Two-photon Excited Fluorescence Research Articles

Photophysical Properties of Mn-Doped InP/ZnS Nanocrystals

InP-based nanocrystals (NCs) have received considerable attention as promising candidates to replace Cd- and Pb-based NCs because they contain no heavy metal elements. However, relevant studies on the photophysical properties of InP-based NCs are still insufficient. In this work, Mn-doped InP/ZnS NCs are synthesized, and their photophysical properties have been investigated. With the help of transient absorption spectrum measurement, it is found that Mn-doped InP/ZnS NCs exhibit efficient exciton–dopant exchange interaction. Through the temperature-dependent fluorescence lifetime measurements it is revealed that some excitons may undergo reverse crossing to the conduction band of the InP core from the long-lived 4T1 state of the Mn2+ ions with increasing temperature, resulting in the exciton fluorescence lifetime of the doped NCs to be less influenced by the temperature. In addition, compared with the Mn fluorescence, the exciton fluorescence shows an opposite intensity tendency to the temperature variation as a result of the greatly decreased energy transfer efficiency at elevated temperature. Importantly, the doped NCs exhibit a bright two-photon excited Mn fluorescence in which no size selection effect is observed. The excellent photophysical properties of Mn-doped InP/ZnS NCs imply that they are promising in the applications of highly effective optoelectronic devices and biological science.

Nicotinamide effects on the metabolism of human fibroblasts and keratinocytes assessed by quantitative, label-free fluorescence imaging.

Alterations in metabolism are central to the aging process. Therefore, understanding the subcellular functional and structural changes associated with metabolic aging is critical. Current established methods for exploring cell metabolism either require the use of exogenous agents or are destructive to the tissue or cells. Two-photon excited fluorescence (TPEF) imaging has emerged as a method for monitoring subtle metabolic changes non-invasively. In this study, we use TPEF imaging to acquire high-resolution fluorescence images from two coenzymes, NAD(P)H (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), within human fibroblasts and keratinocytes in response to B3 (a nicotinamide precursor) supplementation and/or UV irradiation, without addition of exogenous labels. In addition, multi-parametric analysis methods are used to extract functional information of cellular metabolism, including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial organization. Our results demonstrate that such optical metabolic assessments can serve as sensitive, label-free, non-destructive reporters of known effects of B3 to maintain and in some cases even enhance the respiratory function of mitochondria, while lowering oxidative damage. Thus, TPEF imaging, supported by highly-quantitative analysis, can serve as a tool to understand aging-dependent metabolic changes as well as the effect of actives on human epidermal and dermal cells.

Label-free concurrent 5-modal microscopy (Co5M) resolves unknown spatio-temporal processes in wound healing

The non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.

What Mediates Fibrosis in the Tumor Microenvironment of Clear Renal Cell Carcinoma.

Previous studies have demonstrated that direct targeting of interstitial cancer-associated fibroblasts (CAF) and tumor fibrosis alone seemed to be an unpromising treatment option for malignant tumors. Therefore, it is necessary to further explore the mechanism of the influence of collagen and tumor fibrosis on the biological behavior of malignant tumors. The current study aimed to explore the effect of intratumor fibrosis on the prognosis of renal clear cell carcinoma (ccRCC) and its mechanism. With the bioinformatic analysis of The Cancer Genome Atlas (TCGA) database (n = 537), the study showed that high Collagen type I α 1 (COL1A1) mRNA expression indicated the poor prognosis of ccRCC patients compared with low expression ones. We further used the Two-photon-excited fluorescence (TPEF)/second harmonic generation (SHG) microscopy to determine the intratumor fibrosis of 68 patients with surgical resection of ccRCC and confirmed that a high fibrosis level in the tumor was associated with a poor prognosis compared with patients with low expression (Progression-Free Survival: p = 0.030). We further measured the protein chips of 640 cytokines in ccRCC specimens and found that several cytokines, including prolactin (PRL), were associated with the degree of fibrosis in the tumor, as confirmed by the prolactin receptor (PRLR) immunohistochemical method. In addition, the study showed that PRLR expression decreased significantly in the ccRCC compared with adjacent normal tissue (p < 0.05). Our research shows that low expression of PRLR predicted the poor survival of the patient. We used the Cell Counting Kit-8 experiment, the transwell and the plate clone formation assay to evaluate the role of PRL in the 7860 and the ACHN cell lines. We found that PRL promoted ccRCC cell proliferation and migration. JAK-STAT3 activation was found in the high prolactin expression group by mass spectrum analysis. This study delineated the fibrosis-based tumor microenvironment characteristics of ccRCC. PRL/PRLR may be involved in the fibrosis process and are essential prognostic risk factors for ccRCC.

