Two-photon Excitation Conditions Research Articles

Near-infrared two-photon absorption and excited state dynamics of a fluorescent diarylethene derivative

Near-infrared two-photon absorption and excited state dynamics of a fluorescent diarylethene (fDAE) derivative were investigated by time-resolved absorption and fluorescence spectroscopies. Prescreening with quantum chemical calculation predicted that a derivative with methylthienyl groups (mt-fDAE) in the closed-ring isomer has a two-photon absorption cross-section larger than 1000 GM, which was experimentally verified by Z-scan measurements and excitation power dependence in transient absorption. Comparison of transient absorption spectra under one-photon and simultaneous two-photon excitation conditions revealed that the closed-ring isomer of mt-fDAE populated into higher excited states deactivates following three pathways on a timescale of ca. 200 fs: (i) the cycloreversion reaction more efficient than that by the one-photon process, (ii) internal conversion into the S1 state, and (iii) relaxation into a lower state (S1’ state) different from the S1 state. Time-resolved fluorescence measurements demonstrated that this S1’ state is relaxed to the S1 state with the large emission probability. These findings obtained in the present work contribute to extension of the ON–OFF switching capability of fDAE to the biological window and application to super-resolution fluorescence imaging in a two-photon manner.Graphical

Blue-shifted genetically encoded Ca2+ indicator with enhanced two-photon absorption.

Genetically encoded calcium ion () indicators (GECIs) are powerful tools for monitoring intracellular concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy. We describe the development and applications of T-GECO1-a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1. We use protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon imaging in hippocampal slices. The -bound state of T-GECO1 has an excitation peak maximum of 468nm, an emission peak maximum of 500nm, an extinction coefficient of , a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The -dependent fluorescence increase is 15-fold, and the apparent for is 82nM. With two-photon excitation conditions at 850nm, T-GECO1 consistently enabled the detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant. T-GECO1 is a high-performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Near-infrared two-photon excited photoluminescence from Yb3+-doped CsPbClxBr3-x perovskite nanocrystals embedded into amphiphilic silica microspheres.

Nonlinear absorption of metal-halide perovskite nanocrystals (NCs) makes them an ideal candidate for applications which require multiphoton-excited photoluminescence. By doping perovskite NCs with lanthanides, their emission can be extended into the near-infrared (NIR) spectral region. We demonstrate how the combination of Yb3+ doping and bandgap engineering of cesium lead halide perovskite NCs performed by anion exchange (from Cl- to Br-) leads to efficient and tunable emitters that operate under two-photon excitation in the NIR spectral region. By optimizing the anion composition, Yb3+-doped CsPbClxBr3-x NCs exhibited high values of two-photon absorption cross-section reaching 2.3 × 105 GM, and displayed dual-band emission located both in the visible (407-493 nm) and NIR (985 nm). With a view of practical applications of bio-visualisation in the NIR spectral range, these NCs were embedded into silica microspheres which were further wrapped with amphiphilic polymer shells to ensure their water-compatibility. The resulting microspheres with embedded NCs could be easily dispersed in both toluene and water, while still exhibiting a dual-band emission in visible and NIR under both one- and two-photon excitation conditions.

Investigation of the biexciton radiative cascade in a single InAs/GaAs quantum dot embedded in a high-Q microcavity

A biexciton radiative cascade was recorded in the photoluminescence spectra of an InAs/GaAs QD embedded in a λ-cavity with a relatively high-quality factor of 4600, formed in a micropillar with GaAs/AlGaAs distributed Bragg reflectors. The spectrum and kinetics of the radiation, measured under conditions of two-photon excitation, demonstrated a significant effect of the microcavity on the emission process. A possible improvement based on this effect in the generation of entangled photon pairs is discussed.

Entanglement-based quantum key distribution with a blinking-free quantum dot operated at a temperature up to 20 K

Entanglement-based quantum key distribution (QKD) promises enhanced robustness against eavesdropping and compatibility with future quantum networks. Among other sources, semiconductor quantum dots (QDs) can generate polarization-entangled photon pairs with near-unity entanglement fidelity and a multiphoton emission probability close to zero even at maximum brightness. These properties have been demonstrated under resonant two-photon excitation (TPE) and at operation temperatures below 10 K. However, source blinking is often reported under TPE conditions, limiting the maximum achievable photon rate. In addition, operation temperatures reachable with compact cryocoolers could facilitate the widespread deployment of QDs, e.g., in satellite-based quantum communication. We demonstrate blinking-free emission of highly entangled photon pairs from GaAs QDs embedded in a p-i-n diode. High fidelity entanglement persists at temperatures of at least 20 K, which we use to implement fiber-based QKD between two buildings with an average raw key rate of 55 bits / s and a qubit error rate of 8.4%. We are confident that by combining electrical control with already demonstrated photonic and strain engineering, QDs will keep approaching the ideal source of entangled photons for real world applications.

