Multiphoton Excitation Fluorescence Research Articles

Intravital observation of high-scattering and dense-labeling hepatic tissues using multi-photon fluorescence microscopy.

Achieving high-resolution and large-depth microscopic imaging in vivo under conditions characterized by high-scattering and dense-labeling, as commonly encountered in the liver, poses a formidable challenge. Here, through the optimization of multi-photon fluorescence excitation window, tailored to the unique optical properties of the liver, intravital microscopic imaging of hepatocytes and hepatic blood vessels with high spatial resolution was attained. It's worth noting that resolution degradation caused by tissue scattering of excitation light was mitigated by accounting for moderate tissue self-absorption. Leveraging high-quality multi-photon fluorescence microscopy, we discerned structural and functional alterations in hepatocytes during drug-induced acute liver failure. Furthermore, a reduction in indocyanine green metabolism rates associated with acute liver failure was observed using NIR-II fluorescence macroscopic imaging.

Available medical imaging modalities for melanoma screening

The prevalence of melanoma of the skin has seen a significant rise in recent decades, constituting approximately one-third of all diagnosed cancer cases. Melanoma, the most fatal variant among cutaneous malignancies, exhibits a 4% probability of occurrence over an individual’s lifetime. The increasing incidence and mortality rates of skin cancer impose a substantial burden on healthcare resources and the economy. In recent years, several optical modalities, including dermoscopy, reflectance confocal microscopy (RCM), optical coherence tomography, multiphoton excited fluorescence imaging, and dermatofluorescence, have been extensively studied and utilized to improve the non-invasive diagnosis of skin cancer. This review article provides an analysis of the approach employed in the recently developed optical non-invasive diagnostic technologies. It explores the clinical uses of these techniques, while also examining their respective advantages and disadvantages. Furthermore, the paper explores the possibility for additional advancements in these technologies in the future.

Probing delivery of a lipid nanoparticle encapsulated self-amplifying mRNA vaccine using coherent Raman microscopy and multiphoton imaging

The COVID-19 pandemic triggered the resurgence of synthetic RNA vaccine platforms allowing rapid, scalable, low-cost manufacturing, and safe administration of therapeutic vaccines. Self-amplifying mRNA (SAM), which self-replicates upon delivery into the cellular cytoplasm, leads to a strong and sustained immune response. Such mRNAs are encapsulated within lipid nanoparticles (LNPs) that act as a vehicle for delivery to the cell cytoplasm. A better understanding of LNP-mediated SAM uptake and release mechanisms in different types of cells is critical for designing effective vaccines. Here, we investigated the cellular uptake of a SAM-LNP formulation and subsequent intracellular expression of SAM in baby hamster kidney (BHK-21) cells using hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy and multiphoton-excited fluorescence lifetime imaging microscopy (FLIM). Cell classification pipelines based on HS-CARS and FLIM features were developed to obtain insights on spectral and metabolic changes associated with SAM-LNPs uptake. We observed elevated lipid intensities with the HS-CARS modality in cells treated with LNPs versus PBS-treated cells, and simultaneous fluorescence images revealed SAM expression inside BHK-21 cell nuclei and cytoplasm within 5 h of treatment. In a separate experiment, we observed a strong correlation between the SAM expression and mean fluorescence lifetime of the bound NAD(P)H population. This work demonstrates the ability and significance of multimodal optical imaging techniques to assess the cellular uptake of SAM-LNPs and the subsequent changes occurring in the cellular microenvironment following the vaccine expression.