In situ Plasmon-Enhanced CARS and TPEF for Gram staining identification of non-fluorescent bacteria

In this work, we report in situ nonlinear microscopic images on plasmon-enhanced coherent anti-Stokes Raman scattering (CARS) and plasmon-Induced two-photon excited fluorescence （TPEF)of non-fluorescent microorganism. Our unique synthesized Au@Ag nanorods provide with two distinct surface-plasmon resonance (SPR) at 400 and 800 nm, respectively, which can efficiently induce linear fluorescence signals of E. coli but also enhance the nonlinear optical spectroscopy signals of TPEF, and coherent anti-Stokes Raman scattering (CARS) imaging of E. coli and S. aureus. Furthermore, calculations with complete active space self-consistent field (CASSCF) reveals the hot electrons of SPs can efficiently induce the biological fluorescence of non-fluorescent flavin nucleotides on the surface of E. coli. This novel mechanism is expected to guide the development and application of new microbial detection reagents. Gram-negative and Gram-positive bacteria can be well distinguished by nonlinear microscopic imaging of the CARS signal at 1589 cm−1. Benefit by the strong penetrability of non-linear optical signals, it is expected to realize in situ real-time detection and classification of pathogenic microbial infections in vivo.

Three-dimensional characterizations of two-photon excitation fluorescence images of elastic fibers affected by cutaneous scar duration.

The type or duration of a scar determines the choice of therapy available. Traditional detection methods can easily cause secondary trauma, so there is an urgent need for a non-invasive, rapid diagnostic approach. A strategy for quantitative analysis of three-dimensional (3D) elastic fibers in human cutaneous scars was designed, which included 3D reconstruction, skeleton extraction, quantitative analysis, and random forest regression. Four reconstruction methods were used to reconstruct 3D two-photon excitation fluorescence images of elastic fibers for comparison. In the skeleton extraction stage, the 3D thinning algorithm was improved to prepare for accurate quantitative analysis, in which eight parameters comprising branches number (B-NUM), nodes number (N-NUM), averaged branch broken-line length (AB-BL), averaged linear branch length (AB-LL), averaged branch tortuosity (AB-T), branch direction consistency (B-DC), averaged branch volume (AB-V), and averaged branch sectional area (AB-SA) were presented. Six of them, except averaged branch tortuosity (AB-T) and branch direction consistency (B-DC), showed an explicit tendency to change with scar duration. In the random forests regression analysis, the six extracted parameters could be used to predict scar duration with R2=0.981 and RMSE=0.513. The parameters we extracted had a distinct relationship with scar duration, and random forests regression showed better performance in forecasting scar duration than unitary models.

Two-photon absorption characterization and comparison of Au(I) fluorenyl benzothiazole complexes in the visible wavelength regime

We use the two-photon excited fluorescence method to determine the two-photon absorption (2PA) cross sections of three series of (fluorenyl benzothiazole) gold(I) complexes in the visible wavelength range from 570 to 700 nm. We compare the effect of ancillary ligand substitutions on the 2PA magnitudes and find that the ancillary ligand does not drastically affect either the magnitude or the shape of 2PA. Even so, moderate 2PA cross sections were measured that ranged from 10 to 1000 s of GM (Göppert-Mayer, =10-50cm4s/photon), making these types of complexes nonlinear optical materials for two-photon absorbing applications.

