrate that the tendency of N domains to phase separate correlates with weak, nonspecific binding interactions with RNA, suggesting that this weak, nonspecific binding is the primary driver of phase separation.

Time-resolved mechanisms of essential biological pathways.

The diversity of enzyme structure and function in living systems leads to exciting opportunities in biophysical research. Large numbers of these protein machines coordinate across space and time and undergo drastic changes in shape to carry out complex biological processes. My lab focuses on investigating precisely how genetic mutations impact the molecular function of key enzymes involved in DNA repair and/or RNA processing pathways. We use single molecule time-resolved fluorescence spectroscopy as a tool to explore in real time the protein-nucleic acid interactions and dynamic structural rearrangements that drive specific pathways. We are currently studying the detailed mechanisms SARS-CoV-2 viral RNA processing, ribosome assembly, and DNA mismatch repair. I will present recent results from pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) experiments. We aim to completely characterize the impact of mutations linked to disease on the molecular function of enzymes to understand how complex diseases develop at the molecular level and to identify new therapeutic targets.

Post-processing of real-time quantum event measurements for an optimal bandwidth

Single electron tunneling and its transport statistics have been studied for some time using high precision charge detectors. However, this type of detection requires advanced lithography, optimized material systems and low temperatures (mK). A promising alternative, recently demonstrated, is to exploit an optical transition that is turned on or off when a tunnel event occurs. High bandwidths should be achievable with this approach, although this has not been adequately investigated so far. We have studied low temperature resonance fluorescence from a self-assembled quantum dot embedded in a diode structure. We detect single photons from the dot in real time and evaluate the recorded data only after the experiment, using post-processing to obtain the random telegraph signal of the electron transport. This is a significant difference from commonly used charge detectors and allows us to determine the optimal time resolution for analyzing our data. We show how this post-processing affects both the determination of tunneling rates using waiting-time distributions and statistical analysis using full-counting statistics. We also demonstrate, as an example, that we can analyze our data with bandwidths as high as 175 kHz. Using a simple model, we discuss the limiting factors for achieving the optimal bandwidth and propose how a time resolution of more than 1 MHz could be achieved.

Lipid Binding of SH2 Domains.

The Src homology 2 (SH2) domain is a modular protein interaction domain that specifically recognizes the phosphotyrosine (pY) motif of a target molecule. We recently reported that a large majority of human SH2 domains tightly bind membrane lipids, and many show high lipid specificity. Most of them can bind a lipid and the pY motif coincidently because their lipid-binding sites are topologically distinct from pY-binding pockets. Lipid binding of SH2 domain-containing kinases and phosphatases is functionally important because it exerts exquisite spatiotemporal control on protein-protein interaction and cell signaling activities mediated by these proteins. Here, we describe two assays, surface plasmon resonance analysis and fluorescence quenching analysis, which allow quantitative determination of the affinity and specificity of SH2-lipid interaction and high-throughput screening for SH2 domain-lipid-binding inhibitors.

A Leaking-Proof Theranostic Nanoplatform for Tumor-Targeted and Dual-Modality Imaging-Guided Photodynamic Therapy.