Abstract C020: Unconvering the hidden immunosuppressive landscape in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest forms of cancer with an extremely low survival rate. Despite the success of therapeutic T cells in hematologic cancers, many solid tumors, including PDA, remain unresponsive with such therapies, partly due to its mechanically complex, dense, and immunosuppressive stroma. Therefore, understanding the dynamic interplay between therapeutic T cells and the tumor microenvironment (TME) is crucial for revealing engineering targets for next-generation immunotherapies. We approach this problem with genetically engineered KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-1-Cre) mouse models that recapitulates PDA disease progression and stromal dynamics. Within live tumor slices from primary tumors, we are defining how stromal components such as dense collagen fiber networks and tumor-associated macrophages (TAMs) orchestrate an immunosuppressive landscape. We employ multiphoton laser scanning microscopy (MPLSM) to generate multiphoton excitation of fluorescence (cell dyes and immunofluorescence) and second harmonic generation (SHG) for collagen fibers to acquire multidimensional data (x, y, z, c, t) of T cell migration and interaction within the TME. We subsequently reveal T cell migration and interaction as a function of its physical and biological ECM components using advanced computer vision and machine learning algorithms based on the Clustered Random Convolutional Kernel Transform (cROCKET), revealing a hidden landscape in the feature space. Perturbations to the TME in KPC mice with CCR2 inhibitor CCX598 (in vivo) and clodronate liposomes (ex vivo) deplete CD11b+ myeloid cells, resulting in altered behaviors of the infiltrating T cells. Anti-PD-1 monoclonal antibody neutralizes the PD-1/PD-L1 axis in T cells, resulting in enhanced migration and infiltration in tumor slices. Citation Format: Guhan Qian, Hongrong Zhang, Aine Knott, Jon Zettervall, Ingunn Stromnes, Paolo Provenzano. Unconvering the hidden immunosuppressive landscape in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C020.

Fast Diffusion Characterization by Multiphoton Excited Fluorescence Recovery while Photobleaching.

Multiphoton-excited fluorescence recovery while photobleaching (FRWP) is demonstrated as a method for quantitative measurements of rapid molecular diffusion over microsecond to millisecond timescales. Diffusion measurements are crucial in assessing molecular mobility in cell biology, materials science, and pharmacology. Optical and fluorescence microscopy techniques enable non-invasive rapid analysis of molecular diffusion but can be challenging for systems with diffusion coefficients exceeding ∼100 μm2/s. As an example, fluorescence recovery after photobleaching (FRAP) operates on the implicit assumption of a comparatively fast photobleaching step prior to a relatively slow recovery and is not generally applicable for systems exhibiting substantial recovery during photobleaching. These challenges are exacerbated in multiphoton excitation by the lower excitation efficiency and competing effects from local heating. Herein, beam-scanning FRWP with patterned line-bleach illumination is introduced as a technique that addresses FRAP limitations and further extends its application range by measuring faster diffusion events. In FRWP, the recovery of fluorescence is continuously probed after each pass of a fast-scanning mirror, and the upper bound of measurable diffusion rates is, therefore, only limited by the mirror scanning frequency. A theoretical model describing transient fluctuations in fluorescence intensity arising as a result of combined contributions from photobleaching and localized photothermal effect is introduced along with a mathematical framework for quantifying fluorescence intensity temporal curves and recovering room-temperature diffusion coefficients. FRWP is then tested by characterization of normal diffusion of rhodamine-labeled bovine serum albumin, green fluorescence protein, and immunoglobulin G molecules in aqueous solutions of varying viscosity.

Angular Patterns of Nonlinear Emission in Dye Water Droplets Stimulated by a Femtosecond Laser Pulse for LiDAR Applications

Femtosecond laser-induced fluorescence (FLIF) and femtosecond laser-induced optical breakdown spectroscopy (FIBS) are important tools for remote diagnostics of atmospheric aerosols using LiDAR (Light Identification Detection and Ranging) technology. They are based on light emission excitation in disperse media via multiphoton nonlinear processes in aerosol particles induced by high-power optical pulses. To date, the main challenge restraining the large-scale application of FLIF and FIBS in atmospheric studies is the lack of a valued theory of the stimulated light emission in liquid microparticles with a sufficiently broad range of sizes. In this paper, we fill this gap and present a theoretical model of dye water droplet emission under high intensity laser exposure that adequately simulates the processes of multiphoton excited fluorescence and optical breakdown plasma emission in microparticles and gives quantitative estimates of the angular and power characteristics of nonlinear emission. The model is based on the numerical solution to the inhomogeneous Helmholtz equations for stimulating (primary) and nonlinear (secondary) waves provided by the random nature of molecule emission in particles. We show that droplet fluorescence stimulated by multiphoton absorption generally becomes more intense with increasing particle size. Moreover, far-field plasma emission from liquid particles demonstrates a larger angular diversity when changing the droplet radius in comparison with multiphoton excited fluorescence, which is mainly due to the excitation of the internal optical field resonances in spherical particles.