Rational design, facile synthesis, and linear/nonlinear optical properties of novel two-photon absorption stilbene derivatives with different configurations

Four novel two-photon absorption compounds (1MOBI, 2MOHA, 3BrBI, and 4BrHA), which have assembled stilbene π-bridge, different electron acceptors and donors, were synthesized conveniently via the one-step Horner-Wadsworth-Emmons reaction. Their configurations are A-π-D-π-A, D′-π-D-π-D′, A′-π-A-π-A′, and D-π-A-π-D, respectively. Their linear and nonlinear optical properties including linear absorption, one-photon excited fluorescence, two-photon absorption, and two-photon excited fluorescence, were systematically investigated in various solvents. The results demonstrate that the compounds with dihexylamino donors exhibit good optical properties. 2MOHA and 4BrHA exhibit large two-photon absorption cross-sections (2183 and 1737 GM) in DMF, which are larger than those of many known stilbene derivatives. The relationships between the structures and the optical properties were studied aided with the time-dependent density functional theory calculations.

Optical Microscopy and the Extracellular Matrix Structure: A Review.

Biological tissues are not uniquely composed of cells. A substantial part of their volume is extracellular space, which is primarily filled by an intricate network of macromolecules constituting the extracellular matrix (ECM). The ECM serves as the scaffolding for tissues and organs throughout the body, playing an essential role in their structural and functional integrity. Understanding the intimate interaction between the cells and their structural microenvironment is central to our understanding of the factors driving the formation of normal versus remodelled tissue, including the processes involved in chronic fibrotic diseases. The visualization of the ECM is a key factor to track such changes successfully. This review is focused on presenting several optical imaging microscopy modalities used to characterize different ECM components. In this review, we describe and provide examples of applications of a vast gamut of microscopy techniques, such as widefield fluorescence, total internal reflection fluorescence, laser scanning confocal microscopy, multipoint/slit confocal microscopy, two-photon excited fluorescence (TPEF), second and third harmonic generation (SHG, THG), coherent anti-Stokes Raman scattering (CARS), fluorescence lifetime imaging microscopy (FLIM), structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), ground-state depletion microscopy (GSD), and photoactivated localization microscopy (PALM/fPALM), as well as their main advantages, limitations.

Small molecules based Benzothiazole-pyridinium salts with different anions: Two-photon fluorescence regulation and difference in cell imaging application

A series of benzoxazole-pyridinium salt derivatives with two-photon excited fluorescence performance were synthesized and fully characterized. Among them, compounds 2, 3 and 4 were the isomers with different positions of pyridine N, while compounds 4N, 4S, 4P, 4F and 4B were derived from compound 4 through anion exchanging reaction. Their photophysical properties had been systematically investigated, and the results indicated that the position of pyridine N and the type of anions had a significant influence on their optical properties and bio-imaging application. Transforming the position of pyridine N could affect their fluorescence quantum obviously; the maximum fluorescence quantum is 0.21 for compound 3 in DMF. Meanwhile, we found that the altering anions might be used to regulate their two-photon fluorescence performance, only compounds 4N, 4P and 4B possessed excellent two-photon excited fluorescence emissions, and the highest two-photon cross-section was 1868.69 GM for compound 4B in DMF. In addition, two-photon fluorescence cell imaging experiment demonstrated the potential bio-application of partial compounds with good photostability and low cytotoxicity.

Label-free two-photon imaging of mitochondrial activity in murine macrophages stimulated with bacterial and viral ligands

Mitochondria are the metabolic hub of the cell, playing a central role in regulating immune responses. Dysfunction of mitochondrial reprogramming can occur during bacterial and viral infections compromising hosts’ immune signaling. Comparative evaluation of these alterations in response to bacterial and viral ligands can provide insights into a cell’s ability to mount pathogen-specific responses. In this study, we used two-photon excitation fluorescence (TPEF) imaging to quantify reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) and flavin adenine dinucleotide (FAD) levels in the cell and to calculate the optical redox ratio (ORR), an indicator of mitochondrial dysfunction. Analyses were performed on RAW264.7 cells and murine bone marrow derived macrophages (BMM) stimulated with bacterial (LPS) and viral (Poly(I:C)) ligands. Responses were cell type dependent, with primary cells having significantly higher levels of FAD and higher oxygen consumption rates suggesting BMM may be more dependent on mitochondrial metabolism. Our findings also suggest that FAD-TPEF intensity may be a better predictor of mitochondrial activity and localization since it demonstrates unique mitochondrial clustering patterns in LPS vs. Poly(I:C) stimulated macrophages. Collectively, we demonstrate that TPEF imaging is a powerful label-free approach for quantifying changes in mitochondrial function and organization in macrophages following bacterial and viral stimuli.

