Probe Excitation Research Articles

Simultaneous 2-photon and 3-photon excitation with a red fluorescent protein-cyanine dye probe pair in the 1700-nm excitation window for deep in vivo neurovascular imaging

In vivo imaging of the neurovascular network is considered to be one of the most powerful approaches for understanding brain functionality. Nevertheless, simultaneously imaging the biological neural network and blood vessels in deep brain layers in a non-invasive manner remains to a major challenge due to the lack of appropriate labeling fluorescence probe pairs. Herein, we proposed a 2-photon and 3-photon fluorescence probe pair for neurovascular imaging. Specifically, the red fluorescence protein (RFP) with an absorption maximum of around 550 nm is used as a 3-photon excited probe to label neurons, and a cyanine derivative dye Q820@BSA has a NIR absorption maximum of 825 nm as a 2-photon excited probe to label the vasculature, enabling single wavelength excitation at 1650 nm for neurovascular imaging with high emission spectral separation (>250 nm). In particular, the 2-photon action cross-section of Q820@BSA was found to be about 2-fold larger than that of indocyanine green (ICG), a commonly used red 2-photon fluorescence labeling agent, at the same excitation wavelength. Benefiting from the long wavelength advantage in reducing scattering in both 2 and 3-photon excitation of the fluorescence pairs, we demonstrated in vivo neurovascular imaging in intact adult mouse brains through white matter and deep into the hippocampus in the somatosensory cortex.

Dual-responsive two-photon probe for specific lipid droplets near-infrared fluorescence imaging in the brain of epileptic mice

Abnormal lipid metabolism in glial cells is a key pathological feature of epilepsy. The identification of lipid droplets (LDs) is essential for investigating lipid metabolism, disease progression, and potential therapeutic interventions. Two-photon imaging technology enables real-time visualization of the spatial distribution and temporal dynamics of LDs in epilepsy models. In this study, we developed a novel two-photon excited dual-responsive near-infrared fluorescent probe, CabA, based on viscosity and polarity, to monitor dynamic changes in LDs. The fluorescence of CabA at 670 nm exhibits a significant increase in response to low polarity and high viscosity due to the twisted intramolecular charge transfer and intramolecular charge transfer mechanisms. The LDs-targeting capability of CabA at the cellular level and the process of LDs generation between neurons and astrocytes during the pathological advancement of epilepsy have been validated. In situ synchronous imaging experiments in epileptic and normal mice using CabA revealed abnormal LDs accumulation in the brain during seizures. Two-photon fluorescence imaging further demonstrated LDs accumulation in the brains of epileptic mice at a penetration depth of 100 μm. This study offers a valuable tool for enhancing the understanding of LDs in physiological and pathological processes, potentially aiding in the early diagnosis of epilepsy.

I Bind It That Way – Bioorthogonal Unmasking of Profluorescent Quinone Methides

Proof‐of‐concept studies for the bioorthogonally controlled generation of pro‐fluorescent quinone‐methides are presented here. The novel concept relies on a click‐to‐release tetrazine unit that chemically cages a pro‐fluorescent quinone methide precursor. On a synthetically readily accessible model compound we demonstrate that IEDDA reaction of the tetrazine with a TCO leads to the liberation of a pro‐fluorescent quinone methide, which is readily captured by nearby nucleophiles to form a fluorescent scaffold. Unlike in previous approaches to access bioorthogonally activatable fluorogenic probes, where the tetrazine unit acted as a quencher of fluorescence, the role of the tetrazine is redefined here. Such repurposed role of tetrazines is foreseen to address the limitations of tetrazine‐quenched red / NIR excitable fluorogenic probes.

A high‐power coaxial to rectangular waveguide transition

AbstractThe high‐power characteristic of the coaxial to waveguide transition plays an important role in modern communication and radar systems. In this letter, a novel design of a high‐power transition from a 50 Ω coaxial line with an N‐type connector to a WR‐90 rectangular waveguide. The power handling capability (PHC) of the proposed structure is significantly enhanced by a cone‐shaped excitation probe with a bold end, a transitional cavity, and a mode conversion cavity with a curved shorted back wall. The simulated results reveal that the transition possesses high PHC of 257 kW at 8.5 GHz, which is the upper limit of the power tolerance for the N‐type connector. The measured results show that the return loss and insertion loss are better than 15 and 0.15 dB, respectively, in the range of 7.96–9.16 GHz. In addition, experimental validation confirms the normal operation of the structure under a 250 kW power level test, demonstrating the applicability of the proposed transition in high‐power microwave systems.

