Two-photon Fluorescence Research Articles

Hyaluronic acid-modified metal–organic framework for two-photon imaging-guided photodynamic therapy in triple negative breast cancer

Zr-based metal–organic frameworks (MOFs) using thiazolothiazole as an organic ligand exhibit excellent two-photon excited fluorescence performance, particularly in the generation of reactive oxygen species. In this study, a ZrTc nanoMOF was designed and modified with hyaluronic acid (HA) to provide good biocompatibility and cancer cell-specific targeting. In triple negative breast cancer (TNBC), the optimized composite ZrTc@HA exhibited considerable two-photon activity and excellent light-triggered O2·− generation ability at an excitation wavelength of 780 nm. ZrTc@HA can be used as a diagnostic probe for fluorescence imaging and as a therapeutic agent for photodynamic therapy (PDT) with no remarkable toxicity in vitro or in a subcutaneous tumor model in vivo. In summary, we developed a promising two-photon-activated ZrTc@HA for PDT in TNBC, with immense potential for advancements in monotherapy and synergistic phototherapy.

Frequency-encoded two-photon excited fluorescence microscopy.

Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω1, ω2) resulting in excitation energies at 2ω1, 2ω2, and the mixing energy ω1+ω2. Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.

Fluorescent protein chromophore-based photosensitizer modified with anthracenyl for lysosome-targeted photodynamic therapy and two-photon fluorescence imaging

In this work, two cis-anthracenyl fluorescent protein chromophore analogs, AnFP-1 and AnFP-2 were synthesized by [2 + 3] cycloaddition for photodynamic therapy and two-photon fluorescence imaging. We combine the anthracenyl group and fluorescent core imidazolinone to form the dye AnFP-1, and then the morpholine indole structure was introduced via Knoevenagel reaction to obtain the dye AnFP-2. The heavy-atom-free photosensitizing dyes AnFP-1 and AnFP-2 have satisfied yields of singlet oxygen, measured to be 30% and 21% in methanol solution, respectively. The tetrazolium blue (MTT) experiment showed that the survival rate of A549 cells was over 92% without light exposure and under 460 nm light exposure for 10 min, the half-inhibitory concentrations (IC50) of the photosensitizing dyes AnFP-1 and AnFP-2 were 3.7 μmol•L−1 and 4.1 μmol•L−1, respectively. Acridine orange (AO)/Ethidium bromide (EB) double staining experiments showed that the two photosensitizing dyes could generate singlet oxygen and induce apoptosis in cancerous cells under irradiation. In addition, the photosensitizing dye AnFP-2 has high specificity to lysosomes which improve the photodynamic effect. Its Pearson co-localization correlation coefficient is 93%. In biological imaging, AnFP-1 and AnFP-2 showed satisfactory two-photon fluorescence imaging in zebrafish with 800 nm femtosecond laser excitation. According to these results, AnFP-1 and AnFP-2 can be used in photodynamic therapy and two-photon fluorescence imaging, which lays the foundation for developing new photosensitizing dyes.

Assessing pathological features of breast cancer via the multimodal information of multiphoton and Raman imaging

For unveiling the pathological evolution of breast cancer, nonlinear multiphoton microscopic (MPM) and confocal Raman microspectral imaging (CRMI) techniques were both utilized to address the structural and constitutional characteristics of healthy (H), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) tissues. MPM-based techniques, including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), visualized label-free and the fine structure of breast tissue. Meanwhile, CRMI not only presented the chemical images of investigated samples with the K-mean cluster analysis method (KCA), but also pictured the distribution of components in the scanned area through univariate imaging. MPM images illustrated that the cancer cells first arranged around the basement membrane of the duct, then proliferated to fill the lumens of the duct, and finally broke through the basement membrane to infiltrate into the stroma. Although the Raman imaging failed to visualize the cell structure with high resolution, it explained spectroscopically the gradual increase of nucleic acid and protein components inside the ducts as cancer cells proliferated, and displayed the distribution pattern of each biological component during the evolution of breast cancer. Thus, the combination of MPM and CRMI provided new insights into the on-site pathological diagnosis of malignant breast cancer, also ensured technical support for the development of multimodal optical imaging techniques for precise histopathological analysis.