In-depth structural and compositional assessment of aged terpenoid varnish layers by nonlinear optical microscopy

Varnishes are employed in painted artworks for protection from atmospheric pollution and oxidation and for improving the esthetic appearance by providing an even and brilliant surface finish. Also, in some cases, colored varnishes are applied intentionally to modify the chromatic appearance of paintings. Varnishes undergo complex and differentiated structural and chemical changes over time depending on their composition and conservation conditions. The present work investigates the extent of degradation of the outermost layers of varnish coatings due to aging as a function of depth by using nonlinear optical microscopy (NLOM) in the modality of multiphoton excitation fluorescence (MPEF). This non-invasive technique has been employed for the determination of the thickness, with a microscopic axial and lateral resolution, of the affected regions of pictorial terpenoid varnish layers, such as dammar, mastic, shellac and sandarac, subjected to various types and degrees of aging, artificial, natural and a combination of both. A homemade nonlinear optical microscope, based on a tightly focused pulsed femtosecond laser emitting at 800 nm, was used for the investigation. Single-photon laser-induced fluorescence (LIF) measurements served to determine the degree of surface aging and the optimum NLOM-MPEF operating conditions and helped to interpret the results obtained by applying the latter. These results signpost the correlations between the extent of degradation, non-invasively determined by NLOM-MPEF, the type and length of aging, the nature of the varnish and the initial thickness of the varnish layer.

Laser-based techniques for the non-invasive characterisation of grisaille paints on stained-glass windows

Grisaille was the first paint applied on stained-glass panels, used in Europe since the twelfth century. Historical written sources described the use of iron and copper together with a high lead-silica base glass in the grisailles production. This project aims to study the evolution of the grisaille paint composition throughout time and the changes in the raw materials used in their production using non-destructive and non-invasive techniques. To achieve this objective, 23 grisaille samples dated from the 13th to the twentieth centuries from nine different countries (Portugal, Poland, United Kingdom, Sweden, Norway, Belgium, Low Countries, Germany, and France) were studied by means of micro particle-induced X-ray emission (μ-PIXE), micro energy dispersive X-ray fluorescence (μ-EDXRF), laser-induced breakdown spectroscopy (LIBS), laser-induced fluorescence (LIF), non-linear optical microscopy (NLOM) in the modality of multiphoton excitation fluorescence (MPEF) and optical microscopy (OM). The results showed that it was possible to identify compositional differences and patterns throughout the samples when compared with literature results. The preference for using copper in central and south-central European countries and the addition of new compounds (CoO, Cr2O3, MnO) as colouring agents since the nineteenth century was verified. The LIBS analyses allow the identification of boron on two samples, confirming the change of base glass components since the seventeenth century. The NLOM-MPEF showed the capability of this technique to measure the grisaille paint layers’ thickness. This non-invasive multi-analytical and complementary approach proves itself efficient in identifying changes in the grisaille’s composition throughout time, which can be interpreted as changes in the raw materials and manufacture used in the production of these paint materials.

Two-Photon Absorption and Multiphoton Excited Fluorescence of Acetamide-Chalcone Derivatives: The Role of Dimethylamine Group on the Nonlinear Optical and Photophysical Properties.

This work studied the effect of different electron-withdrawing and electron-donating groups on the linear and nonlinear optical properties of acetamide-chalcone derivatives. The results showed that the addition of the dimethylamine group led to a large fluorescence emission (71% of fluorescence quantum yield in DMSO solution) that can be triggered by two and three-photon excitations, which is essential for biological applications. Furthermore, dimethylamine also red-shifts the lower energy state by approximately 90 nm, increasing the two-photon absorption cross-section of the lower energy band by more than 100% compared with the other studied compounds. All compounds presented two-electronic states observed through one and two-photon absorption spectroscopy and confirmed by Quantum Chemistry Calculations (QCCs). QCC results were also used to model the experimental two-photon absorption cross-sectional spectrum by the Sum-Over-States (SOS) approach, revealing a dependence between the coupling of the ground state with the first excited state and the transition dipole moment between these states.