Objective: A protein-based leaking-proof theranostic nanoplatform for dual-modality imaging-guided tumor photodynamic therapy (PDT) has been designed. Impact Statement: A site-specific conjugation of chlorin e6 (Ce6) to ferrimagnetic ferritin (MFtn-Ce6) has been constructed to address the challenge of unexpected leakage that often occurs during small-molecule drug delivery. Introduction: PDT is one of the most promising approaches for tumor treatment, while a delivery system is typically required for hydrophobic photosensitizers. However, the nonspecific distribution and leakage of photosensitizers could lead to insufficient drug accumulation in tumor sites. Methods: An engineered ferritin was generated for site-specific conjugation of Ce6 to obtain a leaking-proof delivery system, and a ferrimagnetic core was biomineralized in the cavity of ferritin, resulting in a fluorescent ferrimagnetic ferritin nanoplatform (MFtn-Ce6). The distribution and tumor targeting of MFtn-Ce6 can be detected by magnetic resonance imaging (MRI) and fluorescence imaging (FLI). Results: MFtn-Ce6 showed effective dual-modality MRI and FLI. A prolonged invivo circulation and increased tumor accumulation and retention of photosensitizer was observed. The time-dependent distribution of MFtn-Ce6 can be precisely tracked in real time to find the optimal time window for PDT treatment. The colocalization of ferritin and the iron oxide core confirms the high stability of the nanoplatform invivo. The results showed that mice treated with MFtn-Ce6 exhibited marked tumor-suppressive activity after laser irradiation. Conclusion: The ferritin-based leaking-proof nanoplatform can be used for the efficient delivery of the photosensitizer to achieve an enhanced therapeutic effect. This method established a general approach for the dual-modality imaging-guided tumor delivery of PDT agents.

Structural, magnetic and optical properties of Fe3O4@NaGdF4 core-shell composite

Nanoparticles as contrast agents for in-vitro and in-vivo imaging is the most astounding, non-invasive method developed by researchers in the present scenario. To improve the multifunctionality of bio-imaging field, both Magnetic resonance imaging (MRI) and fluorescence based nanoconstructs are appreciated. In this regard, current research is dedicated to fabricate Fe3O4@NaGdF4 core–shell structure as a multifunctional, efficient probe to be useful for said purpose. Concerned core–shell is constructed in layer-by-layer approach with Fe3O4 and NaGdF4 served as core and shell respectively. Phase purity of sample is investigated through X-ray diffraction (XRD) data, seen to have inverse spinel phased Fe3O4 core and hexagonal NaGdF4. Field emission scanning electron microscopy (FESEM) images clarified the average dimension of isolated core and shell grains to be 59.86 and 62.20 nm respectively. Formation of Core-shell is verified through HRTEM images. Fourier transform infrared (FTIR) spectroscopy data reveals the functional group associated with the corresponding components. Magnetic criteria of sample are studied through room temperature magnetic field dependent magnetization (M−H) data in the range of ± 1.5 Tesla. Non-magnetic shell is seen to be dominate on magnetic core for reducing the magnetic saturation value to 9.104 emu/g from 37.891 emu/g of isolated Fe3O4 sample. Downshifting luminescence is also detected through photoluminescence (PL) study in Ultraviolet (UV) B range (309 nm) due to excitation in UVC region (271 nm) of Gd3+ energy states.

Frequency stability study of the laser source for iron resonance fluorescence Doppler lidar

Frequency stability study of the laser source for iron resonance fluorescence Doppler lidar

Observation of the Mollow triplet from an optically confined single atom

Resonance fluorescence from atomic systems consists of a single spectral peak that evolves into a Mollow triplet for a strong excitation field. Photons from different peaks of the triplet show distinct timing correlations that make the fluorescence a useful light source for quantum information purposes. We characterize the fluorescence of a single optically trapped $^{87}\mathrm{Rb}$ atom that is excited resonantly at different power levels. Second-order correlation measurements reveal the single photon nature of the fluorescence concurrently with Rabi oscillations of a strongly excited atom. The asymmetry in correlations between photons from two sidebands of the fluorescence spectrum when the atom is exposed to an off-resonant field indicates that there is a preferred time ordering of the emitted photons from different sidebands.

Discerning the transition from electromagnetically induced transparency to Autler–Townes splitting using the decaying dressed state formalism

We use biorthogonal basis consisting of the ‘right’ and ‘left’ eigenvectors of the effective non-Hermitian Hamiltonian of a three-level system driven by a bi-chromatic field, and resolve the probe absorption spectrum into components corresponding to the decaying dressed states of the system. It is observed that the spectrum primarily consists of three components and two of them undergo dramatic changes when the control field Rabi frequency is changed from the low to high field regime. Based on the symmetry properties of a combination of these components, a parameter internal to the dynamics of the system is defined and is used to distinguish between electromagnetically induced transparency (EIT) and Autler–Townes (AT) splitting, and the threshold for transition between them in an objective manner. The formulation is further extended to the resonance fluorescence spectrum on the probe transition in the EIT and AT regimes.

