Time-lapse changes of in vivo injured neuronal substructures in the central nervous system after low energy two-photon nanosurgery

There is currently very little research regarding the dynamics of the subcellular degenerative events that occur in the central nervous system in response to injury. To date, multi-photon excitation has been primarily used for imaging applications; however, it has been recently used to selectively disrupt neural structures in living animals. However, understanding the complicated processes and the essential underlying molecular pathways involved in these dynamic events is necessary for studying the underlying process that promotes neuronal regeneration. In this study, we introduced a novel method allowing in vivo use of low energy（less than 30 m W） two-photon nanosurgery to selectively disrupt individual dendrites, axons, and dendritic spines in the murine brain and spinal cord to accurately monitor the time-lapse changes in the injured neuronal structures. Individual axons, dendrites, and dendritic spines in the brain and spinal cord were successfully ablated and in vivo imaging revealed the time-lapse alterations in these structures in response to the two-photon nanosurgery induced lesion. The energy（less than 30 m W） used in this study was very low and caused no observable additional damage in the neuronal sub-structures that occur frequently, especially in dendritic spines, with current commonly used methods using high energy levels. In addition, our approach includes the option of monitoring the time-varying dynamics to control the degree of lesion. The method presented here may be used to provide new insight into the growth of axons and dendrites in response to acute injury.

Two-photon live imaging of direct glia-to-neuron conversion in the mouse cortex

Over the past decade,a growing number of studies have reported transcription factor-based in situ reprogramming that can directly conve rt endogenous glial cells into functional neurons as an alternative approach for n euro regeneration in the adult mammalian central ne rvous system.Howeve r,many questions remain regarding how a terminally differentiated glial cell can transform into a delicate neuron that forms part of the intricate brain circuitry.In addition,concerns have recently been raised around the absence of astrocyte-to-neuron conversion in astrocytic lineage-tra cing mice.In this study,we employed repetitive two-photon imaging to continuously capture the in situ astrocyte-to-neuron conversion process following ecto pic expression of the neural transcription factor NeuroD1 in both prolife rating reactive astrocytes and lineage-tra ced astrocytes in the mouse cortex.Time-lapse imaging over several wee ks revealed the ste p-by-step transition from a typical astrocyte with numero us short,tapered branches to a typical neuro n with a few long neurites and dynamic growth cones that actively explored the local environment.In addition,these lineage-converting cells were able to migrate ra dially or to ngentially to relocate to suitable positions.Furthermore,two-photon Ca2+imaging and patch-clamp recordings confirmed that the newly generated neuro ns exhibited synchronous calcium signals,repetitive action potentials,and spontaneous synaptic responses,suggesting that they had made functional synaptic connections within local neural circuits.In conclusion,we directly visualized the step-by-step lineage conversion process from astrocytes to functional neurons in vivo and unambiguously demonstrated that adult mammalian brains are highly plastic with respect to their potential for neuro regeneration and neural circuit reconstruction.

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.

A novelα-ketoamide reactivity-based two-photon fluorogenic probe for visualizing peroxynitrite in Parkinson's disease models

Peroxynitrite(ONOO^(-))contributes to oxidative stress and neurodegeneration in Parkinson's disease(PD).Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO^(-)flux and understanding of the ONOO^(-)stress-induced neuropathology of PD.Herein,a novelα-ketoamide-based fluorogenic probe(DFlu)was designed for ONOO^(-)
monitoring in multiple PD models.The results demonstrated that DFlu exhibits a fluorescence turn-on response to ONOO^(-)with high specificity and sensitivity.The efficacy of DFlu for intracellular ONOO^(-)
imaging was demonstrated systematically.The results showed that DFlu can successfully visualize endogenous and exogenous ONOO^(-)in cells derived from chemical and biochemical routes.More importantly,the two-photon excitation ability of DFlu has been well demonstrated by monitoring exogenous/endogenous ONOO^(-)production and scavenging in live zebraflsh PD models.This work provides a reliable and promising
α-ketoamide-based optical tool for identifying variations of ONOO^(-)in PD models.

Two-photon absorption of FAPbBr_(3) perovskite nanocrystals

Perovskite nanocrystals(NCs) with high two-photon absorption(TPA) cross-section are of great interest due to their potential applications in three-dimensional optical data storage and multiphoton fluorescence microscopy. Among various perovskite materials, FAPbBr_(3) NCs show a better development prospect due to their excellent stability. However, there are few reports on their nonlinear optical properties. In this work, the nonlinear optical behavior of FAPbBr_(3) NCs is studied.The methods of multiphoton absorption photoluminescence saturation and open aperture Z-scan technique were applied to determine the TPA cross-section of FAPbBr_(3)NCs, which was around 2.76 × 10^(-45)cm^(4)·s·photon^(-1) at 800 nm. In addition,temperature-dependent photoluminescence induced by TPA was investigated, and the small longitudinal optical phonon energy and electron–phonon coupling strength was obtained, which confirm the weak Pb–Br interaction. Meanwhile, it is found that the exciton binding energy in FAPbBr_(3) NCs was 69.668 me V, which may be ascribed to the strong hydrogen bond interaction. It is expected that our findings will promote the application of FAPbBr_(3) NCs in optoelectronic devices.

