Two-photon System Research Articles

Two-photon polymerization system based on a resonant scanner for high-throughput production of tissue engineering microscaffolds

Two-photon polymerization system based on a resonant scanner for high-throughput production of tissue engineering microscaffolds

Two-photon all-optical neurophysiology for the dissection of larval zebrafish brain functional and effective connectivity

One of the most audacious goals of modern neuroscience is unraveling the complex web of causal relations underlying the activity of neuronal populations on a whole-brain scale. This endeavor, which was prohibitive only a couple of decades ago, has recently become within reach owing to the advancements in optical methods and the advent of genetically encoded indicators/actuators. These techniques, applied to the translucent larval zebrafish have enabled recording and manipulation of the activity of extensive neuronal populations spanning the entire vertebrate brain. Here, we present a custom two-photon optical system that couples light-sheet imaging and 3D excitation with acousto-optic deflectors for simultaneous high-speed volumetric recording and optogenetic stimulation. By employing a zebrafish line with pan-neuronal expression of both the calcium reporter GCaMP6s and the red-shifted opsin ReaChR, we implemented a crosstalk-free, noninvasive all-optical approach and applied it to reconstruct the functional and effective connectivity of the left habenula.

Heralded hyper-CNOT gates for two-photon systems assisted by quantum scattering in waveguides

Photonic hyper-parallel quantum gates play a critical role in high-capacity quantum communication and fast quantum computing. Here, based on photon scattering in one-dimensional (1D) waveguides, we present some heralded schemes for constructing four-qubit hyper-controlled-not (hyper-CNOT) gates in two-photon systems. The qubits are encoded on both the polarization and spatial-mode degrees of freedoms (DOFs) of the photons, which can simplify the quantum circuit and reduce the quantum resource consumption. In our schemes, the faulty scattering events between photons and emitters caused by system imperfections can be filtered out and discarded. That is, our protocols for hyper-CNOT gates work in a heralded way. Our calculations show that, with great progress in the emitter-waveguide systems, our photonic hyper-CNOT gates may be experimentally feasible.

Kinematic analysis of multiple Compton scattering in quantum-entangled two-photon systems

The Stokes–Mueller method is used to analyze the scattering of entangled photon pairs in a two-photon system. This study examines the scenario where one of the photons, part of a pair of maximally entangled annihilation photons, undergoes intermediate Compton scattering before both photons are detected using Compton polarimeters. The method also accounts for potential quantum-decoherence effects resulting from Compton scattering. The analysis investigates the scattering behavior in both parallel and perpendicular planes, identifying variations in the modulation factor that affect azimuthal correlations. These variations include increases, decreases, sign changes, or disappearances at certain intermediate scattering angles. This work aims to provide theoretical results that support the testing and verification of predictions made by quantum field theory.

Physics of bunching and superbunching in superbunching pseudothermal light

Two-photon bunching and superbunching are important to understanding the physics of ghost imaging and second-order interference of light. With the help of the recently proposed superbunching pseudothermal light, the physics of bunching and superbunching in superbunching pseudothermal light is studied in detail. A special two-photon coincidence count detection system is designed to measure the required two-photon coincidence counts. It is concluded that the two-photon superbunching effect cannot be observed without taking the intensity changing process into account. A similar conclusion holds for two-photon bunching of thermal light. The conclusions are helpful for understanding of the physics of two-photon bunching and superbunching in intensity or photon number fluctuation correlation theory.

Simultaneous removal of noise and correction of motion warping in neuron calcium imaging using a pipeline structure of self-supervised deep learning models.