Hollow Core Double-Clad Fiber Coupler for Nonlinear Micro-Endoscopy

We report the design, fabrication and characterization of the first hollow core double-clad fiber coupler for nonlinear micro-endoscopy. It is made of a pure silica negative curvature hollow core double-clad fiber and a highly multimode fiber fused along each other. The fabrication of such a coupler was made possible thanks to the use of a soft (borosilicate) glass multimode fiber which can be fused along the negative curvature fiber without altering its geometry, thanks to its much lower fusion temperature. A 60% power transfer from the double-clad fiber to the multimode one was measured at 500 nm. Demonstration of two-photon fluorescence and second harmonic generation imaging was performed on unlabeled biological tissues with an endoscopic head mounted on the hollow core double-clad fiber coupler to demonstrate its applicability. This device greatly simplifies the integration of nonlinear micro-endoscopes based on hollow core double clad fibers.

A merocyanine dye-based superoxide anion fluorescent probe with second-level response agility enables evaluating interstitial cystitis via two-photon excitation imaging

As a species tightly associated with inflammation, superoxide anion radical (O2·-) is generally recognized a biomarker for interstitial cystitis (IC) but the reliable detection of such species has been remaining a challenge owing to its elusive nature. Herein, we developed a merocyanine dye-based fluorescent probe, MC-O-TBS, capable of detecting O2·- species with a limit of detection down to 49.3 nM, recognition specificity against other potential interfering species and two-photon excitation fluorescence (TPEF) property for bioimaging with satisfactory depth. As another particularly salient feature, MC-O-TBS generates O2·--mediated fluorescence contrast with response time less than 10 s, an unexampled response agility of probe in O2·- fluorescence sensing as compared to the counterpart probes developed to date. The superior performance of MC-O-TBS probe in terms of recognition specificity, detection sensitivity, TPEF for bioimaging with satisfactory depth and response rate as well as the preliminary in vitro experiment results regarding mapping O2·- level in bladder organ unequivocally proclaimed its potential for reliably monitoring the highly elusive O2·- species and clinically diagnosing IC featured with abnormal O2·- level.

Optical coherence tomography-based design for a real-time motion corrected scanning microscope.

While two-photon fluorescence microscopy is a powerful platform for the study of functional dynamics in living cells and tissues, the bulk motion inherent to these applications causes distortions. We have designed a motion tracking module based on spectral domain optical coherence tomography which compliments a laser scanning two-photon microscope with real-time corrective feedback. The module can be added to fluorescent imaging microscopes using a single dichroic and without additional contrast agents. We demonstrate that the system can track lateral displacements as large as 10 μm at 5 Hz with latency under 14 ms and propose a scheme to extend the system to 3D correction with the addition of a remote focusing module. We also propose several ways to improve the module's performance by reducing the feedback latency. We anticipate that this design can be adapted to other imaging modalities, enabling the study of samples subject to motion artifacts at higher resolution.

An acridone-derived fluorescent off-on probe for detection and in vivo imaging of nitroreductase

Understanding the functions of enzymes in various physiological processes is important, but the design of signaling probes for fast analysis of enzymatic activity is particularly challenging. Herein, a fluorescence-enhanced probe, 10-methyl-2-nitro-acridone (MNA), was synthesized and applied to analyze nitroreductase (NTR) activity in vitro and in vivo. The detection mechanism is based on the nitro group in MNA reacting toward NTR with high reactivity and generating 10-methyl-2-amino- acridone (MAA) accompanied by an obvious fluorescence signal enhancement at 525 nm emission. The probe shows low cytotoxicity, fast response, and high selectivity and sensitivity with a limit of detection as low as 150 ng·mL−1. The probe was also employed for two-photon fluorescence imaging of NTR in zebrafish in vivo revealing the distribution of NTR. Versus existing NTR probes, the proposed probe shows favorable analytical performance including near-infrared light excitation with no other byproducts produced after the reaction. The superior properties of this signaling probe allow it to become a fluorescence imaging candidate in other biosystems.

An elastic luminescent organic single crystal with linear and nonlinear optical waveguide properties

In recent years, organic single crystals combining elastic and photoluminescent (PL) properties have attracted extensive attention due to their promising applications in many fields. However, elastic organic crystals with two-photon luminescence (2 PL) waveguiding properties are still rarely found, which is imperative for various applications, including near-infrared (NIR) bioimaging and nonlinear optical (NLO) photonic devices. This work reports an elastic single crystal with a long needle shape, anthracene-9,10-dicarbaldehyde (An-DC). Its crystal (100) face exhibits highly reversible bending by applying the mechanical force, which is facilitated by the van der Waals (C– H···π), H-bond (C–H⋯O), and π···π interactions among molecules. The crystal emits a strong orange-red fluorescence when excited by blue-violet light. Interestingly, it also exhibits excellent two-photon fluorescence emission when stimulated by near-infrared light. Based on these linear and nonlinear optical characteristics, the waveguide applications of the An-DC crystal were implemented. Notably, in the bending state, the luminescence performance and the crystal structure of the An-DC crystal are maintained well, which can be used as a low-loss material for linear and nonlinear optical waveguide devices, thus expanding the potential use of flexible organic crystals.