Cell membrane covered polydopamine nanoparticles with two-photon absorption for precise photothermal therapy of cancer

HypothesisIn view of the photothermal effect of polydopamine (PDA) nanoparticles and their internal D-π-D structures during assembly, the two-photon excited properties of PDA were studied toward the biomedical application. Further, the PDA molecules were coordinated with Mn2+ and the assembled nanoparticles were covered by cancer cell membranes, the complex system could be used directly for the treatment of cancer with photothermal and chemodynamic therapy. ExperimentsThe two-photon excited PDA-Mn2+ nanoparticles were used for the photothermal therapy combined with chemodynamic therapy. The complexes were coated with cancer cell membranes in order to enhance the tumor homologous efficiency. Multi-modal bioimaging and anti-tumor detections were carried out both in vitro and in vivo. FindingsPDA nanoparticles were demonstrated to have both good two-photon excited fluorescence and photothermal efficiency. The assembled nanoparticles modified with Mn2+ and cancer cell membranes have an obvious targeting and synergetic anti-cancer efficiency. The system creates a simple way for a precise operation with multi-modal imaging function.

Molecular Multicolor Multiphoton in Vivo Bioimaging Based on a Direct Diode Pumped Ti:sapphire Oscillator

Multiphoton microscopy (MPM) including two-photon excited fluorescence (TPEF), second harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) is an important tool in biology and medicine to explore the dynamics of cells and to investigate tissue structure in living systems and biopsies. However, MPM still suffers from limited applicability due to the integration of highly sophisticated lasers. In this paper, we introduce our triple-modal multicolor MPM platform employing a single direct diode-pumped femtosecond Kerr-lens-mode-locked Ti:sapphire oscillator and acquire simultaneously intrinsic co-registered multicolor TPEF, TPEF, SHG and CARS signals in the backward direction. The complexity and by that the footprint as well the cost of the laser are considerably reduced by the direct diode-pumping scheme. In combination with an Yb fiber amplifier such a laser is suited for biomedical applications allowing for detailed morphologic and molecular spectroscopic in vivo investigation. The optimized emission wavelengths of our Ti:sapphire laser and Yb fiber amplifier provide simultaneous excitation at 805 nm, 1050 nm and 911 nm for multicontrast information from exogenous and endogenous fluorophores. We perform in vivo imaging of C. elegans and visualize metabolic changes in zebrafish larvae to investigate the tumor microenvironment with spectral focusing CARS and multicolor TPEF of green fluorescent protein.

Label-Free Detection of the Architectural Feature of Blood Vessels in Glioblastoma Based on Multiphoton Microscopy

Glioblastoma (GBM) is a highly angiogenesis malignant tumor with a significant heterogeneity of vascular morphology. The vascular patterns could be served as an indicator of vascular morphological complexity that associated with postoperative prognosis of patients with GBM. Here, multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), is applied for detecting the architectural feature of blood vessels from ex vivo, unfixed, and unstained human GBM specimens. Combined with the image processing algorithms, the morphology, location, orientation, alignment, and density of vascular collagen are qualitatively and quantitatively visualized, revealing the presented method not only contribute to differentiate GBM from normal brain tissue, but also provide important indicators of different vascular patterns, which may serve as a reference to the prognosis more accurately. Therefore, these results can provide prognosis-related histopathological diagnosis of GBM, holding a translational potential to be used as a label-free, quick, and on-site imaging tool for clinicians to determine the extent of excision involving brain functional areas.

Murine Metatarsus Bone and Joint Collagen-I Fiber Morphologies and Networks Studied With SHG Multiphoton Imaging.

