Three-photon Absorption Cross-section Research Articles

Renal-Clearable Organic Probes From D-A-D Type Aza-BODIPY Fluorophores for Multiphoton Deep-Brain Imaging.

Bright near-infrared (NIR) fluorescent probes play an important role in in vivo optical imaging. Here, renal-clearable nanodots prepared from Aza-BODIPY are reported fluorophores for multiphoton brain imaging. The design of donor-acceptor-donor (D-A-D) type conjugated structures endowed the fluorophores with large three-photon absorption cross-section for both 1620 and 2200nm excitation. The side chain modification and lipid encapsulation yield ultrasmall nanodots (≈4nm) and a high fluorescence quantum yield (≈0.35) at 720nm emission in the aqueous phase. The measured three-photon action cross-section of a single Aza-BODIPY fluorophore in the nanodots is ≈30 times higher than the commonly used Sulforhodamine 101 dye. Three-photon deep brain imaging of subcortical structures is demonstrated, reaching a depth of 1900µm below the brain surface in a live mouse study. The nanodots enabled blood flow measurement at a depth of 1617µm using line scanning three-photon microscopy (3PM). This work provides superior fluorescent probes for multiphoton deep-brain imaging.

Molecularly engineered high-performance AIE luminogen for NIR-III excitable deep-brain three-photon fluorescence imaging

Three-photon microscopy (3PM) utilizing near-infrared-III (NIR-III) excitation has emerged as an effective technology for deep-tissue bioimaging. Within this domain, organic small molecules with aggregation-induced emission (AIE) property prove to be promising candidates as high-quality 3PM imaging agents because of their exceptional optical properties and the general merit of biocompatibility that most organic small molecules possess. However, due to the bidirectional effects of the intrinsic structure distortion of AIE luminogens (AIEgens) on the three-photon absorption cross section (σ3) and fluorescence quantum yield (η), two crucial factors that determine 3PM performances, the development of high-powered AIE-active 3PM probes with robust ησ3 remains a formidable challenge. In this work, by synergistically integrating multiple beneficial principles into the molecule design, an innovative AIE-active 3PM probe with a symmetrical quadrupole D–A–D architecture, namely TIT, was successfully constructed. Owing to the harmonized planarity and distortion, as well as strong D–A interaction within TIT, the as-prepared TIT nanoparticles (NPs) exhibited boosted ησ3 under 1665 nm NIR-III excitation. This breakthrough enabled brain vascular imaging at a remarkable depth of 1895 μm after craniotomy, thus positioning TIT among the top three organic 3PM probes in terms of imaging depth. In addition, successful hemodynamic imaging at a depth of 1200 μm within the mouse brain post-craniotomy was also accomplished. These achievements establish TIT NPs as highly promising probes for in vivo deep-brain three-photon imaging.

Improving Three-Photon Fluorescence of Near-Infrared Quantum Dots for Deep Brain Imaging by Suppressing Biexciton Decay.

Three-photon fluorescence microscopy (3PFM) is a promising brain research tool with submicrometer spatial resolution and high imaging depth. However, only limited materials have been developed for 3PFM owing to the rigorous requirement of the three-photon fluorescence (3PF) process. Herein, under the guidance of a band gap engineering strategy, CdTe/CdSe/ZnS quantum dots (QDs) emitting in the near-infrared window are designed for constructing 3PF probes. The formation of type II structure significantly increased the three-photon absorption cross section of QDs and caused the delocalization of electron-hole wave functions. The time-resolved transient absorption spectroscopy confirmed that the decay of biexcitons was significantly suppressed due to the appropriate band gap alignment, which further enhanced the 3PF efficiency of QDs. By utilizing QD-based 3PF probes, high-resolution 3PFM imaging of cerebral vasculature was realized excited by a 1600 nm femtosecond laser, indicating the possibility of deep brain imaging with these 3PF probes.

Third-order nonlinear optical properties of highly electron deficient, nonplanar push-pull porphyrins: β-nitro-hexa-substituted porphyrins bearing bromo, phenyl, and phenylethynyl groups.

