Two-photon Excitation Technique Research Articles

Tetraphenylethylene-based AIE nanoprobes for labeling lysosome by two-photon imaging in living cells

Lysosomes are essential cellular organelles, serving vital functions in cellular metabolism and degradation. The design of specifically targeting lysosomes probes with aggregation-induced emission (AIE) characteristics using two-photon excitation techniques is significance and challenging work. Here we designed and synthesized two tetraphenylethylene (TPE)-based AIE fluorescence probes, naming TPE-Ma and TPE-Py, with TPE as the matrix and morpholine (Ma) or pyrrolidone (Py) as the targeting group. These probes exhibit a significant Stokes shift, low cytotoxicity, two-photo fluorescence imaging and lysosome-specific targeting capability ensuring their suitability for fluorescence imaging applications. To enhance the water solubility and cellular accumulation of TPE-Ma and TPE-Py in tumor cells, we employed a biocompatible polymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) as a nanocarrier. By encapsulating TPE-Ma and TPE-Py within DSPE-mPEG2000, we successfully developed two AIE fluorescent nanoprobes known as DSPE@ TPE-Ma and DSPE@ TPE-Py. The results demonstrated that fluorescent nanoprobes DSPE@ TPE-Ma and DSPE@ TPE-Py possess excellent cell permeability, biocompatibility, superior photostability and specific targeting towards lysosomes in MCF-7 cells. Our findings highlight the potential of these fluorescent nanoprobes as effective tools for two-photon fluorescence imaging and targeted detection of lysosomes in cancer cells.

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser.

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

Luminescence Color Tuning by Regulating Electrostatic Interaction in Light-Emitting Devices and Two-Photon Excited Information Decryption.

It is well-known that the variation of noncovalent interactions of luminophores, such as π-π interaction, metal-to-metal interaction, and hydrogen-bonding interaction, can regulate their emission colors. Electrostatic interaction is also an important noncovalent interaction. However, very few examples of luminescence color tuning induced by electrostatic interaction were reported. Herein, a series of Zn(II)-bis(terpyridine) complexes (Zn-AcO, Zn-BF4, Zn-ClO4, and Zn-PF6) containing different anionic counterions were reported, which exhibit counterion-dependent emission colors from green-yellow to orange-red (549 to 622 nm) in CH2Cl2 solution. More importantly, it was found that the excited states of these Zn(II) complexes can be regulated by changing the electrostatic interaction between Zn2+ and counterions. On the basis of this controllable excited state, white light emission has been achieved by a single molecule, and a white light-emitting device has been fabricated. Moreover, a novel type of data decryption system with Zn-PF6 as the optical recording medium has been developed by the two-photon excitation technique. Our results suggest that rationally controlled excited states of these Zn(II) complexes by regulating electrostatic interaction have promising applications in various optoelectronic fields, such as light-emitting devices, information recording, security protection, and so on.

Poly β-cyclodextrin/TPdye nanomicelle-based two-photon nanoprobe for caspase-3 activation imaging in live cells and tissues.

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. β-cyclodextrin polymer (βCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/βCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

Two-Photon Graphene Oxide/Aptamer Nanosensing Conjugate for In Vitro or In Vivo Molecular Probing

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source have the unique properties of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, higher spatial resolution, and deeper penetration depth (>500 μm). Carbon nanomaterials, for example, graphene oxide (GO), have the advantages of good biocompatibility, efficient transporters into cells, protecting the carried DNA or peptides from enzymatic cleavage, and super fluorescence quenching efficiency. By combination of the nanostructured carbon materials with the TPE technique, herein we have designed an aptamer-two-photon dye (TPdye)/GO TPE fluorescent nanosensing conjugate for molecular probing in biological fluids, living cells, and zebrafish. This approach takes advantage of the exceptional quenching capability of GO for the proximate TP dyes and the higher affinity of single-stranded DNA on GO than the aptamer-target complex. Successful in vitro and in vivo detection of ATP was demonstrated with this sensing strategy. Our results reveal that the GO/Aptamer-TPdye system not only is a robust, sensitive, and selective sensor for quantitative detection of ATP in the complex biological environment but also can be efficiently delivered into live cells or tissues and act as a "signal-on" in vivo sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology model scheme for development of future carbon nanomaterial-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

Submicrometer 3D structures fabrication enabled by one-photon absorption direct laser writing

We demonstrate a new 3D fabrication method to achieve the same results as those obtained by the two-photon excitation technique, by using a simple one-photon elaboration method in a very low absorption regime. Desirable 2D and 3D submicrometric structures, such as spiral, chiral, and woodpile architectures, with feature size as small as 190 nm have been fabricated, by using just a few milliwatts of a continuous-wave laser at 532 nm and a commercial SU8 photoresist. Different aspects of the direct laser writing based on ultralow one-photon absorption (LOPA) technique are investigated and compared with the TPA technique, showing several advantages, such as simplicity and low cost.