Calcium imaging is susceptible to motion distortions and background noises, particularly for monitoring active animals under low-dose laser irradiation, and hence unavoidably hinder the critical analysis of neural functions. Current research efforts tend to focus on either denoising or dewarping and do not provide effective methods for videos distorted by both noises and motion artifacts simultaneously. We found that when a self-supervised denoising model of DeepCAD [Nat. Methods18, 1359 (2021)10.1038/s41592-021-01225-0] is used on the calcium imaging contaminated by noise and motion warping, it can remove the motion artifacts effectively but with regenerated noises. To address this issue, we develop a two-level deep-learning (DL) pipeline to dewarp and denoise the calcium imaging video sequentially. The pipeline consists of two 3D self-supervised DL models that do not require warp-free and high signal-to-noise ratio (SNR) observations for network optimization. Specifically, a high-frequency enhancement block is presented in the denoising network to restore more structure information in the denoising process; a hierarchical perception module and a multi-scale attention module are designed in the dewarping network to tackle distortions of various sizes. Experiments conducted on seven videos from two-photon and confocal imaging systems demonstrate that our two-level DL pipeline can restore high-clarity neuron images distorted by both motion warping and background noises. Compared to typical DeepCAD, our denoising model achieves a significant improvement of approximately 30% in image resolution and up to 28% in signal-to-noise ratio; compared to traditional dewarping and denoising methods, our proposed pipeline network recovers more neurons, enhancing signal fidelity and improving data correlation among frames by 35% and 60% respectively. This work may provide an attractive method for long-term neural activity monitoring in awake animals and also facilitate functional analysis of neural circuits.

Analytical solution and spectral structure of the two-photon anisotropic Rabi-Stark model

Since the realization of the strong coupling between light and matter in experimental setups, the quantum Rabi model and its generalized models describing the interaction between the boson field and the two-level system have attracted extensive interest again. The study of anisotropic generalized Rabi models enables us to better understand the novel physical properties of the interaction between light and matter in the ultra-strong and deep-strong coupling regions. In this work, the two-photon anisotropic Rabi-Stark model (tpARSM) is analytically solved by using the Bogoliubov operator approach and the su(1, 1) Lie algebra. We derive the G-function, whose zeros give the regular spectrum of the system. By studying the pole structure of the G-function and the coefficients in the function, exceptional solutions, including the first-order quantum phase transition points, doubly degenerate exceptional solutions and nondegenerate exceptional solutions, are obtained. By discussing the spectral structure, we give the conditions for the first-order quantum phase transition of tpARSM. Furthermore, we find that the property that all of the lowest doubly degenerate crossing points in the two-photon Rabi-Stark model have the same energy only holds for the special case of the tpARSM in which the anisotropy parameter is equal to 1. Finally, from the perspective of first-order quantum phase transitions, concise conditions for the ground state energy level to collapse to or escape from the collapse point for the tpARSM are presented. A good understanding of the tpARSM will lay a good foundation for studying the extended two-photon systems involving multiple levels and multiple bosonic modes, and even the relevant open quantum systems.

Two-dimensional electro-optical multiphoton microscopy.

The development of genetically encoded fluorescent indicators of neural activity with millisecond dynamics has generated demand for ever faster two-photon (2P) imaging systems, but acoustic and mechanical beam scanning technologies are approaching fundamental limits. We demonstrate that potassium tantalate niobate (KTN) electro-optical deflectors (EODs), which are not subject to the same fundamental limits, are capable of ultrafast two-dimensional (2D) 2P imaging in vivo. To determine if KTN-EODs are suitable for 2P imaging, compatible with 2D scanning, and capable of ultrafast in vivo imaging of genetically encoded indicators with millisecond dynamics. The performance of a commercially available KTN-EOD was characterized across a range of drive frequencies and laser parameters relevant to in vivo 2P microscopy. A second KTN-EOD was incorporated into a dual-axis scan module, and the system was validated by imaging signals in vivo from ASAP3, a genetically encoded voltage indicator. Optimal KTN-EOD deflection of laser light with a central wavelength of 960nm was obtained up to the highest average powers and pulse intensities tested (power: 350mW; pulse duration: 118fs). Up to 32 resolvable spots per line at a 560kHz line scan rate could be obtained with single-axis deflection. The complete dual-axis EO 2P microscope was capable of imaging a by field-of-view at over 10kHz frame rate with lateral resolution. We demonstrate in vivo imaging of neurons expressing ASAP3 with high temporal resolution. We demonstrate the suitability of KTN-EODs for ultrafast 2P cellular imaging in vivo, providing a foundation for future high-performance microscopes to incorporate emerging advances in KTN-based scanning technology.

