Two-photon Luminescence Imaging Research Articles

Near-infrared light-responsive hydrogels for on-demand dual delivery of proangiogenic growth factors

Achieving precise spatiotemporal control over the release of proangiogenic factors is crucial for vasculogenesis, the process of de novo blood vessel formation. Although various strategies have been explored, there is still a need to develop cell-laden biomaterials with finely controlled release of proangiogenic factors at specific locations and time points. We report on the developed of a near-infrared (NIR) light-responsive collagen hydrogel comprised of gold nanorods (GNRs)-conjugated liposomes containing proangiogenic growth factors (GFs). We demonstrated that this system enables on-demand dual delivery of GFs at specific sites and over selected time intervals. Liposomes were strategically formulated to encapsulate either platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF), each conjugated to gold nanorods (GNRs) with distinct geometries and surface plasmon resonances at 710 nm (GNR710) and 1064 nm (GNR1064), respectively. Using near infrared (NIR) irradiation and two-photon (2P) luminescence imaging, we successfully demonstrated the independent release of PDGF from GNR710 conjugated liposomes and VEGF from GNR1064-conjugated liposomes. Our imaging data revealed rapid release kinetics, with localized PDGF released in approximately 4 min and VEGF in just 1 and a half minutes following NIR laser irradiation. Importantly, we demonstrated that the release of each GF could be independently triggered using NIR irradiation with the other GF formulation remaining retained within the liposomes. This light-responsive collagen hydrogels holds promise for various applications in regenerative medicine where the establishment of a guided vascular network is essential for the survival and integration of engineered tissues. Statement of significanceIn this study, we have developed a light-responsive system with gold nanorods (GNRs)-conjugated liposomes in a collagen hydrogel, enabling precise dual delivery of proangiogenic growth factors (GFs) at specific locations and timepoints. Liposomes, containing platelet-derived growth factor (PDGF) or vascular endothelial growth factor (VEGF), release independently under near- infrared irradiation. This approach allows external activation of desired GF release, ensuring high cell viability. Each GF can be triggered independently, retaining the other within the liposomes. Beyond its application in establishing functional vascular networks, this dual delivery system holds promise as a universal platform for delivering various combinations of two or more GFs.

Peptide-Directed Synthesis of Aggregation-Induced Emission Enhancement-Active Gold Nanoclusters for Single- and Two-Photon Imaging of Lysosome and Expressed αvβ3 Integrin Receptors.

This study explores the synthesis and characterization of aggregation-induced emission enhancement (AIEE)-active gold nanoclusters (AuNCs), focusing on their near-infrared luminescence properties and potential applications in biological imaging. These AIEE-active AuNCs were synthesized via the NaBH4-mediated reduction of HAuCl4 in the presence of peptides. We systematically investigated the influence of the peptide sequence on the optical features of the AuNCs, highlighting the role of glutamic acid in enhancing their quantum yield (QY). Among the synthesized peptide-stabilized AuNCs, EECEE-stabilized AuNCs exhibited the maximum QY and a pronounced AIEE effect at pH 5.0, making them suitable for the luminescence imaging of intracellular lysosomes. The AIEE characteristic of the EECEE-stabilized AuNCs was demonstrated through examinations using transmission electron microscopy, dynamic light scattering, zeta potential analysis, and single-particle imaging. The formation of the EECEE-stabilized AuNCs was confirmed by size-exclusion chromatography and mass spectrometry. Spectroscopic and electrochemical examinations uncover the formation process of EECEE-stabilized AuNCs, comprising EECEE-mediated reduction, NaBH4-induced nucleation, complex aggregation, and subsequent cluster growth. Furthermore, we demonstrated the utility of these AuNCs as luminescent probes for intracellular lysosomal imaging, leveraging their pH-responsive AIEE behavior. Additionally, cyclic arginylglycylaspartic acid (RGD)-modified AIEE dots, derived from cyclic RGD-linked peptide-induced aggregation of EECEE-stabilized AuNCs, were developed for single- and two-photon luminescence imaging of αvβ3 integrin receptor-positive cancer cells.

NIR-II Excitable Water-Dispersible Two-Dimensional Conjugated Polymer Nanoplates for In Vivo Two-Photon Luminescence Bioimaging.

