Two-photon excited luminescence of sulfur quantum dots for heavy metal ion detection.

Spectrally-resolved third-order nonlinear optical properties of water-dispersed sulfur quantum dots (SQDs) were investigated in the wavelength range from 740 nm to 820 nm with the two-photon excited emission technique using a tunable femtosecond laser system. The maximum value of the two-photon absorption (TPA) cross-section (σ2) for ∼5.4 nm size SQDs was found to be 185 GM (Goeppert-Mayer unit), while the two-photon brightness (σ2 × Î·) was found to be 1.5 GM at 780 nm, the wavelength being in the first biological transmittance window. The TPA properties are presented here as appropriate cross-sections normalized per molecular weight which enables meaningful comparison of the nonlinear factors of the studied quantum dots with those of various nanomaterials. The optimized TPA properties of these hydrophilic colloidal SQDs may be potentially useful for detection of Fe3+ metal ions. The experimentally determined limit of Fe3+ detection for both one- and two-photon regime was 10 µmol L-1 (0.6 µg mL-1). Förster resonance energy transfer between SQDs as donors and Fe3+ metal ions as acceptors was confirmed as one of the possible detection mechanisms using a time-correlated single photon counting technique.

Third-Order Nonlinear Optical Properties of Aqueous Silver Sulfide Quantum Dots.

Wide spectral wavelength range (500-1600 nm) measurements of nonlinear optical properties of silver sulfide (Ag2S, with 2- or 3-mercaptopropionic acid, 2 or 3MPA ligands) quantum dots (QDs) in aqueous colloidal solutions were performed using the Z-scan technique with tunable ∼55 fs laser pulses at 1 kHz. We have identified regions of the occurrence of various NLO effects including two-photon absorption, nonlinear refraction, as well as saturation of one-photon absorption. At the same time, we evaluated the relationship between the properties of the QDs and the variation of the material that covers their surface. The peak two-photon absorption cross section (σ2) values were determined to be 632 ± 271 GM (at 850 nm) for Ag2S-2MPA QDs and 772 ± 100 GM (at 875 nm) for Ag2S-3MPA QDs. The physicochemical factors influencing the three-dimensional self-organization of Ag2S QDs in water as well as their impact on spectroscopic properties were also investigated.

Strongly Emitting Folic Acid-Derived Carbon Nanodots for One- and Two-Photon Imaging of Lyotropic Myelin Figures.

Non-invasive imaging of morphological changes in biologically relevant lipidic mesophases is essential for the understanding of membrane-mediated processes. However, its methodological aspects need to be further explored, with particular attention paid to the design of new excellent fluorescent probes. Here, we have demonstrated that bright and biocompatible folic acid-derived carbon nanodots (FA CNDs) may be successfully applied as fluorescent markers in one- and two-photon imaging of bioinspired myelin figures (MFs). Structural and optical properties of these new FA CNDs were first extensively characterized; they revealed remarkable fluorescence performance in linear and non-linear excitation regimes, justifying further applications. Then, confocal fluorescence microscopy and two-photon excited fluorescence microscopy were used to investigate a three-dimensional distribution of FA CNDs within the phospholipid-based MFs. Our results showed that FA CNDs are effective markers for imaging various forms and parts of multilamellar microstructures.

Strong fluorescence-detected two-photon circular dichroism of chiral gold nanoclusters.

Progress in syntheses and understanding of the intriguing properties of chiral noble metal nanoclusters sparks interest to extend investigations of their chiroptical response to the nonlinear optics regime. We present a quantitative determination of two-photon circular dichroism of chiral gold nanoclusters with ATT and L- or D-Arg ligands (ATT = 6-aza-2-thiotymine and Arg = arginine). Introduction of arginine ligands enables the formation of two enantiomers of the nanoclusters, with strong chiroptical effects in both linear and nonlinear regime. We present two-photon absorption and luminescent properties measured in a wide range of wavelengths, with the two-photon absorption cross section reaching 1743 GM and two-photon brightness ∼1102 GM at 825 nm. We report strong, 245-fold enhancement of the two-photon circular dichroism of nanoclusters with respect to the one-photon absorption counterpart - the dissymmetry factor. The presence of multiple advantages of nanoclusters: high fluorescence quantum yield, strong nonlinear optical properties and well-controlled chirality is a powerful combination for applications of such clusters in multiphoton microscopy.

