One-photon Excitation Research Articles

Short-wavelength luminescence of Eu3+-doped KGd(WO4)2 crystals

Monoclinic 5 at% Eu3+ doped KGd(WO4)2 (KGW) single crystal was synthesized by top-seeded solution growth method. The spectroscopic properties and especially short-wavelength emission of Eu3+:KGW were investigated. Absorption, emission and excitation spectra were studied at low (10 K) and at room temperature. The strongest observed luminescence band corresponded to 5D0 → 7F2 transition at 610 nm but also intense ultraviolet and visible fluorescence was observed under one-photon excitation. The Ωλ parameters as well as radiative lifetimes of the 5D0,1,2,3 states were calculated based upon the Judd-Ofelt formalism. Branching ratios and electric dipole transition probabilities were also determined, based upon obtained experimental results. Stimulated emission cross sections of the strongest 5D1,2,3 → 7FJ transitions were evaluated for this system. The obtained results indicated that Eu:KGW crystal could potentially be a short wavelength laser material.

Observation of 2D semiconductor P-type dark-exciton lifetime using two-photon ultrafast spectroscopy.

We report direct measurements of intrinsic lifetimes of P-type dark-excitons in MoS2 monolayers. Using sub-gap excitation, we demonstrate two-photon excited direct population of P-type dark excitons, observe their scattering to bright states and decay with femtosecond resolution. In contrast to one-photon excitation schemes, non-monotonic density variation in bright exciton population observed under two-photon excitation shows the indirect nature of its population and competing decay pathways. Detailed modeling of different recombination pathways of bright and dark excitons allows experimental measurement of 2P dark → 1S bright exciton scattering rates. These insights into the dark states in a MoS2 monolayer pave the way for novel devices such as quantum memories and computing.

Local redox conditions in cells imaged via non-fluorescent transient states of NAD(P)H

The autofluorescent coenzyme nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH) are major determinants of cellular redox balance. Both their fluorescence intensities and lifetimes are extensively used as label-free readouts in cellular metabolic imaging studies. Here, we introduce fluorescence blinking of NAD(P)H, as an additional, orthogonal readout in such studies. Blinking of fluorophores and their underlying dark state transitions are specifically sensitive to redox conditions and oxygenation, parameters of particular relevance in cellular metabolic studies. We show that such dark state transitions in NAD(P)H can be quantified via the average fluorescence intensity recorded upon modulated one-photon excitation, so-called transient state (TRAST) monitoring. Thereby, transitions in NAD(P)H, previously only accessible from elaborate spectroscopic cuvette measurements, can be imaged at subcellular resolution in live cells. We then demonstrate that these transitions can be imaged with a standard laser-scanning confocal microscope and two-photon excitation, in parallel with regular fluorescence lifetime imaging (FLIM). TRAST imaging of NAD(P)H was found to provide additional, orthogonal information to FLIM and allows altered oxidative environments in cells treated with a mitochondrial un-coupler or cyanide to be clearly distinguished. We propose TRAST imaging as a straightforward and widely applicable modality, extending the range of information obtainable from cellular metabolic imaging of NAD(P)H fluorescence.

Two-photon excited photoluminescence of single perovskite nanocrystals.

Lead-halide perovskite nanocrystals (NCs) have emerged as a novel type of semiconductor nanostructure, attracting great research interests in both fundamental science and practical applications. Here, we compare the optical properties of single CsPbI3 NCs under both one-photon and two-photon excitations, mainly including the photoluminescence (PL) blinking and PL decay dynamics. By means of the PL saturation effect caused by multi-exciton Auger recombination, we have also estimated a two-photon absorption cross section of ∼6.8 × 106 GM for single CsPbI3 NCs. The ability to realize efficient two-photon excitation of single perovskite NCs with strongly suppressed background fluorescence will help not only to promote their bio-imaging and biolabeling applications but also to reveal and manipulate their delicate electronic structures for potential usage in quantum information processing.