GOALS-JWST: Mid-infrared Molecular Gas Excitation Probes the Local Conditions of Nuclear Star Clusters and the Active Galactic Nucleus in the LIRG VV 114

The enormous increase in mid-IR sensitivity and spatial and spectral resolution provided by the JWST spectrographs enables, for the first time, detailed extragalactic studies of molecular vibrational bands. This opens an entirely new window for the study of the molecular interstellar medium in luminous infrared galaxies (LIRGs). We present a detailed analysis of rovibrational bands of gas-phase CO, H2O, C2H2, and HCN toward the heavily obscured eastern nucleus of the LIRG VV 114, as observed by NIRSpec and the medium resolution spectrograph on the Mid-InfraRed Instrument (MIRI MRS). Spectra extracted from apertures of 130 pc in radius show a clear dichotomy between the obscured active galactic nucleus (AGN) and two intense starburst regions. We detect the 2.3 μm CO bandheads, characteristic of cool stellar atmospheres, in the star-forming regions, but not toward the AGN. Surprisingly, at 4.7 μm, we find highly excited CO (T ex ≈ 700–800 K out to at least rotational level J = 27) toward the star-forming regions, but only cooler gas (T ex ≈ 200 K) toward the AGN. We conclude that only mid-infrared pumping through the rovibrational lines can account for the equilibrium conditions found for CO and H2O in the deeply embedded starbursts. Here, the CO bands probe regions with an intense local radiation field inside dusty young massive star clusters or near the most massive young stars. The lack of high-excitation molecular gas toward the AGN is attributed to geometric dilution of the intense radiation from the bright point source. An overview of the relevant excitation and radiative transfer physics is provided in an appendix.

Resolving conjugated polymer film morphology with polarised transmission and time-resolved emission microscopy

The alignment of chromophores plays a crucial role in determining the optoelectronic properties of materials. Such alignment can make interpretation of fluorescence anisotropy microscopy (FAM) images somewhat ambiguous. The time-resolved emission behaviour can also influence the fluorescence anisotropy. This is particularly the case when probing excitation energy migration between chromophores in a condensed phase. Ideally information concerning the chromophoric alignment, emission decay kinetics and fluorescence anisotropy can be recorded and correlated. We report on the use of polarised transmission imaging (PTI) coupled with both steady-state and time-resolved FAM to enable accurate identification of chromophoric alignment and morphology in thin films of a conjugated polydiarylfluorene. We show that the combination of these three imaging modes presents a comprehensive methodology for investigating the alignment and morphology of chromophores in thin films, particularly for accurately mapping the distribution of amorphous and crystalline phases within the thin films, offering valuable insights for the design and optimization of materials with enhanced optoelectronic performance.

Gold nanoparticle-based two-photon fluorescent nanoprobe for monitoring intracellular nitric oxide levels

SignificanceNitric oxide (NO) is involved in the regulation of physiological and pathological mechanisms of the cardiovascular, nervous and immune systems; and plays an important role in cancer being involved in tumor growth and suppressing processes depending on its concentration. ApproachThe development of a near-infrared excitable nanoprobe, consisting of gold nanoparticles functionalized with a two-photon excitable NO probe, for the detection of intracellular NO is reported. ResultsThe nanoprobe showed good selectivity towards NO over cellular interferences and excellent stability in aqueous-medium over time. The nanoprobe was able to selectively detect endogenous and exogenous NO in different cell lines and it accumulated in the acidic organelles showing negligible toxicity. Importantly, the nanoprobe showed potential to quantify intracellular NO concentrations in breast cancer cells. ConclusionsThe novel gold-based two-photon nanoprobe showed an excellent performance and versatility and could potentially be applied for the spatiotemporal monitoring of in vivo NO levels.

Dual-Emissive Iridium(III) Complexes and Their Applications in Biological Sensing and Imaging.

Phosphorescent iridium(III) complexes are widely recognized for their unique properties in the excited triplet state, making them crucial for various applications including biological sensing and imaging. Most of these complexes display single phosphorescence emission from the lowest-lying triplet state after undergoing highly efficient intersystem crossing (ISC) and ultrafast internal conversion (IC) processes. However, in cases where these excited-state processes are restricted, the less common phenomenon of dual emission has been observed. This dual emission phenomenon presents an opportunity for developing biological probes and imaging agents with multiple emission bands of different wavelengths. Compared to intensity-based biosensing, where the existence and concentration of an analyte are indicated by the brightness of the probe, the emission profile response involves modifications in emission color. This enables quantification by utilizing the intensity ratio of different wavelengths, which is self-calibrating and unaffected by the probe concentration and excitation laser power. Moreover, dual-emissive probes have the potential to demonstrate distinct responses to multiple analytes at separate wavelengths, providing orthogonal detection capabilities. In this concept, we focus on iridium(III) complexes displaying fluorescence-phosphorescence or phosphorescence-phosphorescence dual emission, along with their applications as biological probes for sensing and imaging.