Two-step photoionization of barium atoms through excited levels in optical dense approximation

The kinetics of two-step photoionization of barium atoms through excited levels was studied by numerical simulation method in the optically dense approximation. The first laser at wavelength nm ( nm) pumps transition (). The second laser with nm ( nm) ionizes atoms from the level (). The third probe laser with wavelength nm causes resonant fluorescence of barium ions. It is shown that the metastable from the triplet in barium is strongly populated through levels. Therefore, the applying of the ionizing laser tuned to a continuum from this metastable can yield a significant increase in the ionization efficiency. The laser-induced fluorescence method makes it possible to estimate the photoionization yield of barium ions with an error of up to 8%. The loading rate of the barium ion beam into the ion trap, obtained numerically, is consistent with the experimental data [Opt. Exp. 19(17), 16438 (2011)]. The deceleration of ultracold plasma recombination due to the heating of electrons in superelastic collisions for the characteristic times of a two-stage process is predicted.

The biophysics of disordered proteins from the point of view of single-molecule fluorescence spectroscopy.

Intrinsically disordered proteins (IDPs) and regions (IDRs) have emerged as key players across many biological functions and diseases. Differently from structured proteins, disordered proteins lack stable structure and are particularly sensitive to changes in the surrounding environment. Investigation of disordered ensembles requires new approaches and concepts for quantifying conformations, dynamics, and interactions. Here, we provide a short description of the fundamental biophysical properties of disordered proteins as understood through the lens of single-molecule fluorescence observations. Single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) provides an extensive and versatile toolbox for quantifying the characteristics of conformational distributions and the dynamics of disordered proteins across many different solution conditions, both in vitro and in living cells.

Ultra-low loss quantum photonic circuits integrated with single quantum emitters

The scaling of many photonic quantum information processing systems is ultimately limited by the flux of quantum light throughout an integrated photonic circuit. Source brightness and waveguide loss set basic limits on the on-chip photon flux. While substantial progress has been made, separately, towards ultra-low loss chip-scale photonic circuits and high brightness single-photon sources, integration of these technologies has remained elusive. Here, we report the integration of a quantum emitter single-photon source with a wafer-scale, ultra-low loss silicon nitride photonic circuit. We demonstrate triggered and pure single-photon emission into a Si3N4 photonic circuit with ≈ 1 dB/m propagation loss at a wavelength of ≈ 930 nm. We also observe resonance fluorescence in the strong drive regime, showing promise towards coherent control of quantum emitters. These results are a step forward towards scaled chip-integrated photonic quantum information systems in which storing, time-demultiplexing or buffering of deterministically generated single-photons is critical.

Geometrically engineered two-color time orderings in a laser-dressed atomic antenna: Shaping space-time photon correlations

The space-time connection between the Mollow cascaded transitions with time orderings and the spatially directional correlated emissions of Mollow photons is analyzed theoretically in a strong laser-dressed subwavelength two-atom antenna, which supports only two-level electric dipole transitions. It can be regarded as the most prototypical collective quantum radiator to exploit the space-time quantum characteristics of the Mollow physics of resonance fluorescence. By constructing the photon filtering dynamics from the dressed two-atom antenna to a pair of quantum sensors, the frequency-resolved spatial two-point two-time correlations of different Mollow spectral lines are examined analytically in terms of the quantum sensor modes. It is shown that a rich significance of spatial directionality lies in the time orderings of cascaded emissions between different Mollow spectral lines. This feature enables the time orderings of different Mollow spectral lines to be tailored geometrically and thus prepared deterministically to produce highly directional temporal bunching effects of the Mollow photons with giant nonclassicality only for a specific two-photon detection order. The dressed two-atom system is therefore able to operate as a highly directional atomic antenna that may provide versatile applications in the fields of atom-photon-based quantum precise measurement physics and Mollow spectroscopy.