β-Heptasubstituted porphyrins [MTPP(NO2)X6; M = 2H, NiII, CuII, and ZnII; X = Br, Ph, and PE] were synthesized and their third-order nonlinear optical (NLO) properties explored using the single-beam Z-scan technique with femtosecond, MHz pulses in the visible range. The three-photon absorption (γ), third-order nonlinear optical susceptibility (χ3), three-photon absorption cross-section (σ3), and nonlinear refractive index (n2) have been determined from theoretical fits with experimental results. The sign and magnitude of the nonlinear refractive index (n2) have been obtained from the closed-aperture experiment while the three-photon absorption coefficient and three-photon absorption cross-section were determined from the open-aperture experiment. The magnitudes of the 3PA and σ3 extended in the range of (2.7-3.4) × 10-23 cm3 W-2 and (5.5-7.0) × 10-78 cm6 s2, respectively. The higher magnitude of the NLO coefficients ensures their utility in optical and photonic applications.

Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo.

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.

Two- and three-photon absorption in bulk CuI

We report on photoluminescence emission in copper iodide bulk single crystals induced by two- and three-photon absorption around 1.525 eV. These non-linear optical processes are investigated utilizing density-dependent, steady-state, as well as time-resolved photoluminescence spectroscopy as a function of the excitation energy. Using an excitation energy that corresponds to half of the bandgap energy, the observed photoluminescence intensity dependence on the excitation power shows an almost parabolic behavior. By further reduction of the photon energy, a cubic contribution is observable, which increases with decreasing excitation energy. The experimentally observed behavior can be well described by taking into account two- and three-photon absorption. By a simultaneous analysis of the intensity behavior for all used excitation energies, we determined a ratio between the two- and three-photon absorption cross section on the order of σ0(3)/σ0(2)≈10−28 cm2s.

A Manganese(Ⅱ) complex as high-order multiphoton fluorescent probe for detecting nitric oxide content change in idiopathic pulmonary fibrosis mice

Nitric oxide (NO) is an important gas signaling molecule, is present in the alveoli in correlation with the extent of idiopathic pulmonary fibrosis (IPF) patients. In this work, a high-order multiphoton fluorescent probe FS-MnCl2 is designed and synthesized for detecting NO level in mice model of IPF. FS-MnCl2 has the advantage of low detection limit (11 nM) and resistance to photobleaching in vitro. The three-photon absorption cross section is greatly enhanced after FS-MnCl2 recognizes NO. Moreover, the fluorescence intensity of FS-MnCl2-NO is stronger than that of FS-MnCl2 in the presence of RNA. Biological applications suggest that FS-MnCl2 can monitor changes in NO levels in living cells and IPF mice. This work provides a new strategy for reasonably designing Mn(Ⅱ) complex that can respond to NO for predicting IPF at an early stage.

Band-gap dependence of three-photon absorption in NiO nanoparticles synthesized at different calcination temperatures

The nonlinear optical properties of nickel oxide (NiO) nanoparticles were investigated using Z-scan technique with a fundamental nanosecond Nd:YAG laser. The nanoparticles were prepared using chemical precipitation method and its band-gap was varied by adopting different calcination temperature. We found an analytical result for the three-photon absorption coefficient inversely proportional to the band-gap based on the theory predicted from the wherret's equation for parabolic bands. The three-photon absorption coefficient and the extracted three-photon absorption cross-section were obtained to be 81.60 cm3/GW2 and 3.53 × 10-78 cm6 s2/photon2 at a calcination temperature of 400˚C.

Lipid droplets imaging with three-photon microscopy

Lipid droplets (LDs) participate in many physiological processes, the abnormality of which will cause chronic diseases and pathologies such as diabetes and obesity. It is crucial to monitor the distribution of LDs at high spatial resolution and large depth. Herein, we carried three-photon imaging of LDs in fat liver. Owing to the large three-photon absorption cross-section of the luminogen named NAP-CF3 ([Formula: see text][Formula: see text]cm6 s2), three-photon fluorescence fat liver imaging reached the largest depth of 80[Formula: see text][Formula: see text]m. Fat liver diagnosis was successfully carried out with excellent performance, providing great potential for LDs-associated pathologies research.