Comparative photophysical properties of some widely used fluorescent proteins under two-photon excitation conditions

Understanding the photophysical properties of fluorescent proteins (FPs), such as emission and absorption spectra, molecular brightness, photostability, and photo-switching, is critical to the development of criteria for their selection as tags for fluorescent-based biological applications. While two-photon excitation imaging techniques have steadily gained popularity – due to comparatively deeper penetration depth, reduced out-of-focus photobleaching, and wide separation between emission spectra and two-photon excitation spectra –, most studies reporting on the photophysical properties of FPs tend to remain focused on single-photon excitation. Here, we report our investigation of the photophysical properties of several commonly used fluorescent proteins using two-photon microscopy with spectral resolution in both excitation and emission. Our measurements indicate that not only the excitation (and sometimes emission) spectra of FPs may be markedly different between single-photon and two-photon excitation, but also their relative brightness and their photo-stability. A good understanding of the photophysical properties of FPs under two-photon excitation is essential for choosing the right tag(s) for a desired experiment.

Environment-insensitive two-photon ratiometric probe for in cellulo quantitative measurement of hydrogen peroxide

The quantitative analysis of a biological analyte directly through fluorescence imaging is essential in biomedical sciences but remains as a challenge owing to the environment-sensitive nature of fluorescence intensity. We show that a fluorescent hydrogen peroxide probe based on an in cellulo super-bright benzothiazolyl-benzocoumarin dye of which emission properties are insensitive to environmental changes offers a reliable and practical means for the quantitative analysis of hydrogen peroxide in cells and tissues directly through ratiometric imaging both under one-photon and two-photon excitation conditions. Different cellular hydrogen peroxide concentrations were determined for several cell lines and also for tumour and normal tissues.

Biexciton initialization by two-photon excitation in site-controlled quantum dots: The complexity of the antibinding state case

In this work, we present a biexciton state population in (111)B oriented site-controlled InGaAs quantum dots (QDs) by resonant two photon excitation. We show that the excited state recombines emitting highly pure single photon pairs entangled in polarization. The discussed cases herein are compelling due to the specific energetic structure of pyramidal InGaAs QDs—an antibinding biexciton—a state with a positive binding energy. We demonstrate that resonant two-photon excitation of QDs with antibinding biexcitons can lead to a complex excitation-recombination scenario. We systematically observed that the resonant biexciton state population is competing with an acoustic-phonon assisted population of an exciton state. These findings show that under typical two-photon resonant excitation conditions, deterministic biexciton state initialization can be compromised. This complication should be taken into account by the community members aiming to utilize similar epitaxial QDs with an antibinding biexciton.

A Benzopyronin-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Lysosomal Bisulfite with Complete Spectral Separation.

Bisulfite (HSO3-), which equilibrates with sulfite (SO32-) and sulfur dioxide (SO2) in aqueous media, can be produced endogenously during oxidation of hydrogen sulfide or sulfur-containing amino acids. Lysosomes, known as the scavengers of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. Herein, we disclose a lysosome-targeting, two-photon excitable, and ratiometric signaling (near-infrared/green) fluorescent probe that detects bisulfite through a fast 1,6-conjugate addition reaction. The probe shows excellent selectivity toward bisulfite over other biologically relevant species. Notably, the probe allows ratiometric fluorescence imaging of lysosomal bisulfite with complete spectral separation under one-photon as well as two-photon excitation conditions.

Luminescence spatial characteristics of ZnSe:Fe

Two types of thermal-diffusion-based technological routes have been realized to obtain ZnSe:Fe plates demanded for laser application in the mid-IR spectral range. For the technological routes used, the iron doping process is shown to lead to the formation of auxiliary luminescence bands including (1) luminescence of residual tetrahedral donors and acceptors; (2) unknown bound exciton line located at 459 nm; (3) broad bands with maxima at 490, 520, 670, and 820 nm; and (4) IR band at 960 nm previously assigned to centers related to transition metals. Using the measurements of photoluminescence under conditions of two-photon excitation and measurements of low-temperature microphotoluminescence, the intensity profiles of the bands have been shown to be uncorrelated with the concentration profile of the optically active (Fe2+) iron. This trend does not depend on the technological routes used. Thus, heavily Fe doped areas, as well as adjacent regions in ZnSe:Fe plates produced by thermal-diffusion-based techniques, contain auxiliary centers governing luminescence in visible and near IR spectral ranges. These centers are not directly related to the optically active (Fe2+) iron.