Mechanistic Study on the Photogeneration of Hydrogen by Decamethylruthenocene.

Detailed studies on hydrogen evolution by decamethylruthenocene ([Cp*2 RuII ]) highlighted that metallocenes are capable of photoreducing hydrogen without the need for an additional sensitizer. Electrochemical, gas chromatographic, and spectroscopic (UV/Vis, 1 H and 13 C NMR) measurements corroborated by DFT calculations indicated that the production of hydrogen occurs by a two-step process. First, decamethylruthenocene hydride [Cp*2 RuIV (H)]+ is formed in the presence of an organic acid. Subsequently, [Cp*2 RuIV (H)]+ is reversibly reduced in a heterolytic reaction with one-photon excitation leading to a first release of hydrogen. Thereafter, the resultant decamethylruthenocenium ion [Cp*2 RuIII ]+ is further reduced with a second release of hydrogen by deprotonation of a methyl group of [Cp*2 RuIII ]+ . Experimental and computational data show spontaneous conversion of [Cp*2 RuII ] to [Cp*2 RuIV (H)]+ in the presence of protons. Calculations highlight that the first reduction is endergonic (ΔG0 =108 kJ mol-1 ) and needs an input of energy by light for the reaction to occur. The hydricity of the methyl protons of [Cp*2 RuII ] was also considered.

Red carbon dots as label-free two-photon fluorescent nanoprobes for imaging of formaldehyde in living cells and zebrafishes

Direct, in situ selective detection of intracellular formaldehyde (FA) is of great significance for understanding its function in FA-related diseases. Herein, red carbon dots (RCD) are reported as label-free two-photon fluorescent nanoprobes for detecting and imaging of FA. Upon addition of FA, the -NH2 groups of RCD could quickly and specially react with aldehydes to form Schiff base and then the strong fluorescence of RCD with blue-shift emission is recovery due to the destruction of the hydrogen bond interaction between RCD and water. In addition, the nanoprobes exhibit outstanding photostability, rapid response (<1 min), high sensitivity (∼9.9 μmol/L) and excellent selectivity toward FA over other aldehyde group compounds. Notably, owing to the good cell-membrane permeability and biocompatibility, as well as the large two-photon absorption cross-section, the as-prepared RCD can be used as label-free nanoprobes for selectively detecting and imaging FA in living cells and zebrafishes through one-photon and two-photon excitation. Moreover, RCD could stain the tissue of zebrafishes at depths interval of up to 240 μm under two-photon excitation. This research implied that RCD are promising tools for directly and in situ imaging FA in vivo, thus providing critical insights into FA-related pathophysiological processes.

The origin of the dark S1 state in carotenoids: a comprehensive model.

In carotenoids, by analogy to polyenes, the symmetry of the π-electron system is often invoked to explain their peculiar electronic features, in particular the inactivity of the S0 → S1 transition in one-photon excitation. In this review, we verify whether the molecular symmetry of carotenoids and symmetry of their π-electron system are supported in experimental and computational studies. We focus on spectroscopic techniques which are sensitive to the electron density distribution, including the X-ray crystallography, electronic absorption, two-photon techniques, circular dichroism, nuclear magnetic resonance, Stark and vibrational spectroscopies, and on this basis we seek for the origin of inactivity of the S1 state. We come across no experimental and computational evidence for the symmetry effects and the existence of symmetry restrictions on the electronic states of carotenoids. They do not possess an inversion centre and the C2h symmetry approximation of carotenoid structure is by no means justified. In effect, the application of symmetry rules (and notification) to the electronic states of carotenoids in this symmetry group may lead to a wrong interpretation of experimental data. This conclusion together with the results summarized in the review allows us to advance a consistent model that explains the inactivity of the S0 → S1 transition. Within this model, S1 is never accessible from S0 due to the negative synergy of (i) the contributions of double excitations of very low probability, which elevate S1 energy, and (ii) a non-verticality of the S0 → S1 transition, due to the breaking of Born-Oppenheimer approximation. Certainly, our simple model requires a further experimental and theoretical verification.

Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy

Fiber-optic epifluorescence imaging with one-photon excitation benefits from its ease of use, cheap light sources, and full-frame acquisition, which enables it for favorable temporal resolution of image acquisition. However, it suffers from a lack of robustness against autofluorescence and light scattering. Moreover, it cannot easily eliminate the out-of-focus background, which generally results in low-contrast images. In order to overcome these limitations, we have implemented fast out-of-phase imaging after optical modulation (Speed OPIOM) for dynamic contrast in fluorescence endomicroscopy. Using a simple and cheap optical-fiber bundle-based endomicroscope integrating modulatable light sources, we first showed that Speed OPIOM provides intrinsic optical sectioning, which restricts the observation of fluorescent labels at targeted positions within a sample. We also demonstrated that this imaging protocol efficiently eliminates the interference of autofluorescence arising from both the fiber bundle and the specimen in several biological samples. Finally, we could perform multiplexed observations of two spectrally similar fluorophores differing by their photoswitching dynamics. Such attractive features of Speed OPIOM in fluorescence endomicroscopy should find applications in bioprocessing, clinical diagnostics, plant observation, and surface imaging.

Primer to Voltage Imaging With ANNINE Dyes and Two-Photon Microscopy.

ANNINE-6 and ANNINE-6plus are voltage-sensitive dyes that when combined with two-photon microscopy are ideal for recording of neuronal voltages in vivo, in both bulk loaded tissue and the dendrites of single neurons. Here, we describe in detail but for a broad audience the voltage sensing mechanism of fast voltage-sensitive dyes, with a focus on ANNINE dyes, and how voltage imaging can be optimized with one-photon and two-photon excitation. Under optimized imaging conditions the key strengths of ANNINE dyes are their high sensitivity (0.5%/mV), neglectable bleaching and phototoxicity, a linear response to membrane potential, and a temporal resolution which is faster than the optical imaging devices currently used in neurobiology (order of nanoseconds). ANNINE dyes in combination with two-photon microscopy allow depth-resolved voltage imaging in bulk loaded tissue to study average membrane voltage oscillations and sensory responses. Alternatively, if ANNINE-6plus is applied internally, supra and sub threshold voltage changes can be recorded from dendrites of single neurons in awake animals. Interestingly, in our experience ANNINE-6plus labeling is impressively stable in vivo, such that voltage imaging from single Purkinje neuron dendrites can be performed for 2 weeks after a single electroporation of the neuron. Finally, to maximize their potential for neuroscience studies, voltage imaging with ANNINE dyes and two-photon microscopy can be combined with electrophysiological recording, calcium imaging, and/or pharmacology, even in awake animals.

High-Energy Gain Upconversion in Monolayer Tungsten Disulfide Photodetectors

Photodetectors usually operate in the wavelength range with photon energy above the bandgap of channel semiconductors so that incident photons can excite electrons from valence band to conduction band to generate photocurrent. Here, however, we show that monolayer WS2 photodetectors can detect photons with energy even lying 219 meV below the bandgap of WS2 at room temperature. With the increase of excitation wavelength from 620 to 680 nm, photoresponsivity varies from 551 to 59 mA/W. This anomalous phenomenon is ascribed to energy upconversion, which is a combination effect of one-photon excitation and multiphonon absorption through an intermediate state created most likely by sulfur divacancy with oxygen adsorption. These findings will arouse research interests on other upconversion optoelectronic devices, photovoltaic devices, for example, of monolayer transition metal dichalcogenides (TMDCs).

To Boldly Look Where No One Has Looked Before: Identifying the Primary Photoproducts of Acetylacetone.