Dual-resonant scanning multiphoton microscope with ultrasound lens and resonant mirror for rapid volumetric imaging

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens with a resonant mirror is developed for high-speed volumetric imaging. In the proposed microscope, the pulse train signal of a femtosecond laser is used to trigger an embedded field programmable gate array to sample the multiphoton excited fluorescence signal at the rate of one pixel per laser pulse. It is shown that a frame rate of around 8000 Hz can be obtained in the x–z plane for an image region with a size of 256 × 80 pixels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 343 × 343 × 120 μm3 with an image size of 256 × 256 × 80 voxels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 256 × 256 × 80 voxels, which represents 343 × 343 × 120 μm3 in field-of-view. The rapid volumetric imaging rate eliminates the aliasing effect for observed temporal frequencies lower than 15 Hz. The practical feasibility of the DRSM microscope is demonstrated by observing the mushroom bodies of a drosophila brain and performing 3D dynamic observations of moving 10-μm fluorescent beads.

Developments and Clinical Applications of Noninvasive Optical Technologies for Skin Cancer Diagnosis.

Skin cancer has shown a sharp increase in prevalence over the past few decades and currently accounts for one-third of all cancers diagnosed. The most lethal form of skin cancer is melanoma, which develops in 4% of individuals. The rising prevalence and increased number of fatalities of skin cancer put a significant burden on healthcare resources and the economy. However, early detection and treatment greatly improve survival rates for patients with skin cancer. Since the rising rates of both the incidence and mortality have been particularly noticeable with melanoma, significant resources have been allocated to research aimed at earlier diagnosis and a deeper knowledge of the disease. Dermoscopy, reflectance confocal microscopy, optical coherence tomography, multiphoton-excited fluorescence imaging, and dermatofluorescence are only a few of the optical modalities reviewed here that have been employed to enhance noninvasive diagnosis of skin cancer in recent years. This review article discusses the methodology behind newly emerging noninvasive optical diagnostic technologies, their clinical applications, and advantages and disadvantages of these techniques, as well as the potential for their further advancement in the future.

Control the Single‐, Two‐, and Three‐Photon Excited Fluorescence of Atomically Precise Metal Nanoclusters

AbstractIt remains challenging to control the single‐, two‐, and three‐photon excited fluorescence of metal nanoclusters. In this work, the control over the non‐linear optics of metal nanoclusters as single‐, two‐, and three‐photon excited fluorescence has been accomplished via exploiting the solvent effect. An emissive nanocluster, Au9Ag6(SPhtOMe)4(DPPOE)3Cl3, was synthesized and structurally determined. The solvent effect can not only control the fluorescence of this nanocluster, but more significantly, it can also regulate the photoluminescence nature of the cluster as single‐, two‐, and three‐photon excited fluorescence. We concluded that the increased solution polarity, improved dipole moment, enlarged HOMO–LUMO energy gap, and reduced solution viscosity of the cluster in solutions endow them with excellent high‐order multiphoton excited fluorescence. The results provide an intriguing cluster template that enables us to manipulate the linear and nonlinear optics at the atomic level.

Control the Single-, Two-, and Three-Photon Excited Fluorescence of Atomically Precise Metal Nanoclusters.

It remains challenging to control the single-, two-, and three-photon excited fluorescence of metal nanoclusters. In this work, the control over the non-linear optics of metal nanoclusters as single-, two-, and three-photon excited fluorescence has been accomplished via exploiting the solvent effect. An emissive nanocluster, Au9 Ag6 (SPht OMe)4 (DPPOE)3 Cl3 , was synthesized and structurally determined. The solvent effect can not only control the fluorescence of this nanocluster, but more significantly, it can also regulate the photoluminescence nature of the cluster as single-, two-, and three-photon excited fluorescence. We concluded that the increased solution polarity, improved dipole moment, enlarged HOMO-LUMO energy gap, and reduced solution viscosity of the cluster in solutions endow them with excellent high-order multiphoton excited fluorescence. The results provide an intriguing cluster template that enables us to manipulate the linear and nonlinear optics at the atomic level.