Pr3+ doped NaYF4 and LiYF4 nanocrystals combining visible-to-UVC upconversion and NIR-to-NIR-II downconversion luminescence emissions for biomedical applications.

Lanthanide-doped fluoride nanocrystals (NCs) are known to exhibit unique optical properties, such as upconversion and downconversion luminescence (UCL and DCL), which can be employed for various applications. In this work, we demonstrate that by doping praseodymium(III) and ytterbium(III) ions (Pr3+ and Yb3+) into a nanosized fluoride matrix (i.e. NaYF4 and LiYF4), it is possible to combine their UCL and DCL properties that can be concurrently used for biomedical applications. In particular, the emissive modes combined in a single nanoparticle co-doped with Pr3+ and Yb3+ include DCL emission (excited at 980 nm and peaked at 1320 nm), which can be used for near infrared (NIR) DCL bioimaging in the NIR-II window of biological tissue transparency (∼1000-1350 nm) and UCL emission (excited at 447 nm and peaked at 275 nm) that can be employed for germicide action (via irradiation by light in the UVC range). A possibility of the latter was demonstrated by the denaturation of double-stranded DNA (dsDNA) into single-stranded ones that was caused by the UVC UCL emission from the NCs under 447 nm irradiation; it was evidenced by the hyperchromicity observed in the irradiated dsDNA solution and also by a fluorometric analysis of DNA unwinding (FADU) assay. Concurrently, the possibility of NIR-II luminescence bioimaging through biological tissues (bovine tooth and chicken flesh) was demonstrated. The proposed concept paves a way for NIR-II imaging guided antimicrobial phototherapy using lanthanide-doped fluoride nanocrystals.

Non-toxic Polymeric Dots with the Strong Protein-Driven Enhancement of One- and Two-Photon Excited Emission for Sensitive and Non-destructive Albumin Sensing.

The need for efficient probing, sensing, and control of the bioactivity of biomolecules (e.g., albumins) has led to the engineering of new fluorescent albumins' markers fulfilling very specific chemical, physical, and biological requirements. Here, we explore acetone-derived polymer dots (PDs) as promising candidates for albumin probes, with special attention paid to their cytocompatibility, two-photon absorption properties, and strong ability to non-destructively interact with serum albumins. The PDs show no cytotoxicity and exhibit high photostability. Their pronounced green fluorescence is observed upon both one-photon excitation (OPE) and two-photon excitation (TPE). Our studies show that both OPE and TPE emission responses of PDs are proteinaceous environment-sensitive. The proteins appear to constitute a matrix for the dispersion of fluorescent PDs, limiting both their aggregation and interactions with the aqueous environment. It results in a large enhancement of PD fluorescence. Meanwhile, the PDs do not interfere with the secondary protein structures of albumins, nor do they induce their aggregation, enabling the PD candidates to be good nanomarkers for non-destructive probing and sensing of albumins.

Linear and Nonlinear Optical Properties of Azobenzene Derivatives Modified with an (Amino)naphthalene Moiety.

The design of two-photon absorbing azobenzene (AB) derivatives has received much attention; however, the two-photon absorption (2PA) properties of bis-conjugated azobenzene systems are relatively less explored. Here, we present the synthesis of six azobenzene derivatives and three bis-azobenzenes substituted (or not) at para position(s) with one or two amino group(s). Their linear and nonlinear absorption properties are studied experimentally and theoretically. The switching behavior and thermal stability of the Z-isomer are studied for unsubstituted mono- (1a, 2a) and bis-azobenzene (3a) compounds, showing that when the length of the π system increases, the half-life of the Z-isomer decreases. Moreover, along with the increase of π-conjugation, the photochromic characteristics are impaired and the photostationary state (PSS) related to E-Z photoisomerization is composed of 89% of the Z-isomer for 2a and 26% of the Z-isomer for 3a. Importantly, the 2PA cross-section increases almost five-fold on extending the π-conjugation (2a vs 3a) and by about one order of magnitude when comparing two systems: the unsubstituted π-electron one (2a, 3a) with D-π-D (2c, 3c). This work clarifies the contribution of π-conjugation and substituent effects to the linear and nonlinear optical properties of mono- and bis-azobenzene compounds based on the experimental and theoretical approaches.