Three-Photon AIE Pt(II) Complexes as Cysteine-Targeting Theranostic Agents for Tumor Imaging and Chemotherapy

Herein, we have synthesized a series of three-photon fluorescent Pt(II) complexes targeting a tumor-associated biothiol, cysteine (Cys), which allows it to be detected without any interference from other intracellular proteins. We focused on how to significantly improve the fluorescence response of Cys via regulating the recognition units in probes. The reaction of K2PtCl4 with L-CH3 or L-COOEt in DMSO solution gave Lyso-Pt-CH3 and Lyso-Pt-COOEt, respectively, which present four-coordinated square-planar geometries in mononuclear structures. Lyso-Pt-CH3 consists of a Cys aptamer labeled with typical aggregation-induced emission (AIE) characteristics, which shows strong three-photon absorption cross section (3PA) only in the presence of Cys. It was found that Lyso-Pt-CH3 displayed a perfect signal-to-noise ratio for imaging lysosomes and for rapid detection of Cys. Using Lyso-Pt-CH3, Cys-related cellular mechanisms were proposed. We confirm that cystine (Cyss) could be absorbed in cells through cystine/glutamate antiporters (system xc-) and is then converted to Cys under the effect of enzymes. All of these suggest that Lyso-Pt-CH3 might be a potential candidate as a simple and straightforward biomarker of lysosome-related Cys in vitro. Lyso-Pt-CH3 can effectively identify tumor tissues with excessive levels of Cys. Lyso-Pt-CH3 also showed excellent antitumor activity than cisplatin. This work provides a novel strategy for the rational design of controllably activated and Cys-targeted Pt(II) anticancer prodrugs for clinical diagnosis and treatment.

Frontispiece: Giant Multi‐Photon Absorption by Heptazine Organometalation

Nonlinear Optics. In their Communication (e202208168), Mark G. Humphrey et al. report photo-stable materials with three-photon absorption cross-sections based on organometallic donors covalently attached to an s-heptazine acceptor.

Aggregation-induced emission nanoprobe assisted ultra-deep through-skull three-photon mouse brain imaging

Optical microscopy has enabled in vivo monitoring of brain structures and functions with high spatial resolution. However, the strong optical scattering in turbid brain tissue and skull impedes the observation of microvasculature and neuronal structures at a large depth. Herein, we proposed a strategy to overcome the influence induced by the high scattering effect of both skull and brain tissue via the combination of skull optical clearing (SOC) technique and three-photon fluorescence microscopy (3PM). The visible-NIR-II compatible skull optical clearing agents (VNSOCA) we applied reduced the skull scattering and water absorption in long wavelength by refractive index matching and H2O replacement to D2O respectively. 3PM with the excitation in the 1300-nm window reached 1.5 mm cerebrovascular imaging depth in cranial window assisted by a kind of bright aggregation-induced emission (AIE) nanoprobe we developed with a large three-photon absorption cross section. Combining the two advanced technologies together, we achieved so far the largest cerebrovascular imaging depth of 1.0 mm and neuronal imaging depth of> 700 µm through intact mouse skull. Dual-channel through-skull imaging of both brain vessels and neurons was also successfully realized, giving an opportunity of non-invasively monitoring the deep brain structures and functions at single-cell level simultaneously.

A potent luminogen with NIR-IIb excitable AIE features for ultradeep brain vascular and hemodynamic three-photon imaging

Three-photon excited fluorescence microscopy (3PEFM) has emerged as a promising protocol for visualizing deep-brain vasculature and hemodynamics. However, the current situation is still far from satisfactory, due to small excitation action cross-section and short excitation wavelength of those previously reported 3PEFM luminogens. Herein, we manipulated molecular engineering by subtly regulating structural planarization/twisting to achieve ingenious integration of large three-photon absorption cross-section, high fluorescence quantum yield, ultralong near-infrared IIb excitation, and aggregation-induced emission features. The resulting molecule, namely DPCZ-BT, exhibited as high as 50.6% of fluorescence quantum yield and as large as 2.0 × 10−81 cm6s2/photon2 of three-photon absorption cross-section, which can be excited by 1665 nm fs laser and presents a recorded penetration depth of 1860 μm for deep-brain vascular structural imaging with high spatiotemporal resolution and signal-to-background ratio. Moreover, DPCZ-BT having good photostability and excellent biocompatibility is capable of impressively approaching 1600 μm depth in monitoring red blood cells flow velocity with extraordinary clarity for hemodynamics.