Spatially resolved transcriptomic profiles reveal unique defining molecular features of infiltrative 5ALA-metabolizing cells associated with glioblastoma recurrence

BackgroundSpatiotemporal heterogeneity originating from genomic and transcriptional variation was found to contribute to subtype switching in isocitrate dehydrogenase-1 wild-type glioblastoma (GBM) prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) enables intraoperative visualization of infiltrative tumors outside the magnetic resonance imaging contrast-enhanced regions. The cell population and functional status of tumor responsible for enhancing 5ALA-metabolism to fluorescence-active PpIX remain elusive. The close spatial proximity of 5ALA-metabolizing (5ALA +) cells to residual disease remaining post-surgery renders 5ALA + biology an early a priori proxy of GBM recurrence, which is poorly understood.MethodsWe performed spatially resolved bulk RNA profiling (SPRP) analysis of unsorted Core, Rim, Invasive margin tissue, and FACS-isolated 5ALA + /5ALA − cells from the invasive margin across IDH-wt GBM patients (N = 10) coupled with histological, radiographic, and two-photon excitation fluorescence microscopic analyses. Deconvolution of SPRP followed by functional analyses was performed using CIBERSORTx and UCell enrichment algorithms, respectively. We further investigated the spatial architecture of 5ALA + enriched regions by analyzing spatial transcriptomics from an independent IDH-wt GBM cohort (N = 16). Lastly, we performed survival analysis using Cox Proportinal-Hazards model on large GBM cohorts.ResultsSPRP analysis integrated with single-cell and spatial transcriptomics uncovered that the GBM molecular subtype heterogeneity is likely to manifest regionally in a cell-type-specific manner. Infiltrative 5ALA + cell population(s) harboring transcriptionally concordant GBM and myeloid cells with mesenchymal subtype, -active wound response, and glycolytic metabolic signature, was shown to reside within the invasive margin spatially distinct from the tumor core. The spatial co-localization of the infiltrating MES GBM and myeloid cells within the 5ALA + region indicates PpIX fluorescence can effectively be utilized to resect the immune reactive zone beyond the tumor core. Finally, 5ALA + gene signatures were associated with poor survival and recurrence in GBM, signifying that the transition from primary to recurrent GBM is not discrete but rather a continuum whereby primary infiltrative 5ALA + remnant tumor cells more closely resemble the eventual recurrent GBM.ConclusionsElucidating the unique molecular and cellular features of the 5ALA + population within tumor invasive margin opens up unique possibilities to develop more effective treatments to delay or block GBM recurrence, and warrants commencement of such treatments as early as possible post-surgical resection of the primary neoplasm.

Temperature-Dependent Intensity Modulated Two-Photon Excited Fluorescence Microscopy for High Resolution Mapping of Charge Carrier Dynamics.

We present a temperature-dependent intensity modulated two-photon excited fluorescence microscopy technique that enables high-resolution quantitative mapping of charge carrier dynamics in perovskite microcrystal film. By disentangling the emission into harmonics of the excitation modulation frequency, we analyze the first and second order charge carrier recombination processes, including potential accumulation effects. Our approach allows for a quantitative comparison of different emission channels at a micrometer resolution. To demonstrate the effectiveness of the method, we applied it to a methylammonium lead bromide perovskite microcrystal film. We investigated the temperature-dependent modulated imaging, encompassing the exciton dissociation-association and charge carrier trapping-detrapping equilibrium. Additionally, we explored the potential freezing out of traps and the phase transition occurring at low temperatures.

3D Morphology of Different Crystal Forms in β-Nucleated and Fiber-Sheared Polypropylene: α-Teardrops, α-Teeth, and β-Fans.

Polymorphism of semicrystalline polymers has significant influence on their physical properties, with each form having its advantages and disadvantages. However, real-life polymer processing often results in different coexisting crystal polymorphs, and it remains a challenge to determine their shape, spatial distribution, and volume fraction. Here, i-polypropylene (i-PP) sheets containing both α- and β-forms were prepared either by adding β-nucleating agent or by fiber pulling-induced crystallization. By adding a compatible dye that is partially rejected from the growing crystalline aggregates (spherulites and cylindrites), we visualize the shape of these objects in 3D using two-photon fluorescence confocal microscopy. To distinguish between crystal forms, we take advantage of the difference in dye-retaining ability of the α- and β-aggregates. Even in 2D, fluorescence microscopy (FM) distinguishes the two crystal forms better than polarized microscopy. In 3D imaging, the volume fraction and spatial distribution of α- and β-forms in different morphological types could be determined quantitatively. Morphologies described as α-teeth, β-fans, and α-teardrops were visualized for the first time in 3D. Furthermore, internal and surface microcracks were seen to be associated predominantly with the β-form and around the fiber. Spatial distribution of α- and β-forms was also determined by scanning with a synchrotron X-ray beam. Good agreement was obtained with 3D microscopy, but XRD could not match the detail obtainable by the tomography. The work demonstrates the ability of the 3D imaging method to distinguish different crystal forms and their specific morphologies.