We investigate the gas-phase photochemistry of the enolone tautomer of acetylacetone (pentane-2,4-dione) following S2(ππ*) ← S0 excitation at λ = 266 and 248 nm, using three complementary time-resolved spectroscopic methods. Contrary to earlier reports, which claimed to study one-photon excitation of acetylacetone and found OH and CH3 as the only important gas-phase products, we detect 15 unique primary photoproducts and demonstrate that five of them, including OH and CH3, arise solely by multiphoton excitation. We assign the one-photon products to six photochemical channels and show that the most significant pathway is phototautomerization to the diketone form, which is likely an intermediate in several of the other product channels. Furthermore, we measure the equilibrium constant of the tautomerization of the enolone to diketone on S0 from 320 to 600 K and extract Δ H = 4.1 ± 0.3 kcal·mol-1 and Δ S = 6.8 ± 0.5 cal·mol-1·K-1 using a van't Hoff analysis. We correct the C-OH bond dissociation energy in acetylacetone, previously determined as 90 kcal·mol-1 by theory and experiment, to a new value of 121.7 kcal·mol-1. Our experiments and electronic structure calculations provide evidence that some of the product channels, including phototautomerization, occur on S0, while others likely occur on excited triplet surfaces. Although the large oscillator strength of the S2 ← S0 transition results from the (ππ*) excitation of the C═C-C═O backbone, similar to conjugated polyenes, the participation of triplets in the dissociation pathways of acetylacetone appears to have more in common with ketone photochemistry.

A Threshold-Limited Fluorescence Probe for Viscosity.

Viscosity of body fluid is an established biomarker of pathological conditions. Abnormality of cellular viscosity occurs when cells are challenged with external stresses. Small molecule probes to assess the viscosity are sought after for both disease diagnostics and basic studies. Fluorescence based probes are particular attractive due to their potentials for convenient and high spatiotemporal resolution microscopic monitoring of biological samples. The dyes with a floppy push-pull backbone or dyes with a rotatable substituent exhibits a viscosity responsive fluorescence enhancement and therefore viable viscosity probes. The scaffold of the existing viscosity probes contains typically one such floppy site. Therefore, they typically linearly respond to log(viscosity). We argue that minor viscosity fluctuation could potentially be physiological as the biological system is dynamic. We wish to develop a type of conceptually-new, threshold-limited viscosity probes, to complement the existing probes. Such probes do not exhibit a fluorescence enhancement when challenged with minor and presumably physiological enhancement of viscosity. When the viscosity is higher than a certain threshold, their fluorescence turns on. We hypothesize that a dye with two far-apart floppy sites could potentially yield such a threshold-limited signal and designed VPZ2 and VPZ3. Through spectral titration, VPZ3 was found to yield the desired threshold-limited signal. VPZ3 was suitable for in vitro bioimaging of viscosity under one-photon or two-photon excitation. VPZ3 is potentially useful in many downstream applications. Future work includes fine-tune of the threshold to allow tailored limit for fluorescence turn-on to better meet the need of different applications. Besides the implications in the real-world applications, the design concept could also be translated to design of alternative substrates.

Generation processes of superfluorescence of biexcitons in CuCl quantum dots by one- and two-photon resonant excitation

We investigated the influence of excitation processes of a biexciton on the generation of superfluorescence, which is pulsed radiation from coherently coupled two-level systems, from biexcitons in CuCl quantum dots. Two excitation processes of biexcitons were examined: resonant two-photon excitation of biexcitons, and resonant one-photon excitation of excitons. The excitation density dependence of the time-resolved photoluminescence was measured. The results showed that for one-photon excitation, a stronger peak intensity, longer delay time and wider pulse width were observed compared to resonant two-photon excitation. The high density of the excited dots in the one-photon excitation process results in the strong pulse, while an initial population of excitons results in a suppression of coherent coupling.

Optical Characterization of Zinc Pyrithione.