While two-dimensional conjugated polymers (2DCPs) have shown great promise in two-photon luminescence (TPL) bioimaging, 2DCP-based TPL imaging agents that can be excited in the second near-infrared window (NIR-II) have rarely been reported so far. Herein, we report two 2DCPs including 2DCP1 and 2DCP2, with octupolar olefin-linked structures for NIR-II-excited bioimaging. The 2DCPs are customized with the fully conjugated donor-acceptor (D-A) linkage and aggregation-induced emission (AIE) active building blocks, leading to good two-photon absorption into the NIR-II window with a 2PACS of ∼64.0 GM per choromophore for both 2DCPs. Moreover, 2DCP1 powders can be exfoliated into water-dispersible nanoplates with a Pluronic F-127 surfactant-assisted temperature-swing method, accompanied by both a drastic reduction of 2PACS throughout the range of 780-1080 nm and a sharp increase of photoluminescence quantum yield to 33.3%. The 2DCP1 nanoplates are subsequently proven to be capable of assisting in visualizing mouse brain vasculatures with a penetration depth of 421 μm and good contrast in vivo, albeit that only 19% of previous 2PACS at 1040 nm is preserved. This work not only provides important insights on how to construct NIR-II excitable 2DCPs for TPL bioimaging but also how to investigate the exfoliation-photophysical property correlation of 2DCPs, which should aid in future research on developing highly efficient TPL bioimaging agents.

Rhenium disulfide nanosheets as a promising probe for intracellular two-photon luminescence imaging

Near-infrared (NIR) light-mediated two-photon microscopy has become a promising tool for early cancer therapy. However, exploring novel two-photon probe with high biocompatibility and high photostability to resist photobleaching, remains a key challenge for performing in vivo bioimaging. Herein, a novel two-photon luminescence (TPL) probe named two-dimensional rhenium disulfide (ReS2) nanosheets was firstly reported for performing intracellular bioimaging. Under the illumination of low power NIR femtosecond laser, the prepared ReS2 nanosheets exhibited strong NIR photon absorption, generating a strong TPL band at 640 nm. In addition, in vitro TPL imaging showed that, ReS2 nanosheets possessed excellent photostability, enabling to resist photobleaching. More importantly, ReS2 nanosheets functionalized by polyethylene glycol (PEG) showed negligible cytotoxicity, suitable to perform intracellular TPL imaging. With prolonged 6-hour incubation time, the TPL intensity of PEG-ReS2 nanosheets in three kinds of living cells such as esophageal cancer cells (KYSE150), pancreatic cancer cells (4T1) and HeLa cells, were significantly enhanced by almost 4, 11, and 17 times, respectively. In view of their photothermal effects, the PEG-ReS2 nanosheets could work as an excellent TPL probe for monitoring photothermal therapy and evaluating therapeutic outcomes. This study highlights great potential of PEG-ReS2 nanosheets as a TPL biosensor for intracellular optical imaging and cancer therapy.

Ruthenium(II) complex-based long-lived two-photon luminescence probe for dynamic monitoring of glutathione S-transferases in mouse models of drug-induced liver injury

We report a novel ruthenium(II) complex-based long-lived and two-photon luminescence probe, Ru-NO2, for dynamic monitoring of GSTs fluxes in the process of drug-induced liver injury (DILI). Ru-NO2 shows weak luminescence due to the strong photo-induced electron transfer (PET) process from the Ru(II) center to the electron-withdrawing 3,4-dinitrobenzene (DNB) group. As a result of the specific nucleophilic aromatic substitution triggered by GSTs in the presence of reduced glutathione (GSH), the 4-nitro group of Ru-NO2 is substituted by GSH to form strongly emissive Ru-SG. Ru-NO2 shows high selectivity and sensitivity for responding to GSTs. Notably, one- and two-photon luminescence imaging experiments were conducted to visualize GSTs in living cells and mouse tissues. In addition, Ru-NO2 was applied for background-free time-gated luminescence (TGL) detection and luminescence imaging analysis of GSTs in DILI mouse sera and liver tissues, which showed that GSTs level in sera significantly increased accompanied by the decrease in liver in the DILI process, revealing the possible potential of the probe as an effective tool for clinical diagnosis and treatment monitoring of DILI.