Two-site microendoscopic imaging probe for simultaneous three-dimensional imaging at two anatomic locations in tissues.

Systems that can image in three dimensions at cellular resolution and across different locations within an organism may enable insights into complex biological processes, such as immune responses, for which a single location measurement may be insufficient. In this Letter, we describe an in vivo two-site imaging probe (TIP) that can simultaneously image two anatomic sites with a maximum separation of a few centimeters. The TIP consists of two identical bendable graded index (GRIN) lenses and is demonstrated by a two-photon two-color fluorescence imaging system. Each GRIN lens has a field of view of 162 × 162 × 170 µm3, a nominal numerical aperture of 0.5, a magnification of 0.7, and working distances of 0.2 mm in air for both ends. A blind linear unmixing algorithm is applied to suppress bleedthrough between channels. We use this system to successfully demonstrate two-site two-photon two-color imaging of two biomedically relevant samples, i.e., (1) a mixture of two autofluorescent anti-cancer drugs and (2) a live hybrid tumor consisting of two spectrally distinct fluorescent cell lines.

778.1 nm distributed feedback lasers for Rb two-photon atomic systems with sub-4 kHz linewidths

778.1 nm distributed feedback lasers for Rb two-photon atomic systems with sub-4 kHz linewidths

Terahertz sensing with a 3D meta-absorbing chip based on two-photon polymerization printing

The narrowband meta-absorbers exhibit significantly enhanced electromagnetic confinement capabilities, showcasing broad application prospects in sensing fields. They can be applied for biomarker detection, chemical composition analysis, and monitoring of specific gas in the environment. In this work, we propose a 3D meta-absorber with an out-of-plane plasma mechanism based on a two-photon printing system. Compared to the conventional fabrication of a metal-insulator-metal 2D meta-absorber, the 3D absorber is composed of a metal layer and a resin layer from top to bottom; its manufacturing process is simpler, only including two-photon printing and magnetron sputtering deposition. A noticeable absorbing resonance appears at 0.3142 THz with perfect absorbance with a high Q-factor of 104.67. The theoretical sensitivity to the refractive index of the sensor reaches up to 172.5 GHz/RIU, with a figure of merit (FOM) of 19.56. In the experiments, it was validated as a meta-absorber with high sensitivity for doxycycline (DCH). As the DCH concentration increases from 0 to 4 mg/mL, the absorption intensity decreases around 49%, while the resonant frequency shift is around 70 GHz. It reflects the real-time residual content of DCH, and is potentially applied in trace antibiotic detection. The results showcase a perfect narrowband absorption capability with strong electromagnetic confinement in the terahertz spectrum, along with high-Q sensing characteristics of DCH. Compared to 2D metamaterials, the diversity of 3D metamaterial significantly expands, and introduces additional effects to provide greater flexibility in manipulating electromagnetic waves. The 3D device offers opportunities for the application of terahertz biochemical sensing.

Removing crosstalk signals in neuron activity by time multiplexed excitations in a two-photon all-optical physiology system.

The two-photon all-optical physiology system has attracted great interest in deciphering neuronal circuits in vivo, benefiting from its advantages in recording and modulating neuronal activities at single neuron resolutions. However, the interference, or crosstalk, between the imaging and photostimulation beams introduces a significant challenge and may impede the future application of voltage indicators in two-photon all-optical physiology system. Here, we propose the time multiplexed excitation method to distinguish signals from neuronal activities and crosstalks from photostimulation. In our system, the laser pulses of the imaging beam and photostimulation beam are synchronized, and a time delay is introduced into these pulses to separate the fluorescence signal generated by these two beams. We demonstrate the efficacy of our system in eliminating crosstalk signals from photostimulation and evaluate its influence on both genetically encoded calcium indicators (GECIs) and genetically encoded voltage indicators (GEVIs) through in vivo experiments.

