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 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.

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.

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.

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.

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.

New Thiophene Derivatives with Two-photon Excited Fluorescence

Two new compounds involving a thiophene moiety named as 2,5-bis[4-(N,N- diphenyl- amino)styryl]thiophene (BPST) and 2,5-bis[4-(N,N-diethylamino)styryl]thiophene (BEST) have been synthesized. The two-photon absorption cross section of BPST was measured as large as 256 × 10-50 cm4·s/photon, when it was excited by 800 nm femtosecond laser.

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.

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.

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.

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.

Two-photon Absorption and Optical Power Limiting Properties of Two Novel Dibenzothiophene-based Chromophores

Two novel V-shaped symmetric chromophores： E-2,8-bis（4-vinyl-4-carbazol-9-yl）diben- zothiophene （abbreviated as SK-G1） and E-2,8-bis（4-vinyl-4-triphenylamino） dibenzothiophene （abbreviated as ST-G1） have been synthesized and characterized. Their two photon absorption properties were measured by the open-aperture femtosecond Z-scan technique and the nanosecond nonlinear optical transmission （NLT）, respectively, when pumped by Ti： sapphire laser at 750 nm and 800 nm.

Monitoring microenvironment of Hep G2 cell apoptosis using two-photon fluorescence lifetime imaging microscopy

Apoptosis is very important for the maintenance of cellular homeostasis and is closely related to the occurrence and treatment of many diseases.Mitochondria in cells play a crucial role in programmed cell death and redox processes.Nicotinamide adenine dinucleotide(NAD(P)H)is the primary producer of energy in mitochondria,changing NAD(P)H can directly reflect the physiological state of mitochondria.Therefore,NAD(P)H can be used to evaluate metabolic response.In this paper,we propose a noninvasive detection method that uses two-photon fluorescence lifetime imaging microscopy(TP-FLIM)to characterize apoptosis by observing the binding kinetics of cellular endogenous NAD(P)H.The result shows that the average fluorescence lifetime of NAD(P)H and the fluorescence lifetime of protein-bound NAD(P)H will be affected by the changing pH,serum content,and oxygen concentration in the cell culture environment,and by the treatment with reagents such as H2O2 and paclitaxel.Taxol(PTX).This noninvasive detection method realized the dynamic detection of cellular endogenous substances and the assessment of apoptosis.

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.

Ultra-stable near-infrared Tm^(3+)-doped upconversion nanoparticles for in vivo wide-field two-photon angiography with a low excitation intensity

Two-photon luminescence with near-infrared(NIR)excitation of upconversion nanoparticles(NPs)is of great importance in biological imaging due to deep penetration in high-scattering tissues,low auto-luminescence and good sectioning ability.Unfortunately,common two-photon luminescence is in visible band with an extremely high exciation power density,which limits its application.Here,we synthesized NaYF_(4):Yb/Tm@NaYF_(4)upconversion NPs with strong twophoton NIR emission and a low excitation power density.Furthermore,NaYF_(4):Yb/Tm@NaYF_(4)@SiO_(2)@OTMS@F127 NPs with high chemical stability were obtained by a modified multilayer coating method which was applied to upconversion NPs for thefirst time.In addition,it is shown that the as-prepared hydrophillic upconversion NPs have great biocompatibility and kept stable for 6 hours during in vivo whole-body imaging.The vessels with two-photon luminescence were clear even under an excitation power density as low as 25mW/cm^(2).Vivid visualizations of capillaries and vessels in a mouse brain were also obtained with low background and high contrast.Because of cheaper instruments and safer power density,the NIR two-photon luminescence of NaYF_(4):Yb/Tm@NaYF_(4)upconversion NPs could promote wider application of two-photon technology.The modified multilayer coating method could be widely used for upconversion NPs to increase the stable time of the in vivo circulation.Our work possesses a great potential for deep imaging and imaging-guided treatment in the future.