Production of χc and ηc in Ultra-Peripheral Collisions with Two-Photon Processes

We calculate the production of χc and ηc by the two-photon process in ultra-peripheral heavy ion collisions at Relativistic Heavy Ion Collider (RHIC) and Large Hadron Collider (LHC) energies. The differential cross section of transverse momentum distribution and rapidity distribution for (H = χc and ηc), are estimated by using the equivalent photon flux in the impact parameter space. The numerical results indicate that the study of χc and ηc in ultra-peripheral heavy ion collisions are feasible at RHIC and LHC energies.

Isotropic sintering shrinkage of 3D glass-ceramic nanolattices:backbone preforming and mechanical enhancement

There is a perpetual pursuit for free-form glasses and ceramics featuring outstanding mechanical properties as well as chemical and thermal resistance.It is a promising idea to shape inorganic materials in three-dimensional(3D)forms to reduce their weight while maintaining high mechanical properties.A popular strategy for the preparation of 3D inorganic materials is to mold the organic–inorganic hybrid photoresists into 3D micro-and nano-structures and remove the organic components by subsequent sintering.However,due to the discrete arrangement of inorganic components in the organic-inorganic hybrid photoresists,it remains a huge challenge to attain isotropic shrinkage during sintering.Herein,we demonstrate the isotropic sintering shrinkage by forming the consecutive–Si–O–Si–O–Zr–O–inorganic backbone in photoresists and fabricating 3D glass–ceramic nanolattices with enhanced mechanical properties.The femtosecond(fs)laser is used in two-photon polymerization(TPP)to fabricate 3D green body structures.After subsequent sintering at 1000℃,high-quality 3D glass–ceramic microstructures can be obtained with perfectly intact and smooth morphology.In-suit compression experiments and finite-element simulations reveal that octahedral-truss(oct-truss)lattices possess remarkable adeptness in bearing stress concentration and maintain the structural integrity to resist rod bending,indicating that this structure is a candidate for preparing lightweight and high stiffness glass–ceramic nanolattices.3D printing of such glasses and ceramics has significant implications in a number of industrial applications,including metamaterials,microelectromechanical systems,photonic crystals,and damage-tolerant lightweight materials.

In vivo imaging of the neuronal response to spinal cord injury:a narrative review

Deciphering the neuronal response to injury in the spinal cord is essential for exploring treatment strategies for spinal cord injury(SCI).However,this subject has been neglected in part because appropriate tools are lacking.Emerging in vivo imaging and labeling methods offer great potential for observing dynamic neural processes in the central nervous system in conditions of health and disease.This review first discusses in vivo imaging of the mouse spinal cord with a focus on the latest imaging techniques,and then analyzes the dynamic biological response of spinal cord sensory and motor neurons to SCI.We then summarize and compare the techniques behind these studies and clarify the advantages of in vivo imaging compared with traditional neuroscience examinations.Finally,we identify the challenges and possible solutions for spinal cord neuron imaging.

Attosecond ionization time delays in strong-field physics

Electronic processes within atoms and molecules reside on the timescale of attoseconds. Recent advances in the laserbased pump-probe interrogation techniques have made possible the temporal resolution of ultrafast electronic processes on the attosecond timescale, including photoionization and tunneling ionization. These interrogation techniques include the attosecond streak camera, the reconstruction of attosecond beating by interference of two-photon transitions, and the attoclock. While the former two are usually employed to study photoionization processes, the latter is typically used to investigate tunneling ionization. In this review, we briefly overview these timing techniques towards an attosecond temporal resolution of ionization processes in atoms and molecules under intense laser fields. In particular, we review the backpropagation method, which is a novel hybrid quantum-classical approach towards the full characterization of tunneling ionization dynamics. Continued advances in the interrogation techniques promise to pave the pathway towards the exploration of ever faster dynamical processes on an ever shorter timescale.

Two-photon Excited Fluorescence of Bithiophene Derivatives

Two new bithiophene derivatives named as 5, 5-bis(p-N,N-dimethylaminostyryl)-2, 2 -bithiophene (BMSBT), and 5, 5-bis(p-N,N-diethylaminostyryl)-2, 2-bithiophene (BESBT) have been synthesized. Both compounds can emit strong single-photon excited fluorescence (SPEF) and two-photon excited fluorescence (TPEF) with the emission peaks around ~560 nm and with the lifetime of ~1ns.