Nitriding Effects of Ammonia Flames on Iron-based Metal Walls

Effects of NH3/O2/N2 premixed flame/mixture on iron-based metal walls have been investigated experimentally. A premixed NH3/O2/N2 flame or mixture formed by a quartz tube burner impinged on the disc-shaped metal test plate, of which the temperature was kept at 550 oC by an infrared lamp heater. As materials for the test plate, SACM645, SUS304 and SUS310S, which are widely used as industrial steels, were examined. After being exposed to either the NH3/O2/N2 flame or mixture for 5 hours, the surface hardness was measured by using a nano indenter. Distributions of nitrogen atom diffused into the metal wall were measured by means of the Electron Probe Micro Analyzer (EPMA) and Secondary Ion Mass Spectrometry (SIMS). In addition, spatial distributions of the NH3 concentration in the NH3/O2/N2 flame and mixture were measured by employing a two-photon absorption laser-induced fluorescence (TALIF) technique. It was found that the surface hardness of the test plates increased significantly after being exposed to the NH3/O2/N2 flame/mixture. The distributions of surface hardness and nitrogen atom concentration show that nitriding occurred by the present NH3/O2/N2 flame/mixture with the equivalent level of the conventional gas nitriding method. Moreover, it is also found that NH3 concentration in the vicinity of the wall plays an important role in the surface hardness increase, and a wider distribution of NH3 can result in a broader surface hardness distribution.

A two-photon mitochondria-targeting azo reductase probe for imaging in tumor cells and mice

Herein, a two-photon mitochondria-targeting fluorescent probe (TPNP) which can be activated by the azo reductase (AzoR) has been developed for imaging the hypoxic microenvironment of tumors in vivo. The probe TPNP showed a low detection limit (LOD = 1.85 eq), high selectivity, and considerable steadiness in different pH conditions (3.0–11.0). In living cells, it could co-locate with the commercial dye in the mitochondria (co-localization coefficient: 0.92) and indicate hypoxia in both one-photon and two-photon fluorescence imaging. Its AzoR-activation mechanism was further verified by the docking simulation as well as the experiments of adding AzoR or electron transporter inhibitors. Moreover, the in vivo real-time fluorescence imaging proved that TPNP could monitor hypoxia and the tumor progression in mouse tumor models. Overall, this designed probe exhibited high sensitivity, synthetic ease, and excellent fluorescent properties, which might therefore enlighten new ideas for the diagnosis of tumors in future studies.

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.

Double-clad photonic crystal fibre for two-photon microscopy

Miniaturized and head-mountable scanning nonlinear fibre-optic microscopy offers an exciting opportunity for the visualization of brain activity on freely walking mice. One challenge in the creation of such miniaturized microscopes is the design of optical fibres capable of delivering ultrashort pulses without distortion and collecting fluorescence simultaneously and efficiently. A double-clad photonic crystal fibre (DCPCF) with 37-cell air holes arranged in hexagonal lattice in core is proposed. The full vector finite element method (FEM) is used for numerical analysis. Nearly zero dispersion of −0.34 ps/nm/km at 920 nm wavelength is finally achieved. The single-mode core with an NA of 0.53 at 920 nm wavelength is used to deliver the femtosecond excitation beam and the inner cladding with an NA of 0.54 at 532 nm is used to collect the two-photon fluorescence signal. The nearly zero dispersion, high NA and low nonlinearity will be beneficial to promote its application in two-photon microscopy.

Two-photon fluorescence probe for quantification of cyclosporine.

A novel ratiometric fluorescent sensor was fabricated for the fast and facile determination of cyclosporine A (CsA). Due to the narrow therapeutic index of CsA, its desired therapeutic effects are evident within a limited range of blood concentration, indicating the fundamental role of therapeutic drug monitoring in CsA pharmacological response. In this study, a two-photon fluorescence probe based on the zeolitic imidazolate framework (ZIF-8) and norepinephrine-capped silver nanoparticles (AgNPs@NE) was employed for the quantification of the CsA in human plasma samples. In the presence of CsA, the fluorescent emission intensity of ZIF-8-AgNPs@NE was quenched. Under the optimum conditions, the proposed probe determines CsA in plasma samples in two linear ranges of 0.01 to 0.5 μg mL-1 and 0.5 to 10 μg mL-1. The developed probe demonstrates the advantages of a facile and fast platform with limit of detection as low as 0.007 μg mL-1. At last, this method was applied to find CsA concentration in four patients receiving oral CsA regimen which indicates it as a promising method for on-site detection applications.