Photoinduced Nitric Oxide Release from a Hindered Nitrobenzene Derivative by Two-Photon Excitation

Here, we demonstrated photoinduced NO generation from a 2,6-dimethylnitrobenzene-based compound (Flu-DNB) via a two-photon excitation (TPE) process. After pulse laser irradiation to a solution of Flu-DNB, oxidation products of NO were observed. This is the first account of a non-nitrosyl-chelated metal ion containing NO donor which can be controlled by the TPE technique.

Receptor Actions of Synaptically Released Glutamate: The Role of Transporters on the Scale from Nanometers to Microns

Actions of the excitatory neurotransmitter glutamate inside and outside the synaptic cleft determine the activity of neural circuits in the brain. However, to what degree local glutamate transporters affect these actions on a submicron scale remains poorly understood. Here we focus on hippocampal area CA1, a common subject of synaptic physiology studies. First, we use a two-photon excitation technique to obtain an estimate of the apparent (macroscopic) extracellular diffusion coefficient for glutamate, ∼0.32 μm 2/ms. Second, we incorporate this measurement into a Monte Carlo model of the typical excitatory synapse and examine the influence of distributed glutamate transporter molecules on signal transmission. Combined with the results of whole-cell recordings, such simulations argue that, although glutamate transporters have little effect on the activation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, this does not rule out the occurrence of up to several dozens of transporters inside the cleft. We further evaluate how the expression pattern of transporter molecules (on the 10–100 nm scale) affects the activation of N-methyl-D-aspartic acid or metabotropic glutamate receptors in the synaptic vicinity. Finally, we extend our simulations to the macroscopic scale, estimating that synaptic activity sufficient to excite principal neurons could intermittently raise extracellular glutamate to ∼1 μM only at sparse (microns apart) hotspots. Greater rises of glutamate occur only when <5% of transporters are available (for instance, when an astrocyte fails). The results provide a quantitative framework for a better understanding of the relationship between glutamate transporters and glutamate receptor signaling.

Two-photon spectroscopic behaviors and photodynamic effect on the BEL-7402 cancer cells of the new chlorophyll photosensitizer

The spectroscopic properties of a new chlorophyll derivate photosensitizer (CDP) are studied under the excitation wavelengths at 800 and 400 nm using femtosecond pulses from a Ti: sapphire laser. The damaging effect of CDP on the BEL-7402 cancer cells is also investigated upon two-photon illumination at 800 nm. The normalized fluorescence spectra of CDP in tetrahydrofuran (THF) show that two-photon and one-photon spectra have the same distributions and the same emission bands (675 nm). The lifetimes of two-and one-photon induced fluorescence of this molecule are of the order of 5.0 ns. By comparing the data it is shown that there is some difference between the two lifetimes, but the difference is less than one nanosecond. The two-photon absorption cross section of the molecule is also measured at 800 nm and estimated as about σ′2 ≈ 31.5×10−50 cm4·s·photon−1. The results of two-photon photodynamic therapy (TPPDT) tests show that CDP can kill all of the tested cancer cells according to the usual Eosine assessment. Our results indicate that the two-photon-induced photophysical, photo-chemical and photosensitizing processes of CDP may be basically similar to those of one-photon excitation. These behaviors of the sample suggest that one may find other possible methods to estimate some photosensitizers’ effects in details such as their distribution in cells and the reactive targets of the sub-cellular parts of some tumor cells via two-photon excitation techniques.

One- and two-photon excited luminescence and band-gap assignment inCaWO4

Luminescence properties of ${\mathrm{CaWO}}_{4}$ have been investigated using complementary one- and two-photon excitation techniques. Analysis of the thermal changes in the luminescence spectra indicates that the observed high-energy shift of the ${\mathrm{CaWO}}_{4}$ emission maximum is mainly caused by a change of position of the intrinsic blue band. The thermal broadening and shift of this band can be interpreted satisfactorily in terms of a model of the luminescence center interacting with the vibrating crystalline environment. The characteristic parameters of the phonon system obtained from the experiment agree with those from earlier independent studies. Intense laser stimulation of ${\mathrm{CaWO}}_{4}$ in the spectral region $&lt;505\mathrm{nm}$ $(&gt;2.45\mathrm{eV})$ results in emission with spectral and kinetics features that are characteristic of the radiative decay of a ${\mathrm{WO}}_{4}^{2\ensuremath{-}}$ oxyanion complex in this crystal. The kinetics of luminescence decay under two-photon excitation changes with increasing excitation density due to exchange interaction of the elementary excitations. From a comparative analysis of the optical properties of ${\mathrm{CaWO}}_{4}$ obtained in the course of two-photon and one-photon spectroscopic studies (absorption, reflection, one- and two-photon excitation spectra) it is concluded that the energy gap of the crystal is $5.2\ifmmode\pm\else\textpm\fi{}0.3\mathrm{eV}.$

Chapter 10 Applications of Confocal Microscopy to Studies of Sea Urchin Embryogenesis