Excellent Multiphoton Excitation Fluorescence with Large Multiphoton Absorption Cross Sections of Arginine-Modified Gold Nanoclusters for Bioimaging.

Fluorescent gold nanoclusters (Au NCs) with excellent one-photon and multiphoton properties have been demonstrated as promising candidates in many application fields. However, small multiphoton absorption (MPA) cross sections and weak multiphoton excitation (MPE) fluorescence impede their practical applications under near-infrared (NIR) excitation for biological imaging. Here, we report the regulated one-photon and multiphoton properties and mechanisms of arginine-stabilized 6-aza-2-thiothymine Au NCs (Arg/ATT-Au NCs) and the applications for MPE fluorescence imaging. The introduction of arginine into the capping layer of ATT-Au NCs significantly modifies the electronic structure, the absorption cross sections, and the relaxation dynamics of the lowest excited state, drastically reducing the nonradiative relaxation, suppressing the blinking, and greatly enhancing the fluorescence. Besides the improved one-photon properties, Arg/ATT-Au NCs demonstrate remarkable MPE fluorescence with a large MPA cross section. The two-photon (λex = 850 nm), three-photon (λex = 1400 nm), and four-photon (λex = 1700 nm) absorption cross sections have been determined to be 6.1 × 10-47 cm4 s1 photon-1, 1.5 × 10-78 cm6 s2 photon-2, and 5.5 × 10-108 cm8 s3 photon-3, respectively, much higher than those of conventional organic compounds and previously reported Au NCs. Moreover, Arg/ATT-Au NCs have been successfully applied in two-photon and three-photon excitation fluorescence imaging of living cells with NIR excitation. The manifold advantages of small size, high quantum yield, suppressed blinking, good photostability and cytocompatibility, large MPA cross sections, and excellent MPE fluorescence imaging performances make fluorescent Arg/ATT-Au NCs a great candidate of imaging probes with vis-NIR excitation.

Spectrally Resolved Nonlinear Optical Properties of Doped Versus Undoped Quasi-2D Semiconductor Nanocrystals: Copper and Silver Doping Provokes Strong Nonlinearity in Colloidal CdSe Nanoplatelets

Nonlinear optical processes are crucial for emerging applications including multiphoton-excited fluorescence microscopy and optical power limiting. Therefore, searching for materials of high multiphoton absorption cross sections is essential for the development of these techniques. We present synthesis of 4.5 monolayer CdSe nanoplatelets (NPLs) doped with silver and copper ions along with the evaluation of their two-photon absorption (TPA) and three-photon absorption (3PA) cross sections. Doping significantly increases the TPA cross section of each NPL sample, which reaches up to 1.33 × 107 GM for the most absorbing copper-doped ones. We also detected 1–2 orders of magnitude-enhanced 3PA cross sections for the doped NPLs in comparison with their undoped counterparts. As TPA and 3PA peaks appear, in the first and the second biological transmission windows, respectively, doped NPLs are promising candidates for multiphoton fluorescence microscopy as bioimaging agents. Moreover, the strong nonlinear response suggests application as active optoelectronic materials in optical sensors.

Improved One- and Multiple-Photon Excited Photoluminescence from Cd2+-Doped CsPbBr3 Perovskite NCs.