Two-photon excited luminescence of structural light enhancement in subwavelength SiO2 coating europium ion-doped paramagnetic gadolinium oxide nanoparticle and application for magnetic resonance imaging

BackgroundOxides of lanthanide rare-earth elements show great potential in the fields of imaging and therapeutics due to their unique electrical, optical and magnetic properties. Oxides of lanthanide-based nanoparticles enable high-resolution imaging of biological tissues by magnetic resonance imaging (MRI), computed tomography (CT) imaging, and fluorescence imaging. In addition, they can be used to detect, treat, and regulate diseases by fine-tuning their structure and function. It remains challenging to achieve safer, efficient, and more sensitive nanoparticles for clinical applications through the structural design of functional and nanostructured rare-earth materials.ResultIn this study, we designed a mesoporous silica-coated core–shell structure of europium oxide ions to obtain near-infrared two-photon excitation fluorescence while maintaining high contrast and resolution in MRI. We designed enhanced 800 nm photoexcitation nanostructures, which were simulated by the finite-difference method (FDM) and finite-difference time-domain method (FDTD). The nanoparticle structure, two-photon absorption, up-conversion fluorescence, magnetic properties, cytotoxicity, and MRI were investigated in vivo and in vitro. The nanoparticle has an extremely strong optical fluorescence response and multiple excitation peaks in the visible light band under the 405 nm continuous-wave laser excitation. The nanoparticle was found to possess typical optical nonlinearity induced by two-photon absorption by ultrafast laser Z-scan technique. Two-photon excited fluorescence of visible red light at wavelengths of 615 nm and 701 nm, respectively, under excitation of the more biocompatible near-infrared (pulsed laser at 800 nm). In an in vitro MRI study, a T1 relaxation rate of 6.24 mM−1 s−1 was observed. MRI in vivo showed that the nanoparticles could significantly enhance the signal intensity in liver tissue.ConclusionsThese results suggest that this sample has applied potential in visible light fluorescence imaging and MRI.

Adaptive Optical Two-Photon Fluorescence Microscopy Probes Cellular Organization of Ocular Lenses In Vivo.

The mammalian ocular lens is an avascular multicellular organ that grows continuously throughout life. Traditionally, its cellular organization is investigated using dissected lenses, which eliminates in vivo environmental and structural support. Therefore, in vivo optical imaging methods for studying lenses in their native context in live animals are urgently needed. Here, we demonstrated that two-photon fluorescence microscopy can visualize lens cells in vivo. To maintain subcellular resolution at depth, we used adaptive optics to correct aberrations owing to ocular and lens tissues, which led to substantial signal and resolution improvements. Imaging lens cells up to 980 µm deep, we observed novel cellular organizations including suture-associated voids, enlarged vacuoles, and large cavities, contrary to the conventional view of a highly ordered organization. We tracked these features longitudinally over weeks and observed the incorporation of new cells during growth. Taken together, noninvasive longitudinal in vivo imaging of lens morphology using adaptive optics two-photon fluorescence microscopy will allow us to observe the development or alterations of lens cellular organization in living animals directly.

Accurate Detection of Hcy in Human Serum and Two-Photon Visualization of Atherosclerosis Using a Highly Specific Fluorescent Probe.

As an important metabolic intermediate of sulfur-containing amino acids in human body, homocysteine (Hcy) is regarded as an independent risk factor for atherosclerotic cardiovascular disease. Therefore, real-time monitoring of the fluctuation of Hcy level is of great importance for the early diagnosis as well as the treatment of atherosclerosis. Herein, a new two-photon (TP) fluorescent probe (RH-2) was developed via a hydrogen bond-assisted strategy, which had a high specificity for detecting Hcy over cysteine (Cys) and glutathione (GSH) in solution, cells, and tissue. Probe RH-2 was applied to the quantitative determination of Hcy in human serum successfully. Moreover, the two-photon fluorescence (TPF) imaging of abnormal expression of Hcy in aortic vessels and liver of atherosclerotic model mice were fulfilled by RH-2. Therefore, probe RH-2 can be served as a potential tool to understand the function of Hcy in atherosclerosis, supplying a clinical promise for the early diagnosis of atherosclerosis (AS).