One-Shot Synthesis of B/N-Doped Calix[4]arene Exhibiting Narrowband Multiple Resonance Fluorescence.

A novel macrocycle of B/N-doped calix[4]arene (C-BN) was synthesized by a one-shot double boronation. Owing to the structural tension and electron-donating properties of the nitrogen atoms in the macrocycle, reaction selectively proceeds between the adjacent benzene rings outside the macrocycle. C-BN shows a highly centrosymmetric structure with two multiple resonance (MR) fragments bridged by tertiary amine groups at the 1,3 positions of the benzene ring. Benefiting from the large intermolecular distance (>4.6 Å) between adjacent MR-emitting cores, C-BN also exhibits excellent narrowband emitting features against aggregation-induced quenching and spectrum broadening. Optimized organic light-emitting diode devices based on C-BN exhibit high maximum external quantum efficiencies of 24.7-26.6 % and small full width at half maximums of 25-28 nm over a wide doping range of 1-12 wt %.

One‐Shot Synthesis of B/N‐Doped Calix[4]arene Exhibiting Narrowband Multiple Resonance Fluorescence

AbstractA novel macrocycle of B/N‐doped calix[4]arene (C‐BN) was synthesized by a one‐shot double boronation. Owing to the structural tension and electron‐donating properties of the nitrogen atoms in the macrocycle, reaction selectively proceeds between the adjacent benzene rings outside the macrocycle. C‐BN shows a highly centrosymmetric structure with two multiple resonance (MR) fragments bridged by tertiary amine groups at the 1,3 positions of the benzene ring. Benefiting from the large intermolecular distance (>4.6 Å) between adjacent MR‐emitting cores, C‐BN also exhibits excellent narrowband emitting features against aggregation‐induced quenching and spectrum broadening. Optimized organic light‐emitting diode devices based on C‐BN exhibit high maximum external quantum efficiencies of 24.7–26.6 % and small full width at half maximums of 25–28 nm over a wide doping range of 1–12 wt %.

Alginate-Derivative Encapsulated Carbon Coated Manganese-Ferrite Nanodots for Multimodal Medical Imaging.

Carbon-decorated ferrite nanodots (MNF@Cs) have been enhanced with superparamagnetism and higher fluorescence quantum yield by encapsulation with an alginate derivative to create a cost-effective and less toxic multimodal contrast agent for replacing the conventional heavy metal Gd-containing contrast agent used in MR imaging. The novel surface-engineered particles (MNF@C-OSAs), devoid of labels, can simultaneously provide both longitudinal and transverse relaxation-based magnetic resonance imaging (MRI) and fluorescence emission. According to the findings of in vitro studies, the calculated molar relaxivities and the molar radiant efficiencies are indicative of the multimodal efficacy of MNF@C-OSA as compared with MNF@C particles and conventional contrast agents used in medical imaging. MNF@C-OSAs were shown to be significantly biocompatible and negligibly toxic when assessed against A549 cells and zebrafish embryos, indicating their potential for use as theranostic agents.

Neutral-current neutrino cross section and expected supernova signals for 40Ar from a three-fold increase in the magnetic dipole strength

In view of the great interest in liquid argon neutrino detectors, the 40Ar(γ,γ′)40Ar reaction was revisited to guide a calculation of the neutral current neutrino cross section at supernova energies. Using the nuclear resonance fluorescence technique with a monoenergetic, 99% linearly polarized photon beam, we report a three-fold increase in magnetic dipole strength at around 10 MeV in 40Ar. Based on shell-model calculations, and using the experimentally identified transitions, the neutral current neutrino cross sections for low-energy reactions on 40Ar are calculated.