Plasmon Resonant Two-Photon Luminescence Inducing Photosensitization and Nonlinear Optical Microscopy In Vivo by Near-Infrared Excitation of Au Nanopeanuts

Photodynamic therapy (PDT) provides a potential therapeutic approach for killing malignant cell/solid tumors, but currently approved photosensitizers (PSs) are generally excited by visible light, limiting the penetration depth in tissues. It is necessary to develop a near-infrared (NIR) responsive photodynamic platform, providing maximum tissue penetration. Here, we present a gold nanopeanut platform exhibiting dual functions of NIR PDT and two-photon luminescence imaging. The nanopeanut with a size less than 100 nm exhibits two distinct NIR surface plasmon absorption bands at approximately 1110 and 1300 nm. To perform PDT, we conjugated commercial toluidine blue O (TBO) PS on the surface of the nanopeanuts. With spectral overlap, the 1230-nm femtosecond Cr: forsterite laser can excite the surface plasmons of nanopeanuts, transfer energy to TBO, and generate singlet oxygen to kill cells. Moreover, the plasmon resonance-enhanced two-photon luminescence of nanopeanuts can be used to map their delivery in vivo. These results demonstrate that the PS-conjugated gold nanopeanut is an effective theranostic system for NIR PDT.

Rational design of pyrrole derivatives with aggregation-induced phosphorescence characteristics for time-resolved and two-photon luminescence imaging

Pure organic room-temperature phosphorescent (RTP) materials have been suggested to be promising bioimaging materials due to their good biocompatibility and long emission lifetime. Herein, we report a class of RTP materials. These materials are developed through the simple introduction of an aromatic carbonyl to a tetraphenylpyrrole molecule and also exhibit aggregation-induced emission (AIE) properties. These molecules show non-emission in solution and purely phosphorescent emission in the aggregated state, which are desirable properties for biological imaging. Highly crystalline nanoparticles can be easily fabricated with a long emission lifetime (20 μs), which eliminate background fluorescence interference from cells and tissues. The prepared nanoparticles demonstrate two-photon absorption characteristics and can be excited by near infrared (NIR) light, making them promising materials for deep-tissue optical imaging. This integrated aggregation-induced phosphorescence (AIP) strategy diversifies the existing pool of bioimaging agents to inspire the development of bioprobes in the future.

Multimodal Imaging Iridium(III) Complex for Hypochlorous Acid in Living Systems.

Biomolecule tracing with different imaging methods is of great significance for more accurately unravelling the fundamental processes in living systems. However, considering the different principles of each imaging method for probe design, it is still a great challenge to apply one molecular probe to achieve two or even more imaging analyses for biomarkers. In general, traditional oxime was reported as a recognition group for fluorescence imaging of HOCl. Herein, for the first time, we designed the oxime decorated iridium(III) complex, which can be directly used for chemiluminescence as well as two-photon luminescence and photoluminescence lifetime imaging of HOCl in living systems. Moreover, the novel chemiluminescence mechanism of Ir-CLFLPLIM for HOCl was also proposed and explored by continuously monitoring chemiluminescence peak shapes and mass spectra, inferring the reaction intermediate and calculating the chemical reaction energy range of the reaction process. This strategy could lead us to expand the chemiluminescence application of transition metal complexes and develop more multimodal imaging probes.

Visualization photofragmentation-induced rhodamine B release from gold nanorod delivery system by combination two-photon luminescence imaging with correlation spectroscopy.

Plasmon-enhanced gold nanorod (AuNR) with high photothermal conversion efficiency is a promising light-controllable nanodrug delivery system for cancer therapy. Understanding the mechanism for the light-controllable drug release of AuNR delivery systems is important for the development of nanomedicine. In this study, the rhodamine B (RB) released from AuNR-RB nanodelivery system was quantitated and visualized by using two-photon luminescence (TPL) imaging combined with correlation spectroscopy. The photofragmentation of AuNR induced by femtosecond pulsed laser was revealed by TPL correlation spectroscopy when the laser energy was above the thermal damage threshold of AuNR, and the RB released from this nanodrug delivery system was visualized by TPL imaging. Furthermore, the photofragmentation-induced release of RB from AuNR-RB nanodelivery system was visualized in living MCF-7 breast cancer cells by TPL imaging combined with correlation spectroscopy. These results provided a novel optical approach to quantify the release of drugs from gold nanocarriers in complex biological media.