Level crossing in a two-photon Jaynes-Cummings model

In this paper,the energy spectrum of the two-photon Jaynes-Cummings model（TPJCM） is calculated exactly in the non-rotating wave approximation（non-RWA）,and we study the level-crossing problem by means of fidelity.A narrow peak of the fidelity is observed at the level-crossing point,which does not appear at the avoided-crossing point.Therefore fidelity is perfectly suited for detecting the level-crossing point in the energy spectrum.

Populating ^(229m)Th via two-photon electronic bridge mechanism

The isomer ^(229m)Th is the most promising candidate for clocks based on the nuclear transition because it has the lowest excitation energy of only 8.10±0.17 eV.Various experiments and theories have focused on methods of triggering the transition between the ground state and isomeric state,among which the electronic bridge(EB)is one of the most efficient.In this paper,we propose a new electronic bridge mechanism via two-photon excitation based on quantum optics for a two-level nuclear quantum system.The long-lived 7 s1/2 electronic shell state of^(229m)Th^(3+),with a lifetime of approximately 0.6 s,is chosen as the initial state and the atomic shells(7 s-10 s)could be achieved as virtual states in a two-photon process.When the virtual states return to the initial state 7 s1/2,there is a chance of triggering the nucleus 229Th^(3+),to its isomeric state ^(229m)Th ^(3+),via EB.Two lasers at moderate intensity((10^(10)-10^(14))W/m^(2)),with photon energies near the optical range,are expected to populate the isomer at a saturated rate of approximately 10^(9) s^(-1),which is much higher than that due to other mechanisms.We believe that this twophoton EB scheme can help in the development of nuclear clocks and deserves verification via a series of experiments with ordinary lasers in laboratories.

Two-photon excitation fuorescence lifetime imaging microscopy:A promising diagnostic tool for digestive tract tumors

Digestive tract tumors acount for 15%and 19.3%of the cancer incidence and deaths,respec-tively.Early detection of digestive tract tumors is crucial to the reduction of global cancer burden.Two-photon excitation fuorescence lifetime imaging microscopy(TP-FLIM)allows non-invasive,label free,three-dimensional,high-resolution imaging of living tisues with not only histological but also biochemical characterization ability in both qualitative and quantitative way.Benefiting from these advantages,this technology is protmising for clinical diagnosis of digestive tract tumors.In recent years,many efforts have'been made in this field and some remarkable progress has been achieved.In this paper,we overview the recent progress of TP-FLIM-based researches on digestive tract tumor detection.Among them,our latest results on the gastric cancer and esophageal cancer are elaborately depicted.Finally,we outlook and discuss the potential advantages and challenges of TP-FLIM in future clinical applications.

Crystal Structure, One-and Two-photon Excited Fluorescence and Bioimaging of a D-π-A Structural Triphenylamine Derivative

An electron donor-π-bridge-electron acceptor(D-π-A) optical functional organic compound comprising a triphenylamine moiety as the electron donor and pyridine moiety as the electron acceptor was synthesized. The structure of the compound was solved by single-crystal X-ray diffraction analysis. It crystallizes in monoclinic, space group P21, with a = 9.753(5), b = 8.815(5), c = 25.554(5) ?, β = 96.315(5)°, V = 2184(2) ?~3, Z = 2, D_c = 1.136 g/m^3, F(000) = 792, Μr = 746.92, μ = 0.069 mm^(-1), the final R = 0.0658 and wR = 0.1730 for 6790 observed reflections with I > 2(I). Study of nonlinear optical properties shows that the compound exhibits excellent two-photon excited fluorescence with the two-photon absorption cross-section value of 116 GM. The structure-property relationship was researched in detail through X-ray crystallography and quantum chemical calculation. Result of living cell imaging experiment shows its potential in fluorescence microscopy bioimaging.

Preparation of optimal entropy squeezing state of atomic qubit inside the cavity via two-photon process and manipulation of atomic qubit outside the cavity

Considering two atomic qubits initially in Bell states, we send one qubit into a vacuum cavity with two-photon resonance and leave the other one outside. Using quantum information entropy squeezing theory, the time evolutions of the entropy squeezing factor of the atomic qubit inside the cavity are discussed for two cases, i.e., before and after rotation and measurement of the atomic qubit outside the cavity. It is shown that the atomic qubit inside the cavity has no entropy squeezing phenomenon and is always in a decoherent state before the operating atomic qubit outside the cavity. However,the periodical entropy squeezing phenomenon emerges and the optimal entropy squeezing state can be prepared for the atomic qubit inside the cavity by adjusting the rotation angle, choosing the interaction time between the atomic qubit and the cavity, controlling the probability amplitudes of subsystem states. Its physical essence is cutting the entanglement between the atomic qubit and its environment, causing the atomic qubit inside the cavity to change from the initial decoherent state into maximum coherent superposition state, which is a possible way of recovering the coherence of a single atomic qubit in the noise environment.