Two-photon absorption of 28-hetero-2,7-naphthiporphyrins: expanded carbaporphyrinoid macrocycles.

The one- and two-photon absorption (1PA and 2PA) properties of three expanded aceneporphyrinoids, 28-thia-, 28-selena- and 28-tellura-2,7-naphthiporphyrin, have been studied. The open-aperture Z-scan technique was used to determine two-photon absorption cross-sections in the near infrared range using an amplified femtosecond laser system. The maximum values of the cross sections were found to be 99, 200 and 650 GM at 900 nm and 1, 13 and 31 GM at 1400 nm for the three investigated compounds, respectively. These results demonstrate enhanced 2PA properties compared with well-known porphyrin photosensitizers, such as Foscan®, showing the potential of porphyrin core modification for optimizing infrared nonlinear absorbers.

One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids.

Autofluorescence properties of amyloid fibrils are of much interest but, to date, the attention has been given mostly to one-photon excited fluorescence (1PEF), while the two-photon excited fluorescence (2PEF) properties of amyloids are much less explored. We investigate 1PEF and 2PEF of hen egg-white lysozyme (HEWL) in the form of monomers and fibrils. HEWL monomers feature some autofluorescence, which is enhanced in the case of fibrils. Moreover, by varying NaCl content, we introduce changes to fibrils morphology and show how the increase of the salt concentration is linked with an increase of 1PEF and 2PEF intensities. Interestingly, we observe 2PEF emission red-shifted in comparison to 1PEF. We confirm the presence of different relaxation pathways upon one- or two-photon excitation by different lifetimes of the fluorescence decays. Finally, we correlate the changes in optical properties of HEWL fibrils and monomers with salt-mediated changes in their morphology and the secondary structure.

Nonlinear Optical Absorption in Nanoscale Films Revealed through Ultrafast Acoustics.

Herein we describe a novel spinning pump-probe photoacoustic technique developed to study nonlinear absorption in thin films. As a test case, an organic polycrystalline thin film of quinacridone, a well-known pigment, with a thickness in the tens of nanometers range, is excited by a femtosecond laser pulse which generates a time-domain Brillouin scattering signal. This signal is directly related to the strain wave launched from the film into the substrate and can be used to quantitatively extract the nonlinear optical absorption properties of the film itself. Quinacridone exhibits both quadratic and cubic laser fluence dependence regimes which we show to correspond to two- and three-photon absorption processes. This technique can be broadly applied to materials that are difficult or impossible to characterize with conventional transmittance-based measurements including materials at the nanoscale, prone to laser damage, with very weak nonlinear properties, opaque, or highly scattering.

Prospects for More Efficient Multi-Photon Absorption Photosensitizers Exhibiting Both Reactive Oxygen Species Generation and Luminescence.

The use of two-photon absorption (TPA) for such applications as microscopy, imaging, and photodynamic therapy (PDT) offers several advantages over the usual one-photon excitation. This creates a need for photosensitizers that exhibit both strong two-photon absorption and the highly efficient generation of reactive oxygen species (ROS), as well as, ideally, bright luminescence. This review focuses on different strategies utilized to improve the TPA properties of various multi-photon absorbing species that have the required photophysical properties. Along with well-known families of photosensitizers, including porphyrins, we also describe other promising organic and organometallic structures and more complex systems involving organic and inorganic nanoparticles. We concentrate on the published studies that provide two-photon absorption cross-section values and the singlet oxygen (or other ROS) and luminescence quantum yields, which are crucial for potential use within PDT and diagnostics. We hope that this review will aid in the design and modification of novel TPA photosensitizers, which can help in exploiting the features of nonlinear absorption processes.