Wideband metasurface-loaded rectenna for azimuth-insensitive electromagnetic energy absorption using characteristic mode analysis

In this paper, a wideband metasurface-loaded (MTS-L) rectenna system is proposed to capture electromagnetic (EM) energy at arbitrary azimuth angles. The radiation patterns of different modes in the original MTS configuration are analyzed using the characteristic mode theory, and potential modes with omnidirectional radiation are screened out. By the arrangement of patches, the roundness performance of the radiation pattern can be ameliorated, and the omnidirectional characteristic is obtained over a wide frequency band. Subsequently, the surface current density of the selected mode is carefully and artificially designed to facilitate probe excitation as well as refrain from introducing complex power-combining networks. A wideband rectifier circuit is designed as the load of the proposed antenna. Eventually, measured results show that it operates from 4.6 to 9.6 GHz with a fractional bandwidth of 70.4%, and the peak system efficiency is 52.2%. The proposed system demonstrates excellent potential for wireless power transmission and EM energy harvesting in indoor environments.

Nonlinear imaging-based bubble cloud size estimates compared with histotripsy treatment zone: An in vitro study

Volterra filtering combined with subharmonic imaging was previously reported to enhance the visualization of the histotripsy bubble cloud. In this study, we compared the bubble cloud size obtained by imaging to the histotripsy ablation region using receiver operating characteristic (ROC) analysis. Histotripsy was performed in a red blood cell (RBC)-doped agarose phantom using a 1 MHz transducer. Ultrasound imaging data was acquired using a C5-2V probe and chirp-coded excitation (bandwidth: 2–5 MHz). These ultrasound acquisitions were then processed by subharmonic (SH) matched filtering alone, SH with quadratic Volterra filtering, and SH with cubic Volterra filtering. The therapy pulse and imaging sequence were interleaved (N = 100 frames), and a video camera was used to visualize the damaged region. A mean bubble cloud image was generated across all treatment sequences and co-registered with the camera image. A ROC curve was formed using binary classification of the mean bubble cloud versus the ablation zone, and the area under the ROC curve was determined. Filtering by quadratic and cubic Volterra filters reduced artifacts significantly [over 20 and 30 dB contrast-to-tissue enhancement (p < 0.01), respectively], along with achieving a high area under the ROC curve (0.97). These findings highlight the potential of Volterra filtering for histotripsy guidance.

Nondestructive testing method for internal defects in ferromagnetic materials under weak bias magnetization

Magnetic flux leakage (MFL) testing is effective in detecting internal defects in ferromagnetic materials. However, it requires the saturation magnetization of materials, which is not always practicable in complex inspection environments. Therefore, this study presents a novel dynamic permeability testing (DPT) method, in which internal defects will induce dynamic permeability variations on the surface of weakly magnetized ferromagnetic materials, and the variations are then detected by alternating current detection coils with magnetic cores, enabling the identification of internal defects. The principle of DPT is analyzed through simulations, which are subsequently verified by experiments. Furthermore, the effects of excitation frequency, probe lift-off, and defect size on the DPT signal are investigated experimentally. Finally, the comparison with conventional MFL demonstrates that the DPT method can effectively identify the internal defect using a weak magnetization current of 0.1 A (about 0.25 T in the steel plate), whereas MFL requires 0.5 A (about 1.32 T) to achieve a similar detection capability under the same probe lift-off values. This new method has significant practical value, especially in situations where saturation magnetization is not feasible.

Free-electron interaction with nonlinear optical states in microresonators.

The short de Broglie wavelength and strong interaction empower free electrons to probe structures and excitations in materials and biomolecules. Recently, electron-photon interactions have enabled new optical manipulation schemes for electron beams. In this work, we demonstrate the interaction of electrons with nonlinear optical states inside a photonic chip-based microresonator. Optical parametric processes give rise to spatiotemporal pattern formation corresponding to coherent or incoherent optical frequency combs. We couple such "microcombs" to electron beams, demonstrate their fingerprints in the electron spectra, and achieve ultrafast temporal gating of the electron beam. Our work demonstrates the ability to access solitons inside an electron microscope and extends the use of microcombs to spatiotemporal control of electrons for imaging and spectroscopy.

Singlet Oxygen Detection by Chemiluminescence Probes in Living Cells.

Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.