Several Orders-of-Magnitude Enhancement of Multiphoton Absorption Property for CsPbX3 Perovskite Quantum Dots by Manipulating Halide Stoichiometry

Two-photon absorption (2PA) and three-photon absorption (3PA) processes feature many technological applications for fluorescence microscopy, photodynamic therapy, optical data storage, and so on, Herein, we reveal that the giant 2PA and 3PA properties for all-inorganic CsPbX3 (X = Cl, Br, I, and mixed Cl/Br and Br/I) perovskite quantum dots (PQDs) can be enhanced several orders of magnitude, respectively, by simply changing the halide stoichiometry at the X site. Notably, reported data show excellent 2PA and 3PA properties for CsPbI3 (σ2 ∼ 2.1 × 106 GM and σ3 ∼ 1.1 × 10–73 cm8 s3/photon3), which is 2–4 orders of magnitude higher than those of conventional red-emitting QDs and 5–7 orders of magnitude higher than well documented organic molecules. Experimental results show multiphoton absorption (MPA) cross sections can be adjusted 2–3 orders of magnitude by band gap engineering in a predictable manner, via increasing the Pauling electronegativity of the halide. Two-photon luminescence imaging data show tha...

Synergistic Nanozymetic Activity of Hybrid Gold Bipyramid-Molybdenum Disulfide Core@Shell Nanostructures for Two-Photon Imaging and Anticancer Therapy.

In recent years, the concept of combined therapy using gold hybrid nanomaterials has been broadly adopted to pioneer new anticancer treatments. However, their synergistic anticancer effects have yet to be thoroughly investigated. Herein,a hybrid gold nanobipyramid nanostructure coated with molybdenum disulfide (MoS2) semiconductor (AuNBPs@MoS2) was proposed as a smart nanozyme for anticancer therapy and two-photon bioimaging. The hybrid material showed dramatically enhanced localized surface plasmon resonance property under excitation owing to its anisotropic nature, coupled with the rich electron density in MoS2, resulting in the superior in situ photogeneration of reactive oxidative species (ROS - 1O2, •OH). We demonstrated that the synergistic effect of enhanced photothermal conversion and generation of ROS could increase the anticancer effect of AuNBPs@MoS2. Two-photon luminescence imaging confirmed that AuNBPs@MoS2 was successfully internalized in cancer cells and that simultaneous anticancer treatments based on catalytic and photothermal therapy could be achieved. This study highlighted, for the first time, a novel approach of plasmon-mediated powerful anticancer therapy and imaging via the unprecedented combination of anisotropic AuNBPs and two-dimensional MoS2 material.

Large-Scale High-Yield Synthesis of PdCu@Au Tripods and the Quantification of their Luminescence Properties for Cancer Cell Imaging

The synthesis of anisotropic branched gold nanoparticles remines to be challenging as their arm number and arm length could hardly be controlled, greatly limited their biomedical application. We report the large-scale high-yield synthesis of PdCu@Au tripods, and, for the first time, their two-photon luminescence properties with quantitative characterization of the two-photon action cross section as well as quantum yield. By introducing nitrogen protection to the synthesis of the PdCu bimetallic cores, this approach eliminates the oxidative etching caused by oxygen in the air, providing a 2.5 times higher synthetic yield of 70.4 %, which enables the large-scale preparation of PdCu@Au at ca. 380 mg per batch. By the conformal coating of PdCu bimetallic cores, the PdCu@Au tripods are prepared with a purity of >90 % with average arm length 45.3 ± 5.6 nm that is ideal for biomedical applications. The PdCu@Au tripods demonstrate a much brighter two-photon luminescence than that from Au nanorods, with a 3.6 ± 0.9 times larger two-photon action cross section and comparable quantum yield. Our result also shows the two-photon luminescence property of PdCu@Au tripods could be tuned by their distinct localized surface plasmon resonance property and, in turn, the different amount of Au coating. This tunability could be explained by the recently-proposed two-step excitation mechanism of two-photon luminescence in Au nanoparticle. The folate-targeted in vitro two-photon luminescence imaging of MDA-MB-435 breast cancer cells were also demonstrated to show the great potential using PdCu@Au tripods as novel multi-functional platforms for cancer theranostics.