Two-photon absorption properties of multipolar triarylamino/tosylamido 1,1,4,4-tetracyanobutadienes.

The synthesis and characterization of four new tetracyanobutadiene (TCBD) derivatives (1, 3c and 4b-c) incorporating tosylamido and 4-triphenylamino moieties are reported. Along with those of five closely related or differently branched TCBDs derivatives (2, 3a-b, 4c and 5), their linear and (third-order) nonlinear optical properties were investigated by electronic absorption spectroscopy and Z-scan measurements. Among these compounds, the tri-branched compounds 3c and 5 are the most active two-photon absorbers, with effective cross-sections of 275 and 350 GM at 900 nm, respectively. These properties are briefly discussed with the help of DFT calculations, focussing on structural and electronic factors, and contextualized with results obtained previously for related compounds.

Two-Photon Excited Polarization-Dependent Autofluorescence of Amyloids as a Label-Free Method of Fibril Organization Imaging.

Amyloids are broadly investigated protein misfolding products with characteristic ß-sheet assemblies that have an important role in neurodegenerative diseases (e.g., Alzheimer's disease). While they are usually visualized by staining with Thioflavin-T, Congo Red, or other fluorescent markers, it still arouses a controversy over possible staining molecule influence on the amyloid structure or aggregation process. In this work we present, for the first time, the polarization analysis of two-photon excited autofluorescence of amyloids and confirm that polarization dependence of the observed emission can be correlated with the orientation of fibrils. We show the potential of two-photon excited autofluorescence for resolution of molecular organization of fibrils within amyloid superstructures. This label-free method is compatible with two-photon imaging already applied in investigation of neurodegeneration model in mice.

One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues.

The solvatochromic fluorophore Nile Red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is one of the most commonly used stains to enhance contrast of lipid-rich areas of microscopic biosamples. Quite surprisingly, relatively little is known about the spectrally-resolved two-photon absorption (2PA) properties of this dye despite its promising features for two-photon microscopy of biological matter. For this reason, the two-photon solvatochromism of Nile Red still remains an uncharted territory as well. Also, no study has yet reported on how electron-withdrawing substituents attached to the Nile Red backbone affect its solvatochromic properties and two-photon brightness. In this paper, we demonstrate how solvent polarity influences the one- and two-photon absorption spectra of Nile Red as well as its fluorescence parameters, and we present new analogues that contain -CF3, -F and -Br substituents on its eastern side. Two-photon excited fluorescence experiments in a broad spectral range (780-1240 nm) and electronic structure calculations show that both the nature and location of the substituent have particular influence on the strength of 2PA, peaking in all cases at approx. 860 and 1050 nm. 2PA cross sections are higher at 1050 nm than at 860 nm, which suggests that Nile Red and its analogues are best suited for two-photon imaging employing excitation in the NIR-II optical transparency window of biological tissues.

Nonlinear Optical Pigments. Two-Photon Absorption in Crosslinked Conjugated Polymers and Prospects for Remote Nonlinear Optical Thermometry.