Rational design of anthocyanidins-directed near-infrared two-photon fluorescent probes.

Recently, two-photon fluorescent probes based on anthocyanidin molecules have attracted extensive attention due to their outstanding photophysical properties. However, there are only a few two-photon excited fluorescent probes that really meet the requirements of relatively long emission wavelengths (>600 nm), large two-photon absorption (TPA) cross-sections (300 GM), significant Stokes shift (>80 nm), and high fluorescence intensity. Herein, the photophysical properties of a series of anthocyanidins with the same substituents but different fluorophore skeletons are investigated in detail. Compared with b-series molecules, a-series molecules with a six-membered ring in the backbone have a slightly higher reorganization energy. This results in more energy loss upon light excitation, enabling the reaction products to detect NTR through a larger Stokes shift. More importantly, there is very little decrease in fluorescence intensity as the Stokes shift increases. These features are extremely valuable for high-resolution NTR detection. In light of this, novel 2a-n (n = 1-5) compounds are designed, which are accomplished by inhibiting the twisted intramolecular charge transfer (TICT) effect through alkyl cyclization, azetidine ring and extending π conjugation. Among them, 2a-3 gains a long emission spectrum (λem = 691.4 nm), noticeable TPA cross-section (957 GM), and large Stokes shift (110 nm), indicating that it serves as a promising candidate for two-photon fluorescent dyes. It is hoped that this work will offer some insightful theoretical direction for the development of novel high performance anthocyanin fluorescent materials.

All-optical photoacoustic detection of SF6 decomposition component SO2 based on fiber-coupled UV-LED

An all-optical photoacoustic sensor for measuring the concentration of SF6 decomposition derivative SO2 was designed, whose detection limit was in the order of ppb-level. An UV-fiber and a single-mode fiber were utilized to transmit excitation light and probe light, respectively. The technical scheme of all-optical gas sensing has realized the competitive advantages of long-distance detection and anti-electromagnetic interference. As an UV-LED was the excitation light, the challenge of designing all-optical SO2 sensor was to effectively couple UV-LED with large divergence angle into an UV- fiber. Theoretical calculation and experiment have proved that the lens assembly as an effective strategy to realize the efficient coupling and beam collimation, which can achieve high excitation power and low solid photoacoustic background interference. The lens assembly with high focusing property was the key to improve the signal-to-noise ratio (SNR) by 5.2 times. To detect photoacoustic pressure with high sensitivity, a fiber optic cantilever acoustic sensor based on F-P interference was designed. Based on the demodulation algorithm of white light interference, dynamic cavity length of F-P was demodulated with high-resolution. Allan-Werle analysis result indicate that the detection limit of SO2 gas was 48 ppb with an averaging time of 100 s

Deep ultraviolet 266 nm laser excitation for flow cytometry.

High dimensional flow cytometry relies on multiple laser sources to excite the wide variety of fluorochromes now available for immunophenotyping. Ultraviolet lasers (usually solid state 355 nm) are a critical part of this as they excite the BD Horizon™ Brilliant Ultraviolet (BUV) series of polymer fluorochromes. The BUV dyes have increased the number of simultaneous fluorochromes available for practical high-dimensional analysis to greater than 40 for spectral cytometry. Immunologists are now seeking to increase this number, requiring both novel fluorochromes and additional laser wavelengths. A laser in the deep ultraviolet (DUV) range (from ca. 260 to 320 nm) has been proposed as an additional excitation source, driven by the on-going development of additional polymer dyes with DUV excitation. DUV lasers emitting at 280 and 320 nm have been previously validated for flow cytometry but have encountered practical difficulties both in probe excitation behavior and in availability. In this article, we validate an even shorter DUV 266 nm laser source for flow cytometry. This DUV laser provided minimal excitation of the BUV dyes (a desirable characteristic for high-dimensional analysis) while demonstrating excellent excitation of quantum nanoparticles (Qdots) serving as surrogate fluorochromes for as yet undeveloped DUV excited dyes. DUV 266 nm excitation may therefore be a viable candidate for expanding high-dimensional flow cytometry into the DUV range and providing an additional incidental excitation wavelength for spectral cytometry. Excitation in a spectral region with strong absorption by nucleic acids and proteins (260-280 nm) did result in strong autofluorescence requiring care in fluorochrome selection. DUV excitation of endogenous molecules may nevertheless have additional utility for label-free analysis applications.

On the description of conical intersections between excited electronic states with LR-TDDFT and ADC(2).