Chronic inflammatory disease of bones and joints (e.g., rheumatoid arthritis, gout, etc.), but also acute bone injury and healing, or degenerative resorptive processes inducing osteoporosis, are associated with structural remodeling that ultimately have impact on function. For instance, bone stability is predominantly orchestrated by the structural arrangement of extracellular matrix fibrillar networks, i.e., collagen-I, -IV, elastin, and other proteins. These components may undergo distinct network density and orientation alterations that may be causative for decreased toughness, resilience and load bearing capacity or even increased brittleness. Diagnostic approaches are usually confined to coarse imaging modalities of X-ray or computer tomography that only provide limited optical resolution and lack specificity to visualize the fibrillary collagen network. However, studying collagen structure at the microscopic scale is of considerable interest to understand the mechanisms of tissue pathologies. Multiphoton Second Harmonic Generation (SHG) microscopy, is able to visualize the sterical topology of the collagen-I fibrillar network in 3D, in a minimally invasive and label-free manner. Penetration depths exceed those of conventional visible light imaging and can be further optimized through employing decalcification or optical clearing processing ex vivo. The goal of this proof-of-concept study was to use SHG and two-photon excited fluorescence (2-PEF) imaging to mainly characterize the fibrillary collagen organization within ex vivo decalcified normal mouse metatarsus bone and joint. The results show that the technique resolved the fibrillar collagen network of complete bones and joints with almost no artifacts and enabled to study the complex collagen-I networks with various fiber types (straight, crimped) and network arrangements of mature and woven bone with high degree of detail. Our imaging approach enabled to identify cavities within both cortical and trabecular bone architecture as well as interfaces with sharply changing fiber morphology and network structure both within bone, in tendon and ligament and within joint areas. These possibilities are highly advantageous since the technology can easily be applied to animal models, e.g., of rheumatoid arthritis to study structural effects of chronic joint inflammation, and to many others and to compare to the structure of human bone.

Crystal structure and optical property of a Cadmium(II) complex based on triphenylamine derivative—Theoretical and experimental investigation

A triphenylamine derivative (E)-N,N-diphenyl-4-(2-(pyridin-2-yl)vinyl)benzenamine (L) was synthesized, and the corresponding cadmium complex CdI2L2 crystal was obtained and solved by single crystal X-ray diffraction analysis. The experimental resutls of one photon absorption (OPA) and one photon excited fluorescence (OPEF) indicate that coordination of CdI2 only has slight effect on the spectrum peak position relative to the free ligand. Besides the Stokes shift, the full width half maximum of OPEF and the variation of the “peak span between solvents” from OPA to OPEF can also indicate the electron transition direction of the excitation process. The laser experiments show that both the ligand and complex have considerable two photon excited fluorescence (TPEF) emission. The experimental Stokes shift is correlated with the computational solvent polarity rMPI, and the results illustrate that the curve of “experimental Stokes shift vs. solvent rMPI” is exactly the Boltzmann S-curve. The computational results also indicate that the static interaction between the CdI2L2 molecules in crystal has strong effect on OPA properties. Considering the weak coordination bond strength of the complex CdI2L2, the S1→S0′ fluorescence emission may occur on both ligands, although the S0→S1′ excitation only occurs on the right ligand.

Heterogeneity and Development of Fine Astrocyte Morphology Captured by Diffraction-Limited Microscopy.

The fine processes of single astrocytes can contact many thousands of synapses whose function they can modulate through bi-directional signaling. The spatial arrangement of astrocytic processes and neuronal structures is relevant for such interactions and for the support of neuronal signaling by astrocytes. At the same time, the geometry of perisynaptic astrocyte processes is variable and dynamically regulated. Studying these fine astrocyte processes represents a technical challenge, because many of them cannot be fully resolved by diffraction-limited microscopy. Therefore, we have established two indirect parameters of astrocyte morphology, which, while not fully resolving local geometry by design, provide statistical measures of astrocyte morphology: the fraction of tissue volume that astrocytes occupy and the density of resolvable astrocytic processes. Both are straightforward to obtain using widely available microscopy techniques. We here present the approach and demonstrate its robustness across various experimental conditions using mainly two-photon excitation fluorescence microscopy in acute slices and in vivo as well as modeling. Using these indirect measures allowed us to analyze the morphology of relatively large populations of astrocytes. Doing so we captured the heterogeneity of astrocytes within and between the layers of the hippocampal CA1 region and the developmental profile of astrocyte morphology. This demonstrates that volume fraction (VF) and segment density are useful parameters for describing the structure of astrocytes. They are also suitable for online monitoring of astrocyte morphology with widely available microscopy techniques.