Metal halide perovskite nanocrystals (NCs) attract much attention for light-emitting applications due to their exceptional optical properties. More recently, perovskite NCs have begun to be considered a promising material for nonlinear optical applications. Numerous strategies have recently been developed to improve the properties of metal halide perovskite NCs. Among them, B-site doping is one of the most promising ways to enhance their brightness and stability. However, there is a lack of study of the influence of B-site doping on the nonlinear optical properties of inorganic perovskite NCs. Here, we demonstrate that Cd2+ doping simultaneously improves both the linear (higher photoluminescence quantum yield, larger exciton binding energy, reduced trap states density, and faster radiative recombination) and nonlinear (higher two- and three-photon absorption cross-sections) optical properties of CsPbBr3 NCs. Cd2+ doping results in a two-photon absorption cross-section, reaching 2.6 × 106 Goeppert-Mayer (GM), which is among the highest reported for CsPbBr3 NCs.

An AIE triggered fluorescence probe with three-photon absorption and its biological applications

Three-photon absorption (3 PA) in the near IR region is among the most prominent nonlinear optical (NLO) effects and has attractive applications in chemical/biological sensing and imaging. Yet, rationally constructed molecules with small molecular weight and reasonable 3 PA cross-section has been rarely reported. Herein, we designed a novel three-photon absorption photostable luminogen (namely X1) with enhanced aggregation induced emission (AIE) and the ability to achieve multi-photon imaging with femtosecond laser excitation. X1 was constructed from diaminobenzene and diethylamino salicylaldehyde forming a novel di-Schiff base. It possesses a large conjugated delocalization which exhibits large three-photon absorption (3 PA) cross-section values. We also showed that by using a suitable delivery vector, X1 compound could applied as a live cell imaging probe thus providing a valuable tool to study lipid droplets/lysosome interaction in depth tissues.

Design, multiphoton optical properties and cell imaging of D-π-A pyridine salt near-infrared materials

Accurate calibration of nucleoli in cells is of great significance for disease diagnosis and treatment. In this paper, a simple method was used to synthesize a new nuclear fluorescent probe TpsPym with excellent nonlinear optical properties. TpsPym has excellent three-photon behavior, and the three-photon absorption cross section can reach 11.49 × 10-89 cm6s2photon-2, located at the second near-infrared window (1200 nm). In vitro experimental studies have shown that TpsPym has an obvious response to RNA, mainly through hydrogen bonding and π-π stacking, inserting into RNA. TpsPym can specifically target nucleoli in cells with high specificity.

Multi-photon properties of branched chromophores derived from indenoquinoxaline and oxadiazole heterocyclic units.

Two chromophoric congeners derived from indenoquinoxaline and oxadiazole are designed, synthesized and characterized for their multi-photon properties in the femtosecond time domain. These two model structures are experimentally found to exhibit strong and widely distributed two- and three-photon activities within the spectral range of 680–1500 nm and the larger congener manifests maximum two- and three-photon absorption cross-section values of 2120 GM (at 750 nm) and ∼85 × 10−80 cm6 s2 (at 1280 nm), respectively. Both two- and three-photon absorption-based optical power-limiting performance of a representative model compound are also evaluated and demonstrated.

Role of water vapour in the absorption of nanosecond 266-nm laser pulses by atmospheric air

The absorption of the Nd : YAG fourth harmonic in air and binary mixtures of water vapour with nitrogen and oxygen at atmospheric pressure has been measured as a function of pulse energy (peak intensity). The mixtures obtained by adding equal amounts of water vapour to dry nitrogen and oxygen have been found to differ significantly in absorption. Preliminary quantitative data have been obtained for two- and three-photon absorption cross sections of water and oxygen molecules: σ(2)(H2O) = (4 ± 1) ± 10–49 cm4 s and σ(3)(O2) = (5.6 ± 1.4) ± 10–78 cm6 s2. The absorption of 266-nm pulses with peak intensities from 0.05 to 2 GW cm–2 in the near-surface atmosphere has been shown to be determined by two-photon absorption in water vapour and three-photon absorption in oxygen. In moist air containing 1 % water vapour, the absorption coefficient for 266-nm laser pulses exceeds that in dry air by four to five times. There is no absorption in nitrogen. We have developed a technique for photoacoustic measurements of multiphoton absorption cross sections in single-component gases and gas mixtures.