Nonlinear optical (NLO) pigments are compounds insoluble in solvents that exhibit phenomena related to nonlinear optical susceptibilities (χ(n) where n = 2,3,...), e.g., two-photon absorption (2PA) which is related to the imaginary part of χ(3). Determination of spectrally-resolved 2PA properties for NLO pigments of macromolecular nature, such as coordination polymers or crosslinked polymers, has long been a challenging issue due to their particulate form, precluding characterizations with standard techniques such as Z-scan. In this contribution, we investigate thus far unknown spectrally-resolved 2PA properties of a new subclass of NLO pigments-crosslinked conjugated polymers. The studied compounds are built up from electron-donating (triphenylamine) and electron-withdrawing (2,2'-bipyridine) structural fragments joined by vinylene (Pol1) or vinyl(4-ethynylphenyl) (Pol2) aromatic bridges. 2PA properties of these polymers have been characterized in broad spectral range by specially modified two-photon excited fluorescence (TPEF) techniques: solid state TPEF (SSTPEF) and internal standard TPEF (ISTPEF). The impact of self-aggregation of aromatic backbones on the 2PA properties of the polymers has been evaluated through extended comparisons of NLO parameters, i.e., 2PA cross sections (σ2) and molar-mass normalized 2PA merit factors (σ2/M) with those of small-molecular model compounds: Mod1 and Mod2. By doing this, we found that the 2PA response of Pol1 and Pol2 is improved 2-3 times versus respective model compounds in the solid state form. Further comparisons with 2PA results collected for diluted solutions of Mod1 and Mod2 supports the notion that self-aggregated structure contributes to the observed enhancement of 2PA response. On the other hand, it is clear that Pol1 and Pol2 suffer from aggregation-caused quenching phenomenon, well reflected in time-resolved fluorescence properties as well as in relatively low values of quantum yield of fluorescence. Accordingly, despite improved intrinsic 2PA response, the effective intensity of two-photon excited emission for Pol1 and Pol2 is slightly lower relative to Mod1 and Mod2. Finally, we explore temperature-resolved luminescence properties under one- (377 nm), two- (820 nm), and three-photon excitation (1020 nm) conditions of postsynthetically Eu3+-functionalized material, Pol1-Eu, and discuss its suitability for temperature sensing applications.

The Two-Photon Absorption Cross-Section Studies of CsPbX3 (X = I, Br, Cl) Nanocrystals.

The CsPbX3 nanocrystals (NCs) with X = I, Br, Cl, or the mixture of Br:I and Br:Cl in a 1:1 ratio were synthesized and characterized by TEM, DLS, and XRD. Recrystallization of the small luminescent NCs in the metastable cubic phase into bigger orthorhombic nanocrystals was monitored by XRD and identified as the main cause of the nanocolloid coagulation. The recrystallization also leads to a decrease in the photoluminescence quantum yield (QY) of the colloidal solution and shortening of the emission lifetime. The two-photon absorption cross-section σ2 values calculated from femtosecond Z-scan measurements were compared with those obtained based on the two-photon excited emission technique. These two techniques were shown to be equivalent with the cross-section values calculated per molar mass of CsPbX3 perovskite being in the range of 10-200 GM depending on the halide anions X-. The σ2 values recalculated for the mole of the NCs were in the range of 104-105 GM, which is in good agreement with values previously reported elsewhere and the σ2/M parameter was in the range of 0.01 to 0.33. This study shows the perovskite NCs to be a good nonlinear material with the third-order nonlinear optical susceptibility χ(3) of the NCs on the order of 10-11 esu.

Functional CdS-Au Nanocomposite for Efficient Photocatalytic, Photosensitizing, and Two-Photon Applications.

We demonstrate a low-temperature synthesis of hydrophilic, penicillamine-stabilized hybrid CdS-Au nanoparticles (NPs) utilizing different Au concentrations. The obtained hybrid nanomaterials exhibit photoluminescence quenching and emission lifetime reduction in comparison with their raw semiconductor CdS NPs counterparts. An increase of concentration of Au present at the surface of CdS leads to lower photoluminescence intensity and faster emission decays, suggesting more efficient charge separation when larger Au domains are present. For photocatalysis studies, we performed methylene blue (MB) absorption measurements under irradiation in the presence of CdS-Au NPs. After 1 h of light exposure, we observed the absorbance decrease to about 35% and 10% of the initial value for the CdS-5Au and CdS-7.5Au (the hybrid NPs obtained in a presence of 5.0 and 7.5 mM Au), respectively, which indicates MB reduction caused by electrons effectively separated from holes on metal surface. In further similar photocatalysis experiments, we measured bovine serum albumin (BSA) integrated photoluminescence intensity quenching in the presence of CdS-Au NPs, with a 50% decrease being obtained for CdS-2.5Au NPs and CdS-5Au NPs, with a faster response rate detected for the system prepared with a higher Au concentration. The results suggest hole-driven reactive oxygen species (ROS) production, causing BSA degeneration. Finally, we performed two-photon excited emission (TPEE) measurements for CdS-5Au NPs, obtaining their two-photon absorption (TPA) cross-section values up to 15.8 × 103 GM (Goeppert-Mayer units). We conclude that the obtained water-soluble CdS-Au NPs exhibit potential triple functionalities as photocatalysts for reduction and oxidation reactions as well as materials for two-photon absorption applications, so that they may be considered as future theranostics.