Rational design of two-photon absorbing dicyanomethylene-4H-chromene derivatives and their application in bioimaging

In vitro and in vivo fluorescence imaging of cells and animals plays a significant role in studying biological systems. However, autofluorescence becomes a serious issue in vivo imaging under excitation at UV−vis wavelengths where biological molecules cause interference with the chromophore. In order to address this issue, a novel class of dicyanomethylene-4H-chromene (DCM) derivatives (PCM, PCV, BPCM and BPCV) that can be excited at ∼900 nm under two-photon excitation conditions and emit in the red wavelength region (≥600 nm) have been developed. Particularly, one of the DCM derivatives (PCM) shows several advantages, including remarkable two-photon absorption (2PA) properties in the near-infrared (NIR) region, superior photostability, large Stokes-shift and pronounced solvent-sensitive emission behavior, which is favorable for in vivo fluorescence labelling with a high signal-to-noise ratio.

A Dipolar Anthracene Dye: Synthesis, Optical Properties and Two-photon Tissue Imaging.

Two-photon microscopy is a powerful tool for studying biological systems. In search of novel two-photon absorbing dyes for bioimaging, we synthesized a new anthracene-based dipolar dye (anthradan) and evaluated its two-photon absorbing and imaging properties. The new anthradan, 9,10-bis(o-dimethoxy-phenyl)-anthradan, absorbs and emits at longer wavelengths than acedan, a well-known two-photon absorbing dye. It is also stable under two-photon excitation conditions and biocompatible, and thus used for two-photon imaging of mouse organ tissues to show bright, near-red fluorescence along with negligible autofluorescence. Such an anthradan thus holds promise as a new class of two-photon absorbing dyes for the development of fluorescent probes and tags for biological systems.

Two-Photon Absorbing Dyes with Minimal Autofluorescence in Tissue Imaging: Application to in Vivo Imaging of Amyloid-β Plaques with a Negligible Background Signal.

Fluorescence imaging of tissues offer an essential means for studying biological systems. Autofluorescence becomes a serious issue in tissue imaging under excitation at UV-vis wavelengths where biological molecules compete with the fluorophore. To address this critical issue, a novel class of fluorophores that can be excited at ∼900 nm under two-photon excitation conditions and emits in the red wavelength region (≥600 nm) has been disclosed. The new π-extended dipolar dye system shows several advantageous features including minimal autofluorescence in tissue imaging and pronounced solvent-sensitive emission behavior, compared with a widely used two-photon absorbing dye, acedan. As an important application of the new dye system, one of the dyes was developed into a fluorescent probe for amyloid-β plaques, a key biomarker of Alzheimer's disease. The probe enabled in vivo imaging of amyloid-β plaques in a disease-model mouse, with negligible background signal. The new dye system has great potential for the development of other types of two-photon fluorescent probes and tags for imaging of tissues with minimal autofluorescence.

Pulse-shaping based two-photon FRET stoichiometry.

Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

Nanometer-scale optical imaging of collagen fibers using gold nanoparticles.

We describe 3D single particle tracking of gold nanoparticles (AuNPs) moving along collagen fibers in aqueous environment with two-photon excitation conditions. The photoacoustic effect at the collagen fiber caused by the irradiation with ultrashort, near-infrared laser pulses propels the particles adsorbed to the surface of the collagen fibers. We report the tracking of individual AuNPs in three dimensions with high spatial and temporal resolution, of few nanometers and milliseconds, respectively. Due to the emission signal caused by the interaction between the AuNPs and the weak chromophores in the collagen fiber, the trajectories of individual AuNPs reveal the fiber topography with nanometric resolution. The intensity along the trajectory shows that we are sensitive to the distribution of the weak chromophores on the fiber.

Fluorescence Decoration of Defects in Carbon Nanotubes

Well-dispersed and functionalized carbon nanotubes exhibit visible fluorescence emissions due to passivated defects on the nanotube surface. It was found in this study that the defects in nanotubes could be decorated by an inorganic salt, which augmented the passivation effect of organic functionalization to result in dramatically enhanced emission intensities under both one- and two-photon excitation conditions. The structures and properties of the functionalized carbon nanotubes with inorganic coating were thoroughly characterized by using spectroscopy and microscopy techniques. The fluorescence decoration with the coating may serve as a tool in the study of surface defects in carbon nanotubes, and these brightly fluorescent pseudo-one-dimensional nanomaterials may be exploited for optical applications.