Pulse-Picking Multimodal Nonlinear Optical Microscopy.

Nonlinear optical microscopy techniques can map chemical compositions in biological samples in a label-free manner. Commonly used nonlinear optical processes for imaging include multiphoton excitation fluorescence (MPEF), second harmonic generation (SHG), and coherent Raman scattering (CRS). Femtosecond lasers are typically used for MPEF and SHG due to the requirement of high peak power for excitation, while picosecond lasers are preferred for CRS due to the need for high spectral resolution. Therefore, it is challenging to integrate CRS with MPEF and SHG for chemical imaging. We develop a pulse-picking strategy based on an acousto-optic modulator that can program the duty cycle of the laser pulse train, significantly increasing the pulse peak power at low input average power. This approach offers strong enhancement of nonlinear optical signals and makes hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy compatible with MPEF and SHG for multimodal imaging at low laser average power. The pulse-picking method also enables the evaluation and comparison of phototoxicity of laser pulses at different average and peak power levels. The photo-perturbations to biological samples are evaluated using cellular dynamics and sample morphological changes, allowing the selection of optimal laser power for the best sensitivity and minimal phototoxicity.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Real-time adaptive ultrashort pulse compressor for dynamic group delay dispersion compensation.

The optical dispersion effect in ultrafast pulse laser systems broadens the laser pulse duration and reduces the theoretical peak power. The present study proposes an adaptive ultrashort pulse compressor for compensating the optical dispersion using a direct optical-dispersion estimation by spectrogram (DOES) method. The DOES has fast and accurate computation time which is suitable for real time controller design. In the proposed approach, the group delay dispersion (GDD) and its polarity are estimated directly from the delay marginal of the trace obtained from a single-shot frequency-resolved optical gating (FROG). The estimated GDD is then processed by a closed-loop controller, which generates a command signal to drive a linear deformable mirror as required to achieve the desired laser pulse compression. The dispersion analysis, control computation, and deformable mirror control processes are implemented on a single field programmable gate array (FPGA). It is shown that the DOES dispersion computation process requires just 0.5 ms to complete. Moreover, the proposed pulse compressor compensates for both static dispersion and dynamic dispersion within five time steps when closed-loop controller is performed at a frequency of 100 Hz. The experimental results show that the proposed pulse compressor yields an effective fluorescence intensity improvement in a multiphoton excited fluorescence microscope (MPEFM).

Embedding Multiphoton Active Units within Metal-Organic Frameworks for Turning on High-Order Multiphoton Excited Fluorescence for Bioimaging.

The fabrication of high-order multiphoton excited fluorescent materials for second near-infrared (NIR-II, 1000-1700 nm) light-induced fluorescence imaging has always been an intractable challenge. In this study, a reasonable strategy guided by theoretical calculations was employed to fabricate a novel high-order multiphoton excited fluorescence (H-MPEF)-responsive UiO-type metal-organic framework (MOF, ZrTc). Strategically, the functionalization of the photonic-responsive thiazolothiazole-based organic ligand gave rise to amplified H-MPEF (four-photon activity) performance and prolonged excitation wavelength (1550 nm) of ZrTcI. The results reveal that the extended π-electron system, enhanced charge transfer, improved dipole moment, and weakened π-π stacking interactions within the MOFs endow them with excellent H-MPEF performance for NIR-II light-induced fluorescence imaging. It is an extremely rare report on H-MPEF bioimaging using MOFs and provide a universal strategy for the fabrication of H-MPEF-responsive materials.