Conical intersections constitute the conceptual bedrock of our working understanding of ultrafast, nonadiabatic processes within photochemistry (and photophysics). Accurate calculation of potential energy surfaces within the vicinity of conical intersections, however, still poses a serious challenge to many popular electronic structure methods. Multiple works have reported on the deficiency of methods like linear-response time-dependent density functional theory within the adiabatic approximation (AA LR-TDDFT) or algebraic diagrammatic construction to second-order [ADC(2)]-approaches often used in excited-state molecular dynamics simulations-to describe conical intersections between the ground and excited electronic states. In the present study, we focus our attention on conical intersections between excited electronic states and probe the ability of AA LR-TDDFT and ADC(2) to describe their topology and topography, using protonated formaldimine and pyrazine as two exemplar molecules. We also take the opportunity to revisit the performance of these methods in describing conical intersections involving the ground electronic state in protonated formaldimine-highlighting in particular how the intersection ring exhibited by AA LR-TDDFT can be perceived either as a (near-to-linear) seam of intersection or two interpenetrating cones, depending on the magnitude of molecular distortions within the branching space.

FRET Luminescent Probe for the Ratiometric Imaging of Peroxynitrite in Rat Brain Models of Epilepsy-Based on Organic Dye-Conjugated Iridium(III) Complex.

Epilepsy is a chronic neurological disorder characterized by recurrent seizures globally, imposing a substantial burden on patients and their families. The pathological role of peroxynitrite (ONOO-), which can trigger oxidative stress, inflammation, and neuronal hyperexcitability, is critical in epilepsy. However, the development of reliable, in situ, and real-time optical imaging tools to detect ONOO- in the brain encounters some challenges related to the depth of tissue penetration, background interference, optical bleaching, and spectral overlapping. To address these limitations, we present Ir-CBM, a new one-photon and two-photon excitable and long-lived ratiometric luminescent probe designed specifically for precise detection of ONOO- in epilepsy-based on the Förster resonance energy transfer mechanism by combining an iridium(III) complex with an organic fluorophore. Ir-CBM possesses the advantages of rapid response, one-/two-photon excitation, and ratiometric luminescent imaging for monitoring the cellular levels of ONOO- and evaluating the effects of different therapeutic drugs on ONOO- in the brain of an epilepsy model rat. The development and utilization of Ir-CBM offer valuable insights into the design of ratiometric luminescent probes. Furthermore, Ir-CBM serves as a rapid imaging and screening tool for antiepileptic drugs, thereby accelerating the exploration of novel antiepileptic drug screening and improving preventive and therapeutic strategies in epilepsy research.

Naphthylamine-based ratiometric fluorescence probe with a large Stokes shift for monitoring mitochondrial peroxynitrite

The design of ratiometric fluorescence probes with large Stokes shift is greatly significant in addressing the major challenges of detecting and imaging target substances in complex organisms. Such probes with a self-calibration function can minimize the interference of probe concentration and excitation light. In this study, a novel fluorophore of NA-Py with large Stokes shift of 158 nm, was easily obtained in one step. As a proof of concept, based on NA-Py, a fluorescence probe NA-BOH with strong intramolecular charge transfer (ICT) structural characteristics is designed for detecting peroxynitrite (ONOO−). NA-BOH exhibits red emission (627 nm) and a larger Stokes shift of 178 nm. Furthermore, it demonstrats rapid response (within 20 min) and high selectivity towards ONOO−, with ratiometric quantitative response (0–20 μM), and high sensitivity (182 nM). Alongside excellent biocompatibility and remarkable mitochondrial targeting ability, NA-BOH is successfully utilized for the proportional imaging of ONOO− in living cells and zebrafish.

Non-destructive evaluation of corrosion in steel liner plates embedded in concrete using nonlinear ultrasonics

Nuclear containment structures employ a leak-tight steel liner to avoid any unwanted particle release into the exterior environment. The steel liner can be embedded in concrete which creates large continuous areas where the steel plate is in direct contact with surrounding concrete. The impact of the aging of concrete structures due to the presence of structural discontinuities, such as the presence of foreign matter and a delamination gap at the embedded steel–concrete interface, is investigated for small laboratory-scale specimens manufactured with a novel inlay technique by nonlinear ultrasonic evaluation. A Total Damage Index (TDI) which combines several parameters related to acoustic wave distortion into a single damage index to rank the specimens is introduced. The findings in this work indicate that severe corrosion, the presence of foreign matter, and delamination gap in the steel–concrete interface can be detected using nonlinear ultrasonic techniques based on higher-harmonic analysis and modulation intensity using continuous ultrasonic probe excitation and an impactor to modulate the signal. The results obtained using the TDI are consistent with attenuation, a conventional parameter used in corrosion detection.