A scheme for two-photon lasing with two coupled flux qubits in circuit quantum electrodynamics

We theoretically study the system of a superconducting transmission line resonator coupled to two interacting super- conducting flux qubits. It is shown that under certain conditions the resonator mode can be tuned to two-photon resonance between the ground state and the highest excited state while the middle excited states are far-off resonance. Furthermore, we study the steady-state properties of the flux qubits and resonator, such as the photon statistics, the spectrum and squeezing of the resonator, and demonstrate that two-photon laser can be implemented with current experimental technology.

Computational design of ratiometric two-photon fluorescent Zn2+probes based on quinoline and di-2-picolylamine moieties

To improve two-photon absorption(TPA)response of a newly synthesized probe,a series of ratiometric two-photon fluorescent Zn^(2+) sensors based on quinoline and DPA moieties have been designed.The one-photon absorption,TPA,and emission properties of the experimental and designed probes before and after coordination with Zn^(2+) are investigated employing the density functional theory in combination with response functions.The design consists of two levels.In the first level of design,five probes are constructed through using several electron acceptors or donors to increase accepting or donating ability of the fluorophores.It shows that all the designed probes have stronger TPA intensities at longer wavelengths with respect to the experimental probe because of the increased intra-molecular charge transfer.Moreover,it is found that the probe 4 built by adding an acyl unit has the largest TPA cross section among the designed structures due to the form of longer conjugated length and more linear backbone.One dimethylamino terminal attached along the skeleton can improve TPA intensity more efficiently than two side amino groups.Therefore,in the second level of design,a new probe 7 is formed by both an acyl unit and a dimethylamino terminal.It exhibits that the TPA cross sections of probe 7 and its zinc complex increase dramatically.Furthermore,the fluorescence quantum yields of the designed probes4 and 7 are calculated in a new way,which makes use of the relation between the computed difference of dipole moment and the measured fluorescence quantum yield.The result shows that our design also improves the fluorescence quantum yield considerably.All in all,the designed probes 4 and 7 not only possess enhanced TPA intensities but also have large differences of emission wavelength upon Zn^(2+) coordination and strong fluorescence intensity,which demonstrates that they are potential ratiometric two-photon fluorescent probes.

Label-free imaging immune cells and collagen in atherosclerosis with two-photon and second harmonic generation microscopy

Atherosclerosis has been recognized as a chronic inflammation disease,in which many types of cells participate in this process,including lymphocytes,macrophages,dendritic cells(DCs),mast cells,vascular smooth muscle cells(SMCs).Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions.The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques.Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation(SHG)were developed to image mast cells,SMCs and collagen in plaque ex vivo using endogenous optical signals.Mast cells were imaged with two-photon tryptophan autofluorescence,SMCs were imaged with two-photon NADH auto fluorescence,and collagen were imaged with SHG.This development paves the way for further study of mast cell degranulation,and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases(MMPs)which participate in the degradation of collagen.

Synthesis, Structure of a Symmetrically Substituted Stilbene with Strong Two-photon Absorption and Up-converted Blue Fluorescence

Efficient Ti-catalyzed reductive coupling methodology was first employed to synthesize the symmetrical bis-donor stilbene, trans-4, 4'-bis[diphenyl amino] stilbene (BDPAS). X-ray diffraction analyses reveal that this new crystal belongs to the triclinic crystal system of centro-symmetric P-1 space group. The DBPAS solution, with the linear transmission at wavelength of greater than or equal to 450 nm, possesses large two-photon absorption cross section as high as 39.4x10(-48) cm(4).s/photon resulting in strong two-photon induced blue fluorescence of 460 nm, pumped by 740 nm laser irradiation.

Multidither coherent optical adaptive technique for deep tissue two-photon microscopy

Two-photon microscopy normally suffers from the scattering of the tissue in biological imaging.Multidither coberent optical adaptive technique(COAT)can correct the scattered wavefront in parallel.However,the determination of the corrective phases may not be completely accurate using conventional method,which undermines the performance of this technique.In this paper,we theoretically demonstrate a method that can obtain more accurate corrective phases by determining the phase values from the square root of the fuorescence signal.A numnerical simulation model is established to study the performance of adaptive optics in two-photon micros-copy by combining scalar diffraction theory with vector diffraction theory.The results show that the distortion of the wavefront can be corrected more thoroughly with our method in two-photon imaging.In our simulation,with the scattering from a 450-mn-thick mouse brain tissue,excitation focal spots with higher peak-to background ratio(PBR)and images with higher contrast can be obtained.Hence,further enhancement of the multidither COAT correction performance in two-photon imaging can be expected.