Stokes-parameter representation for Compton scattering of entangled and classically correlated two-photon systems

A Stokes parameter representation for two-photon systems is developed to calculate cross sections for both entangled and classically correlated mixed states scattering off unpolarized and polarized Compton electrons. The cross section of Compton scattering for pairs of maximally entangled annihilation photons, generated by the disintegration of para-positronium, is compared with classical analogs. An analysis of the symmetrical properties and basis independence of these systems is conducted to elucidate the observed differences. We propose a method to establish an upper bound for identifying correlations influenced by entanglement. Furthermore, we calculate the cross section for Compton scattering of annihilation photons from spin-polarized electrons. A qualitative analysis reveals a contradiction to the contemporary assumption that Compton scattering acts as an entanglement kill switch. These findings contribute to a broader understanding of photon systems and their behavior in various scattering scenarios. Although the primary focus of this article is on Compton scattering, the formalism can be adapted to other interactions, provided that the Mueller matrix representation of the interaction is available. Published by the American Physical Society 2024

Opsin-Free Activation of Bmp Receptors by a Femtosecond Laser.

Bone morphogenetic protein (BMP) signaling plays a vital role in differentiation, organogenesis, and various cell processes. As a member of TGF-β superfamily, the BMP initiation usually accompanies crosstalk with other signaling pathways and simultaneously activates some of them. It is quite challenging to solely initiate an individual pathway. In this study, an opsin-free optical method to specifically activate BMP receptors (BMPR) and subsequent pSmad1/5/8 cascades by a single-time scan of a tightly-focused femtosecond laser in the near infrared range is reported. Via transient two-photon excitation to intrinsic local flavins near the cell membrane, the photoactivation drives conformational changes of preformed BMPR complexes to enable their bonding and phosphorylation of the GS domain in BMPR-I by BMPR-II. The pSmad1/5/8 signaling is initiated by this method, while p38 and pSmad2 are rarely perturbed. Based on a microscopic system, primary adipose-derived stem cells in an area of 420 × 420µm2 are photoactivated by a single-time laser scanning for 1.5s and exhibit pSmad1/5/8 upregulation and osteoblastic differentiation after 21 days. Hence, an opsin-free, specific, and noninvasive optical method to initiate BMP signaling, easily accomplished by a two-photon microscope system is reported.

Integration of nonlinear two-photon excited fluorescence and photocatalysis boosts overall water splitting performance.

A nonlinear two-photon excited fluorescence photocatalytic system was constructed for the first time by integrating (ZnO)1-x(GaN)x photocatalyst and a fluorescence solution of phenanthridine derivatives. This work offers a strategy for increasing the photocatalytic solar spectral utilization rate and boosting the expectation for photocatalytic solar-to-hydrogen efficiencies.

Two-Photon Light Trigger siRNA Transfection of Cancer Cells Using Non-Toxic Porous Silicon Nanoparticles.

The concept of using two-photon excitation in the NIR for the spatiotemporal control of biological processes holds great promise. However, its use for the delivery of nucleic acids has been very scarcely described and the reported procedures are not optimal as they often involve potentially toxic materials and irradiation conditions. This work prepares a simple system made of biocompatible porous silicon nanoparticles (pSiNP) for the safe siRNA photocontrolled delivery and gene silencing in cells upon two-photon excitation. PSiNP are linked to an azobenzene moiety, which possesses a lysine group (pSiNP@ICPES-azo@Lys) to efficiently complex siRNA. Non-linear excitation of the two-photon absorber system (pSiNP) followed by intermolecular energy transfer (FRET) to trans azobenzene moiety, result in the photoisomerization of the azobenzene from trans to cis and in the destabilization of the azobenzene-siRNA complex, thus inducing the delivery of the cargo siRNA to the cytoplasm of cells. Efficient silencing in MCF-7 expressing stable firefly luciferase with siRNAluc against luciferase is observed. Furthermore, siRNA against inhibitory apoptotic protein (IAP) leads to over 70% of MCF-7 cancer cell death. The developed technique using two-photon light allows a unique high spatiotemporally controlled and safe siRNA delivery in cells in few seconds of irradiation.