Carbazole tricationic salt: A novel potential two-photon fluorescent DNA probe for nucleic imaging of cells

A new carbazole tricationic salt, 4,4′-(1E,1′E)-2,2′-(9-(2-(1-(2-hydroxyethyl)pyridinium-4-yl)ethyl)-9H-carbazole-3,6-diyl) bis(ethane-2,1-diyl) bis(1-(2-hydroxyethyl)pyridinium) iodide (THEPC) was synthesized. Photophysical experiments have shown that THEPC has large two-photon excited fluorescence action cross-sections (33 GM in the presence of DNA), which ranks THEPC as a good biological fluorophore. The results from electronic absorption, circle dichroism and single-/two-photon fluorescence emission spectra suggest that THEPC can strongly bind to DNA, with an intrinsic binding constant of 5.79 × 106 L mol−1. THEPC has better photostability under one- or two-photon excitation conditions. Finally, the staining photos from two-photon fluorescence microscopy (TPM) show that THEPC can exclusively label the nucleus with high contrast and without image distortion. These remarkable properties and optimized imaging ability make THEPC an attractive DNA probe in TPM.

Sensing of Mycotoxin Producing Fungi in the Processing of Grains

Due to the growing demand for rapid, sensitive and inexpensive detection of contaminations in food and feed, both in the field and after harvest, non-invasive and non-destructive spectroscopic methods are reviewed for the direct identification of fungi and mycotoxins on grain and in grain products. Different model samples containing defined contaminations of fungi and/or mycotoxins as well as real-world samples were investigated using spectroscopic methods (steady-state and time-resolved fluorescence spectroscopy, ultraviolet/visible/near infrared (NIR) spectroscopy, two-photon-induced fluorescence (TPIF)). To evaluate the capability of TPIF for on-line and in-line mycotoxin analysis in real-world samples, fundamental parameters of different mycotoxins, such as two-photon cross-sections, were determined in beer and wine. A comparison showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon excitation conditions, and TPIF can be applied for a qualitative and quantitative analysis of mycotoxins in wine and beer. The combination of data obtained from different spectroscopic methods (such as optimised excitation and emission wavelengths, fluorescence decay times and fluorescence quantum efficiency on the one hand side and NIR spectroscopy on the other side) is promising for the qualitative as well as quantitative identification of fungi and mycotoxins. Moreover, NIR reflection spectra yield additional information on ingredients, moisture content and the presence (or absence) of fungi in the sample. The spectroscopic techniques are complemented by chemometric tools to extract the desired chemical information, e.g. with respect to the presence of contaminations.

Two-photon absorbing nanocrystal sensors for ratiometric detection of oxygen.

Two nanocrystal-osmium(II) polypyridyl (NC-Os(II)PP) conjugates have been designed to detect oxygen in biological environments. Polypyridines appended with a single free amine were linked with facility to a carboxylic acid functionality of a semiconductor NC overlayer to afford a biologically stable amide bond. The Os(II)PP complexes possess broad absorptions that extend into the red spectral region; this absorption feature makes them desirable acceptors of energy from NC donors. Fluorescence resonance energy transfer (FRET) from the NC to the Os(II)PP causes an enhanced Os(II)PP emission with a concomitant quenching of the NC emission. Owing to the large two-photon absorption cross-section of the NCs, FRET from the NC to the Os(II)PP can be established under two-photon excitation conditions. In this way, two-photon processes of metal polypyridyl complexes can be exploited for sensing. The emission of the NC is insensitive to oxygen, even at 1 atm, whereas excited states of both osmium complexes are quenched in the presence of oxygen. The NC emission may thus be used as an internal reference to correct for fluctuations in the photoluminescence intensity signal. These properties taken together establish NC-Os(II)PP conjugates as competent ratiometric, two-photon oxygen sensors for application in biological microenvironments.

Energy level structure and excited-state absorption properties of Er^3+-doped KPb_2Cl_5

We present a detailed study of the spectroscopy and fluorescence properties of the Er:KPb2Cl5 laser crystal, together with complementary data to complete the existing literature in the visible and near UV spectral domain. It deals with fluorescence decays, with anti-Stokes emission spectra and of calibration of excited-state absorption and emission spectra in the cross-section unit in the region of optical excitation around 800 nm. It brings new data for analyzing laser operation under one- and two-photon excitation conditions and for characterizing the involved energy transfer processes.