Triarylisocyanurate-Based Fluorescent Two-Photon Absorbers.

The synthesis and characterization of six triarylisocyanurates, featuring 2,7-fluorenyl or 9,10-anthracenyl groups incorporated in their peripheral arms are reported. Photophysical studies reveal that these new octupolar derivatives are more fluorescent (ΦF ≥0.60 for all new compounds except for 1,3,5-tri(9H-fluoren-2-yl)-1,3,5-triazinane-2,4,6-trione 3) and present a red-shifted lowest absorption and emission compared to their known phenyl analogues of comparable size. Depending on the nature of the terminal substituent, fast intramolecular energy transfer among the three arms or localization of the excitation on a single branch occurs after population of their first singlet excited state. The latter effect was only observed in the presence of strongly electron-releasing substituents in polar media. These new chromophores are also better two-photon absorbers than the 1,4-phenylene-based isocyanurates reported so far, with cross sections σ2 ≥500 GM at 770 nm for 4-NPh2 the fluorenyl group containing (13) and the anthracenyl group containing (14) chromophores. All these spectroscopic features, analyzed with the help of quantum chemical calculations, are crucial for the design of new biphotonic fluorescent dyes.

Acetone-derived luminescent polymer dots: a facile and low-cost synthesis leads to remarkable photophysical properties.

Carbon-based dots have been attracting much attention as potentially superior alternatives to more conventional semiconductor nanoparticles, due to their fascinating optical properties, chemical and photochemical stability, unique environmental-friendliness, and the versatility of fabrication routes. Many commercial materials and organic compounds have been considered so far as carbon precursors but in many cases the fabrication required high-temperature conditions or led to inhomogeneous final products. Here we report on a simple low-cost synthesis of non-conjugated carbon-rich polymer dots (PDs) that uses acetone as carbon precursor. Both hydrophilic and hydrophobic fractions of PDs were obtained, with the respective average diameters of 2-4 nm and ca. 6 nm. The as-obtained PDs reveal greenish-blue photoluminescence (PL) and high quantum yields (∼5-7%) and complex kinetics of the decays with the average lifetime of ∼3.5 ns. Such luminescent acetone-derived PDs may find application in several fields, including sensing and bioimaging.

Two-photon absorption and two-photon-induced isomerization of azobenzene compounds.

The process of two-photon-induced isomerization occurring in various organic molecules, among which azobenzene derivatives hold a prominent position, offers a wide range of functionalities, which can be used in both material and life sciences. This review provides a comprehensive description of nonlinear optical (NLO) properties of azobenzene (AB) derivatives whose geometries can be switched through two-photon absorption (TPA). Employing the nonlinear excitation process allows for deeper penetration of light into the tissues and provides opportunities to regulate biological systems in a non-invasive manner. At the same time, the tight focus of the beam needed to induce nonlinear absorption helps to improve the spatial resolution of the photoinduced structures. Since near-infrared (NIR) wavelengths are employed, the lower photon energies compared to usual one-photon excitation (typically, the azobenzene geometry change from trans to cis form requires the use of UV photons) cause less damage to the biological samples. Herein, we present an overview of the strategies for optimizing azobenzene-based photoswitches for efficient two-photon excitation (TPE) and the potential applications of two-photon-induced isomerization of azobenzenes in biological systems: control of ion flow in ion channels or control of drug release, as well as in materials science, to fabricate data storage media, optical filters, diffraction elements etc., based on phenomena like photoinduced anisotropy, mass transport and phase transition. The extant challenges in the field of two-photon switchable azomolecules are discussed.