Embedding Multiphoton Active Units within Metal–Organic Frameworks for Turning on High‐Order Multiphoton Excited Fluorescence for Bioimaging

AbstractThe fabrication of high‐order multiphoton excited fluorescent materials for second near‐infrared (NIR‐II, 1000–1700 nm) light‐induced fluorescence imaging has always been an intractable challenge. In this study, a reasonable strategy guided by theoretical calculations was employed to fabricate a novel high‐order multiphoton excited fluorescence (H‐MPEF)‐responsive UiO‐type metal–organic framework (MOF, ZrTc). Strategically, the functionalization of the photonic‐responsive thiazolothiazole‐based organic ligand gave rise to amplified H‐MPEF (four‐photon activity) performance and prolonged excitation wavelength (1550 nm) of ZrTcI. The results reveal that the extended π‐electron system, enhanced charge transfer, improved dipole moment, and weakened π‐π stacking interactions within the MOFs endow them with excellent H‐MPEF performance for NIR‐II light‐induced fluorescence imaging. It is an extremely rare report on H‐MPEF bioimaging using MOFs and provide a universal strategy for the fabrication of H‐MPEF‐responsive materials.

Abstract 2472: Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy

Abstract We have developed a live tumor fragment (LTF) platform for predicting clinical response to cancer drugs. The success of this approach depends on screening tissue fragments derived from biopsies and excisions before drug treatment to select those that have acceptable levels of viability and tumor content. To enable this screening, we are developing label-free methods for integrated assessment of LTF histology, viability, and metabolic status using intrinsic multiphoton-excited fluorescence lifetime microscopy (MP-FLIM). We cut live EMT6 tumors and mammary fat pad into rectangular fragments that were then sorted and cultured in glass-bottomed multi-well plates. To induce cancer cell death, we treated one group with a therapeutic agent, e.g., the multi-kinase inhibitor, staurosporine, or the alkylating agent, cisplatin. Before and after treatment, we imaged LTF structure and metabolic status based on the intrinsically fluorescent metabolic co-factors nicotinamide dinucleotides (NAD(P)H) and flavin adenine dinucleotides (FAD) fluorescence intensity and lifetime using dual-excitation multiphoton microscopy. As a ground truth viability reference, we then stained and re-imaged the fragments using nuclear and cell viability probes such as Hoechst, SYTOX, TMRE and/or propidium iodide (PI). We analyzed the data by fitting fluorescence decay curves with dual or triple exponents to generate images of lifetime parameters, including the mean and individual component lifetimes and amplitudes. Finally, we co-registered the extrinsically labeled and autofluorescent lifetime images for 3D spatial analysis using commercial and custom software. We verified the methods using monolayer cell cultures and ATP luminescence and flow cytometry viability assays. Image spatial heterogeneity was quantified utilizing entropy metrics. Intrinsic contrast from multiphoton imaging revealed cellular and tissue structures. The median entropy was higher and its distribution negatively skewed for EMT6 (p<0.01), allowing us to distinguish tumor from its corresponding normal tissue of origin. In viable cells, nuclei were distinguishable from the cytoplasm via higher cytoplasmic NADH signal. Upon loss of cell viability, nuclear NADH signals increased, with characteristically short lifetimes, and the overall NADH intensity decreased, reducing the contrast between nuclei and their surrounding cytoplasm. We detected an increase in the whole fragment mean lifetime (τm p<0.002) and concomitant decrease in the short lifetime component (α1 p< 0.002) within 24 hours of staurosporine exposure. We have demonstrated that MP-FLIM can distinguish tumor from normal and live from dead in LTFs without labels. This approach will facilitate selection of fragments with acceptable viability and tumor content for subsequent drug treatment as part of a platform built for personalizing cancer therapy. Citation Format: Jonathan Ouellette, John Rafter, Kelsey Tweed, Leung Kau Tang, Christina Scribano, Pichet Adstamongkonkul, Mikaela Schultz, Justine Coburn, Maged Aldeeb, Neil Anthony, Christin Johnson, Kevin Eliceiri, Jonathan Oliner, Tomasz Zal. Tumor/normal and live/dead classification in live tumor fragments using label-free multiphoton microscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2472.

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.