White Light Generation and Anisotropic Damage in Gold Films near Percolation Threshold

Strongly enhanced and confined electromagnetic fields generated in metal nanostructures upon illumination are exploited in many emerging technologies by either fabricating sophisticated nanostructures or synthesizing colloid nanoparticles. Here we study effects driven by field enhancement in vanishingly small gaps between gold islands in thin films near the electrically determined percolation threshold. Optical explorations using two-photon luminescence (TPL) and near-field microscopies reveals super-cubic TPL power dependencies with white-light spectra, establishing unequivocally that the strongest TPL signals are generated with close to the percolation threshold films, and occurrence of extremely confined (~ 30 nm)and strongly enhanced (~ 100 times) fields at the illumination wavelength. For linearly polarized and sufficiently powerful light, we observe pronounced optical damage with TPL images being sensitive to both wavelength and polarization of illuminating light. We relate these effects to thermally induced morphological changes observed with scanning electron microscopy images. Fascinating physics involved in light interaction with near-percolation metal films along with their straightforward and scalable one-step fabrication procedure promises a wide range of fascinating developments and technological applications within diverse areas of modern nanotechnology, from bio-molecule optical sensing to ultra-dense optical data storage.

Phase mask-based multimodal superresolution microscopy.

We demonstrate a multimodal superresolution microscopy technique based on a phase masked excitation beam in combination with spatially filtered detection. The theoretical foundation for calculating the focus from a non-paraxial beam with an arbitrary azimuthally symmetric phase mask is presented for linear and two-photon excitation processes as well as the theoretical resolution limitations. Experimentally this technique is demonstrated using two-photon luminescence from 80 nm gold particle as well as two-photon fluorescence lifetime imaging of fluorescent polystyrene beads. Finally to illustrate the versatility of this technique we acquire two-photon fluorescence lifetime, two-photon luminescence, and second harmonic images of a mixture of fluorescent molecules and 80 nm gold particles with > 120 nm resolution (λ/7). Since this approach exclusively relies on engineering the excitation and collection volumes, it is suitable for a wide range of scanning-based microscopies.

Power‐ and polarization dependence of two photon luminescence of single CdSe nanowires with tightly focused cylindrical vector beams of ultrashort laser pulses

AbstractWe investigate two‐photon luminescence (TPL) of single CdSe nanowires (NWs) excited by tightly focused cylindrical vector beams of 150 fs pulses, achieving an optical resolution better than λ/4. Comparing the TPL images recorded by scanning the nanowires through the focal fields created either by a radially or an azimuthally polarized vector beam we observe a distinct anisotropic excitation efficiency which depends on the orientation of the electric field component with respect to the nanowire. The excitation anisotropy of the linear photoluminescence (PL) and TPL can directly be derived from the respective image patterns. Our results show that the highest TPL signal from a single NW is detected when the electric excitation field is parallel to the long axis of the NW, i.e. about one order of magnitude stronger than that excited along the short axis. We attribute the TPL to an emission mechanism based on the recombination of free carriers, which exhibits a fourth order excitation power dependence in low power regime and a second order power dependence in high power regime. image

Modulating the Morphology of Gold Graphitic Nanocapsules for Plasmon Resonance-Enhanced Multimodal Imaging.

With their unique optical properties and distinct Raman signatures, graphitic nanomaterials can serve as substrates for surface-enhanced Raman spectroscopy (SERS) or provide signal amplification for bioanalysis and detection. However, a relatively weak Raman signal has limited further biomedical applications. This has been addressed by encapsulating gold nanorods (AuNRs) in a thin graphitic shell to form gold graphitic nanocapsules. This step improves plasmon resonance, which enhances Raman intensity, and has the potential for integrating two-photon luminescence (TPL) imaging capability. However, changing the morphology of gold graphitic nanocapsules such that high quality and stability are achieved remains a challenge. To address this task, we herein report a confinement chemical vapor deposition (CVD) method to prepare the construction of AuNR-encapsulated graphitic nanocapsules with these properties. Specifically, through morphological modulation, we (1) achieved higher plasmon resonance with near-IR incident light, thus achieving greater Raman intensity, and (2) successfully integrated two-photon luminescence dual-modal (Raman/TPL) bioimaging capabilities. Cancer-cell-specific aptamers were further modified on the AuNR@G graphitic surface through simple, but strong, π-π interactions to achieve imaging selectivity through differential cancer cell recognition.