Enhanced electric field sensitivity of quantum dot/rod two-photon fluorescence and its relevance for cell transmembrane voltage imaging

Abstract The optoelectronic properties of semiconductor nanoparticles make them valuable candidates for the long-term monitoring of transmembrane electric fields in excitable cells. In this work, we show that the electric field sensitivity of the fluorescence intensity of type-I and quasi-type-II quantum dots and quantum rods is enhanced under two-photon excitation compared to single-photon excitation. Based on the superior electric field sensitivity of the two-photon excited fluorescence, we demonstrate the ability of quantum dots and rods to track fast switching E-fields. These findings indicate the potential of semiconductor nanoparticles as cellular voltage probes in multiphoton imaging.

Fate of Antibody-Targeted Ultrasmall Gold Nanoparticles in Cancer Cells after Receptor-Mediated Uptake.

Nanoparticles with ultrasmall sizes (less than 10 nm) offer many advantages in biomedical applications compared to their bigger counterparts, including better intratumoral distribution, improved pharmacokinetics (PK), and efficient body clearance. When functionalized with a biocompatible coating and a target-specific antibody, ultrasmall nanoparticles represent an attractive clinical translation platform. Although there is a tremendous body of work dedicated to PK and the biological effects of various nanoparticles, little is known about the fate of different components of functionalized nanoparticles in a biological environment such as in live cells. Here, we used luminescence properties of 5 nm gold nanoparticles (AuNPs) to study the intracellular trafficking and fate of the AuNPs functionalized with an organic layer consisting of a polyethylene glycol (PEG) coating and epidermal growth factor receptor (EGFR)-targeting antibody. We showed that intracellular uptake of the targeted 5 nm AuNPs results in a strong two-photon luminescence (TPL) that is characterized by broad emission and very short lifetimes compared to the fluorescence of the nanoparticle-conjugated fluorophore-tagged antibody, thereby allowing selective imaging of these components using TPL and two-photon excited fluorescence lifetime microscopy (2P-FLIM). Our results indicate that the nanoparticle’s coating is detached from the particle’s surface inside cells, leading to formation of nanoparticle clusters with a strong TPL. Furthermore, we observed an optically resolved spatial separation of the gold core and the antibody coating of the particles inside cells. We used data from two-photon microscopy, 2P-FLIM, electron microscopy, and in vitro assays to propose a model of interactions of functionalized 5 nm AuNPs with live cells.

Functionalized Au15 nanoclusters as luminescent probes for protein carbonylation detection

Atomically precise, ligand-protected gold nanoclusters (AuNCs) attract considerable attention as contrast agents in the biosensing field. However, the control of their optical properties and functionalization of surface ligands remain challenging. Here we report a strategy to tailor AuNCs for the precise detection of protein carbonylation—a causal biomarker of ageing. We produce Au15SG13 (SG for glutathione) with atomic precision and functionalize it with a thiolated aminooxy moiety to impart protein carbonyl-binding properties. Mass spectrometry and molecular modelling reveal the key structural features of Au15SG12-Aminooxy and its reactivity towards carbonyls. Finally, we demonstrate that Au15SG12-Aminooxy detects protein carbonylation in gel-based 1D electrophoresis by one- and two-photon excited fluorescence. Importantly, to our knowledge, this is the first application of an AuNC that detects a post-translational modification as a nonlinear optical probe. The significance of post-translational modifications in life sciences may open avenues for the use of Au15SG13 and other nanoclusters as contrast agents with tailored surface functionalization and optical properties.