Zinc pyrithione is ubiquitous in commercial products particularly antidandruff shampoos. For the efficacy of zinc pyrithione therapeutic cleansers to be assessed accurately, the distribution of particles on the scalp needs to be visualized. Currently, no technique is available which provides the chemical specificity and sensitivity required. Here, we report application of fluorescence-lifetime imaging microscopy (FLIM) for high-contrast mapping of zinc pyrithione distribution on the scalp. Characterization of the zinc pyrithione emission by using both one-photon excitation at five specific wavelengths and two-photon excitation in the range of 740-820 nm revealed its FLIM fingerprint-a characteristic short average time-weighted emission lifetime of ΤZnPT = 250 ps. Bandpass-filtering FLIM signals at ΤZnPT enabled an efficient discrimination between the zinc pyrithione and major endogenous skin species in comparison with that of the conventional reflectance confocal microscopy. Our findings provide means for in vivo high-sensitivity assaying and high-contrast imaging of zinc pyrithione in biological systems.

Two-Photon Supramolecular Nanoplatform for Ratiometric Bioimaging.

Two-photon fluorescent imaging that utilizes two near-infrared photons as an excitation source affords higher penetration depth of tissue for biomedical research, compared with one-photon fluorescent imaging. However, the high laser power levels of the excitation source may induce photobleaching of two-photon dyes and photodamage to biosamples, which hampers its wide application for in vivo imaging. Inspired by supramolecular chemistry, we have developed a two-photon excited nanoprobe (TPFN) via host-guest interaction with excellent sensitivity, selectivity, biocompatibility, water solubility, and imaging penetration depth. Notably, this supramolecular assembly can significantly amplify the fluorescence intensities of guest molecules (21-fold increase), thereby affording a detection limit of 0.127 μM for sensing H2O2, which is greatly lower than that of free guest molecules (11.98 μM). In particular, ratiometric fluorescent imaging provides more accurate analysis of intracellular H2O2 via the built-in correction of the internal reference. Importantly, TPFN excited by a two-photon laser provides higher penetration depth for visualizing H2O2 in deeper liver tissues, compared with that of one-photon excitation. Thus, TPFN can serve as a powerful nanoplatform for ratiometric imaging of various species via this facile supramolecular self-assembly strategy.

Ultrafast bulk carrier dynamics in various GaN crystals at near-infrared wavelengths under one- and two-photon absorption

Femtosecond transient absorption (TA) as a probe of ultrafast carrier dynamics was conducted at near-infrared wavelengths in a series of GaN crystals. The TA kinetics in all the GaN crystals appeared to be single exponential under one-photon (1P) excitation but biexponential under two-photon (2P) excitation, which was inconsistent with previous experimental reports and model predictions. Surface recombination and carrier diffusion could be eliminated and the TA responses were identified as phonon-assisted indirect free-carrier absorption. Modelling the bulk carrier dynamics with a simplified model revealed that, at a 1P high carrier injection level, the carrier recombination was limited by the slow capture rate of electrons via deep defects, while at a 2P low carrier injection level, the initial carrier lifetime decreased remarkably due to fast hole capturing and could be further controlled by the inherent carrier and/or dislocation concentrations.

Three-Photon Absorption of Coordination Polymer Transforms UV-to-VIS Thermometry into NIR-to-VIS Thermometry.

Lanthanide-based metal-organic frameworks (MOFs) and coordination polymers (CPs) attract much attention as candidates for optical ratiometric thermometry applications. Thus far, excitation of these materials was mainly performed in the ultraviolet that drastically limits their applicability as sensors, e.g., in tissue biological thermometry. As a remedy for this constraint, for the first time, we leverage a nonlinear optical process, the three-photon absorption property of Eu,Tb-CPs to shift the excitation wavelength from ultraviolet into near-infrared region. Experiments demonstrate that three-photon induced thermometric responses of Eu,Tb-CPs follow excellent optical characteristics similar to those determined for one-photon excitation, yet are not identical. The relative sensitivity reaches a very high value of 2.91%K-1 in the physiological temperature region. We put forward a notion that utilizing multiphoton absorption is a general strategy for realizing NIR-to-VIS remote temperature sensing in practically any CP that is designed for UV-to-VIS thermometry.

Optical properties of adult Drosophila brains in one-, two-, and three-photon microscopy.