The advent of confocal microscopy and three-dimensional reconstruction methods has further enhanced the capabilities of researchers in various fields of developmental biology. The chapter discusses the methodology required for microscopic analyses and applications of confocal microscopy. Protocols are described for using confocal microscopy and three-dimensional visualizations to investigate nuclear divisions in embryos that were fixed during cleavage. The chapter also describes time-lapse analyses of calcium dynamics, four-dimensional studies of embryogenesis, and supravital staining of nuclei as visualized by confocal microscopy employing the two-photon excitation technique. The methods for investigating the relationship between cell lineage and patterns of gene expression are discussed. Although the methods described in this chapter directly pertain to sea urchin embryos, many of the techniques can be adapted for use on other cellular and developmental systems. The greatest limitations of confocal microscopy at present are relatively slow acquisition rates and a limited choice of organelle- or molecule-specific probes that can be applied in conjunction with the most widely used types of laser illumination systems.

Depletion spectroscopy of the Sr 2 B 1Π u ← X 1Σ +g system

Spectroscopic studies have been performed on Sr 2 molecules produced in a seeded molecular beam. Two-photon excitation techniques were used to investigate excited states of Sr 2: the A←X system has been observed using two-photon ionization while depletion spectroscopy allowed us to characterize a new state of Sr 2, B 1Π u, dissociating in (5s 2) 1S+ (5s5p) 1P. Preliminary spectroscopic constants are deduced and the dissociation mechanism allowing depletion is discussed.

Temperature effect on inverse intersystem crossing of anthracenes

Abstract The temperature and heavy-atom effects on the quantum yield Φ TS of the inverse (T n → S 1 ) intersystem crossing of several anthracenes are studied by the stepwise two-photon excitation technique. The first effect is explained in terms of the relative positions of the S 1 and T 2 levels and the second effect in terms of the spin—orbit coupling between S 1 and T 2 . It was confirmed that the T n → S 1 inverse intersystem crossing occurs from T 2 to S 1 after rapid T n → T 2 internal conversion.

Vibrational overtone spectroscopy of bound and predissociative states of hydrogen peroxide cooled in a supersonic expansion

The vibrational overtone excitation spectra of both bound and predissociative states of hydrogen peroxide molecules cooled in a supersonic expansion show features that are obscured otherwise. Spectra of predissociative states are measured by detecting the decomposition product following excitation of an overtone vibration. Spectra of bound states are obtained by a two-photon excitation technique in which a second photon excites the molecule from its bound vibrational overtone state to a dissociative state. The features in the bound state (4νOH) spectrum are 0.08 to 0.13 cm−1 wide, reflecting small inhomogeneous broadening, but those to the predissociative state (6νOH) are 1.5±0.3 cm−1 wide. This width, which corresponds to a lifetime of about 3.5 ps, reflects coupling into the dissociative continuum.

The Sm ← T 1 absorption spectrum of 9,10-dibromoanthracene

The excitation spectrum for the S 1 → S 0 fluorescence of 9,10-dibromoanthracene caused by exciting the T 1 state was measured in ethanol with a stepwise two-photon excitation technique. It was compared with the T n ← T 1] absorption spectrum and the S m ← T 1] absorption spectrum was observed for the first time.

Identification of a new ion-pair state of Br2 by a sequential two-photon excitation technique

A new ion-pair state of Br2 has been identified by a sequential two-photon excitation technique through the B 3Π0u+ state. The associated UV emission spectra are analyzed as a function of excitation frequency and the new state can be assigned to f(0+g) which correlates with Br−(1S)+Br+(3P0). The spectroscopic constants for 79Br2 are Te=53 101.7 cm−1, ωe=152.8 cm−1, ωexe=0.42 cm−1, Be=0.042 60 cm−1, and re=3.167 Å.

Temperature effect on inverse (T n → S 1) intersystem crossing

The temperature effects on the quantum yields φ TS of the inverse (T n → S 1 intersystem crossings of anthracene, 9-methylanthracene and acridine are studied by the stepwise two-photon excitation technique. Φ TS of anthracene in ethanol increases with increasing temperature, while that of 9-methylanthracene does not depend on temperature, Φ TS of acridine in alkaline water decreases with increasing temperature.

Distribution of Excitonic Molecules under the Giant Two-Photon Excitation in CuCl

Line shapes of the luminescence, coexisting with the Raman scattering, of excitonic molecules (EM) have been studied in the case of the resonant excitation. The EM's are resonantly created with wave vectors, such as k =0, k =2 k 0 and k =2.3 ∼12.2 k 0 ( k 0 =4.44 ×10 5 cm -1 ), by the two-photon excitation technique. By the use of model calculations based on the Gaussian distribution in the k-space, the EM's are inferred to re-distribute anisotropically in the range | k -2 k 0 | ≤2.5 k 0 and | k | ≤1.5 k 0 for the excitation into k =2 k 0 and k =0, respectively. EM's created at large k re-distribute isotropically, and the emitted light is shown to be due to the hot luminescence during the cooling process.