Sensitivity comparison of two-photon vs three-photon Rydberg electrometry

We investigate the sensitivity of co-linear three-photon electromagnetically induced transparency (EIT) in 133Cs Rydberg atoms to radio frequency electric fields and compare against the conventional two-photon system. Specifically, we model the 4-level and 5-level atomic systems and compare how the transmission of the probe changes with different laser powers and RF field strengths. In this model, we define a sensitivity metric that relates to the current best experimental implementation and assumes photon shot noise limited detection. We find that the three-photon system boasts much narrower linewidths compared to the conventional two-photon EIT. These narrow line features, however, do not align with the regions of the best sensitivity. In addition to this, we calculate the expected sensitivity for the two-photon Rydberg sensor and find that the best achievable sensitivity is over an order of magnitude better than the current measured values of 5 μVm−1Hz−1/2. However, by accounting for additional noise sources in the experiment and the quantum efficiency of the photo-detectors, the values are in good agreement.

Determining Single Photon Quantum States through Robust Waveguides on Chip

Waveguided beam splitters were microfabricated by using a commercial two-photon lithography system (Nanoscribe), Ip-Dip as the waveguides and fused silica as the substrate, and they were covered with Loctite. The gap between the waveguides in the coupler was used to determine the transmission and reflection coefficients, and our results were compared with simulation results (using OptiFDTD software). The input and output ports of the beam splitters were spliced with multimode optical fibers in a robust system that can easily be handled. Then, they were tested by leading single photons (from an SPDC) to the beam splitters to produce different quantum statistics that were rated using the Fano factor.

A simple Bessel module with tunable focal depth and constant resolution for commercial two-photon microscope

The volumetric imaging of two-photon microscopy expands the focal depth and improves the throughput, which has unparalleled superiority for three-dimension samples, especially in neuroscience. However, emerging in volumetric imaging is still largely customized, which limits the integration with commercial two-photon systems. Here, we analyzed the key parameters that modulate the focal depth and lateral resolution of polarized annular imaging and proposed a volumetric imaging module that can be directly integrated into commercial two-photon systems using conventional optical elements. This design incorporates the beam diameter adjustment settings of commercial two-photon systems, allowing flexibility to adjust the depth of focus while maintaining the same lateral resolution. Further, the depth range and lateral resolution of the design were verified, and the imaging throughput was demonstrated by an increase in the number of imaging neurons in the awake mouse cerebral cortex.

Short-wavelength excitation two-photon intravital microscopy of endogenous fluorophores.

The noninvasive two-photon excitation autofluorescence imaging of cellular and subcellular structure and dynamics in live tissue could provide critical in vivo information for biomedical studies. However, the two-photon microscopy of short-wavelength endogenous fluorophores, such as tryptophan and hemoglobin, is extremely limited due to the lack of suitable imaging techniques. In this study, we developed a short-wavelength excitation time- and spectrum-resolved two-photon microscopy system. A 520-nm femtosecond fiber laser was used as the excitation source, and a time-correlated single-photon counting module connected with a spectrograph was used to provide time- and spectrum-resolved detection capability. The system was specially designed for measuring ultraviolet and violet-blue fluorescence signals and thus was very suitable for imaging short-wavelength endogenous fluorophores. Using the system, we systematically compared the fluorescence spectra and fluorescence lifetimes of short-wavelength endogenous fluorophores, including the fluorescent molecules tyrosine, tryptophan, serotonin (5-HT), niacin (vitamin B3), pyridoxine (vitamin B6), and NADH and the protein group (keratin, elastin, and hemoglobin). Then, high-resolution three-dimensional (3D) label-free imaging of different biological tissues, including rat esophageal tissue, rat oral cheek tissue, and mouse ear skin, was performed in vivo or ex vivo. Finally, we conducted time-lapse imaging of leukocyte migration in the lipopolysaccharide injection immunization model and a mechanical trauma immunization model. The results indicate that the system can specifically characterize short-wavelength endogenous fluorophores and provide noninvasive label-free 3D visualization of fine structures and dynamics in biological systems. The microscopy system developed here can empower more flexible imaging of endogenous fluorophores and provide a novel method for the 3D monitoring of biological events in their native environment.