A Self-Assembled DNA Origami-Gold Nanorod Complex for Cancer Theranostics.

A self-assembled DNA origami (DO)-gold nanorod (GNR) complex, which is a dual-functional nanotheranostics constructed by decorating GNRs onto the surface of DNA origami, is demonstrated. After 24 h incubation of two structured DO-GNR complexes with human MCF7 breast cancer cells, significant enhancement of cell uptake is achieved compared to bare GNRs by two-photon luminescence imaging. Particularly, the triangle shaped DO-GNR complex exhibits optimal cellular accumulation. Compared to GNRs, improved photothermolysis against tumor cells is accomplished for the triangle DO-GNR complex by two-photon laser or NIR laser irradiation. Moreover, the DO-GNR complex exhibits enhanced antitumor efficacy compared with bare GNRs in nude mice bearing breast tumor xenografts. The results demonstrate that the DO-GNR complex can achieve optimal two-photon cell imaging and photothermal effect, suggesting a promising candidate for cancer diagnosis and therapy both in vitro and in vivo.

Dual-modality fiber-based OCT-TPL imaging system for simultaneous microstructural and molecular analysis of atherosclerotic plaques.

New optical imaging techniques that provide contrast to study both the anatomy and composition of atherosclerotic plaques can be utilized to better understand the formation, progression and clinical complications of human coronary artery disease. We present a dual-modality fiber-based optical imaging system for simultaneous microstructural and molecular analysis of atherosclerotic plaques that combines optical coherence tomography (OCT) and two-photon luminescence (TPL) imaging. Experimental results from ex vivo human coronary arteries show that OCT and TPL optical contrast in recorded OCT-TPL images is complimentary and in agreement with histological analysis. Molecular composition (e.g., lipid and oxidized-LDL) detected by TPL imaging can be overlaid onto plaque microstructure depicted by OCT, providing new opportunities for atherosclerotic plaque identification and characterization.

Two-Photon Luminescence of Gold Nanorods Mediated by Higher Order Plasmon Modes

Metallic nanorod antennas can be considered as an analogue to classical half-wave dipole antennas, constituting an important tool for manipulating linear and nonlinear light-matter interactions in nanoscale volumes. Using two-photon luminescence (TPL) scanning laser microscopy, we investigate such optical antennas beyond their fundamental dipole mode. The antenna mode dispersion is extracted from the nonlinear TPL measurement and reveals a TPL process that is dominated by plasmon-induced enhancement of the two-photon absorption in the metal. Additionally, a clear signature of the mode parity is observed in the TPL images. TPL maxima are observed outside the antenna boundaries for even parity modes, whereas they are located inside for odd modes. It is concluded that for even modes the two-photon luminescence emission is strongly mediated by retardation of the excitation field, a consequence of their zero net-dipole moment. This selective excitation of different mode parities is highly relevant for nanoscale enhanced nonlinear optics, as well as plasmonic nanosensor applications and tuning of radiative properties of quantum emitters.

Three-photon luminescence of gold nanorods and its applications for high contrast tissue and deep in vivo brain imaging.

Gold nanoparticles can be used as contrast agents for bio-imaging applications. Here we studied multi-photon luminescence (MPL) of gold nanorods (GNRs), under the excitation of femtosecond (fs) lasers. GNRs functionalized with polyethylene glycol (PEG) molecules have high chemical and optical stability, and can be used as multi-photon luminescent nanoprobes for deep in vivo imaging of live animals. We have found that the depth of in vivo imaging is dependent upon the transmission and focal capability of the excitation light interacting with the GNRs. Our study focused on the comparison of MPL from GNRs with two different aspect ratios, as well as their ex vivo and in vivo imaging effects under 760 nm and 1000 nm excitation, respectively. Both of these wavelengths were located at an optically transparent window of biological tissue (700-1000 nm). PEGylated GNRs, which were intravenously injected into mice via the tail vein and accumulated in major organs and tumor tissue, showed high image contrast due to distinct three-photon luminescence (3PL) signals upon irradiation of a 1000 nm fs laser. Concerning in vivo mouse brain imaging, the 3PL imaging depth of GNRs under 1000 nm fs excitation could reach 600 μm, which was approximately 170 μm deeper than the two-photon luminescence (2PL) imaging depth of GNRs with a fs excitation of 760 nm.