Drosophila is widely used in connectome studies due to its small brain size, sophisticated genetic tools, and the most complete single-neuron-based anatomical brain map. Surprisingly, even the brain thickness is only 200-μm, common Ti:sapphire-based two-photon excitation cannot penetrate, possibly due to light aberration/scattering of trachea. Here we quantitatively characterized scattering and light distortion of trachea-filled tissues, and found that trachea-induced light distortion dominates at long wavelength by comparing one-photon (488-nm), two-photon (920-nm), and three-photon (1300-nm) excitations. Whole-Drosophila-brain imaging is achieved by reducing tracheal light aberration/scattering via brain-degassing or long-wavelength excitation at 1300-nm. Our work paves the way toward constructing whole-brain connectome in a living Drosophila.

Rationally designed azobenzene photoswitches for efficient two-photon neuronal excitation

Manipulation of neuronal activity using two-photon excitation of azobenzene photoswitches with near-infrared light has been recently demonstrated, but their practical use in neuronal tissue to photostimulate individual neurons with three-dimensional precision has been hampered by firstly, the low efficacy and reliability of NIR-induced azobenzene photoisomerization compared to one-photon excitation, and secondly, the short cis state lifetime of the two-photon responsive azo switches. Here we report the rational design based on theoretical calculations and the synthesis of azobenzene photoswitches endowed with both high two-photon absorption cross section and slow thermal back-isomerization. These compounds provide optimized and sustained two-photon neuronal stimulation both in light-scattering brain tissue and in Caenorhabditis elegans nematodes, displaying photoresponse intensities that are comparable to those achieved under one-photon excitation. This finding opens the way to use both genetically targeted and pharmacologically selective azobenzene photoswitches to dissect intact neuronal circuits in three dimensions.

Impact of heat treatment on NBOHC luminescence of OH-containing and H2-impregnated fused silica for deep-ultraviolet applications

OH-containing and H2-impregnated fused silica is the optical materials of choice for deep-ultraviolet (DUV) applications. Intrinsic and UV laser irradiation induced non-bridging oxygen hole center (NBOHC) defects play a central role in the optical properties of fused silica in the DUV spectra range. Understanding the NBOHC generation mechanism is of great importance to the performance improvement. In this paper, NBOHC-related 650 nm photoluminescence (PL) band is investigated in details by heat-treating the fused silica at different temperature and exciting the PL band at different wavelength. From the 650 nm PL characteristics of un-treated and heat-treated fused silica samples excited at 193 nm, 266 nm, 355 nm, and 532 nm, it is found that the 650 nm PL band of un-treated fused silica is excited at 193 nm and 266 nm mainly via a two-photon process, and cannot be excited with 355 nm and 532 nm. The un-treated fused silica also presented a reversible time delay (annealing) in 650 nm PL intensity following an abrupt change of excitation fluence at 193 nm, due to the recombination of NBOHCs with impregnated H2. On the other hand, for the heat-treated fused silica, the 650 nm PL band is excited by a combination of one- and two-photon processes at 193 nm and 266 nm with the one-photon process dominating, and by the one-photon excitation process at 355 nm and 532 nm. The quantum yield of 650 nm PL band decreases with the decreasing excitation photon energy. From these experimental results it is confirmed with no doubt that in un-treated fused silica NBOHCs are produced by 193 nm and 266 nm laser irradiation induced photolysis of an Si‒OH group via a two-photon process, with a minimum photolysis energy between 7.0 eV and 7.9 eV. In heat-treated fused silica NBOHCs are produced by heat treatment induced breakage of Si‒OH bonds. Heat treatment also removed totally the impregnated H2 in fused silica, that makes the heat-treatment produced NBOHCs become stable and show one-photon excited 650 nm PL band when excited at 193 nm, 266 nm, 355 nm, and 532 nm. In contrary to the un-treated fused silica, the heat-treated fused silica presented no reversible time delay in 650 nm PL intensity following the abrupt change of excitation fluence at 193 nm, due to the total removal of H2 in fused silica and the absence of recombination of NBOHCs with H2.