Two-photon Fluorescence Imaging Research Articles

Excitation and emission dual-wavelength confocal metalens designed directly in the biological tissue environment for two-photon micro-endoscopy.

With the advantages of completely controlling the phase, amplitude, and polarization in subwavelength range, metalenses have drawn intensive attentions in high resolution two-photon micro-endoscopic fluorescence imaging system. However, chromatic dispersion and severe scattering of biological tissue significantly reduce excitation-collection efficiency in the traditional two-photon imaging system based on traditional metalenses designed in the air background. Here, an excitation and emission dual-wavelength confocal and polarization-insensitive metalens designed in the biological tissue environment was proposed by adopting the composite embedding structure and spatial multiplexing approach. The metalens with numerical aperture (NA) of 0.895 can focus the excitation (915 nm) and emission (510 nm) beams to the same focal spot in the mouse cortex. According to the theoretical simulation of two-photon fluorescence imaging, the lateral resolution of the collected fluorescent spots via the proposed metalens can be up to 0.42 µm. Compared to the metalens designed in the air environment, the collection efficiency of fluorescent spot is improved from 5.92% to 14.60%. Our investigation has opened a new window of high resolution and minimally invasive imaging in deep regions of biological tissues.

Ultrabright bimetallic AuAg complex: From luminescence mechanism to biological application

Metal clusters have attracted wide interests due to their unique electronic and optical properties, but the low luminescence quantum yield (QY) prevents them from potential biomedical applications. In this work, silver-doped Au nanoclusters (NCs) are shown to be able to improve the QY of metal clusters. We succeeded in synthesizing ultrabright glutathione (GSH) protected AuAg clusters with 10.8% QY by a one-pot route. Their florescence is about 7.5 times brighter than pure Au NCs, with super photostability and good biocompatibility in physiological environment. Based on density functional theory (DFT) calculations, we investigated the electronic structures and optical properties of the AuAg NCs. The results show that the increase of the density of states of the lowest unoccupied molecular orbital (LUMO) leads to the fluorescence enhancement. In addition, two-photon excitation fluorescence imaging has been performed to show their great potential for biomedicine.

Two-photon imaging of oxidative stress in living erythrocytes as a measure for human aging.

According to the "oxidative stress theory" of aging, this process is accompanied by a progressive and irreversible accumulation of oxidative damage caused by reactive oxygen species (ROS). This, in turn, has a deleterious impact on molecular mechanisms in aging thereby altering the physiological function of the organism, increasing the risk of different aging-related diseases, as well as impacting the life span. The aim of the current study was to investigate oxidative stress in living red blood cells (RBCs) in human aging as an oxidative stress-related pathological condition. Two-photon laser scanning and light microscopy techniques were applied to analyze the oxidative stress in RBCs and the cell viability. Spectrophotometric analyzes were performed to determine the percentage of RBC hemolysis, activities of superoxide dismutase and catalase in RBCs, as well as the ferroxidase activities of ceruloplasmin in blood plasma samples. The studies included three human aging groups, young, middle-aged, and elderly. According to the results, the two-photon fluorescence of carboxy-DCFDA, indicating the intensity of oxidative stress, significantly increase in RBCs by the increase of age (P < 0.05), and these intensities are in statistically significant positive correlation with age (P < 0.001) and a strong negative correlation (P < 0.05) with the activity of catalase in RBCs and ferroxidase activity of ceruloplasmin in plasma. In conclusion, two-photon fluorescent imaging of oxidative stress in human living RBCs is a valuable and accurate method for the determination of aging processes in humans and can be suggested as a novel indicator for human aging processes in individual aging.

Femtosecond two-photon laser-induced fluorescence imaging of atomic hydrogen in a laminar methane–air flame assisted by nanosecond repetitively pulsed discharges

Sustainable and low-emission combustion is in need of novel schemes to enhance combustion efficiency and control to meet up with new emission standards and comply with varying quality of renewable fuels. Plasma actuation is a promising candidate to achieve this goal but few detailed experiments have been carried out that target how specific combustion and plasma related species are affected by the coupling of plasma and combustion chemistry. Atomic hydrogen is such a species that here is imaged by using the two-photon absorption laser induced fluorescence (TALIF) technique as an atmospheric pressure methane–air flame is actuated by nanosecond repetitively pulsed (NRP) discharges. Atomic hydrogen is observed both in the flame and in the discharge channel and plasma actuation results in a wide modification of the flame shape. A local 50% increase of fluorescence occurs at the flame front where it is crossed by the discharge. Atomic hydrogen in the discharge channel in the fresh-gases is found to decay with a time constant of about 2.4 μs. These results provide new insights on the plasma flame interaction at atmospheric pressure that can be further used for cross-validation of numerical calculations.

Two-Photon Dual-Emissive Carbon Dot-Based Probe: Deep-Tissue Imaging and Ultrasensitive Sensing of Intracellular Ferric Ions.

Carbon dots (CDs)-based nanoparticles have been extensively explored for biological applications in sensing and bioimaging. However, the major translational barriers to CDs for imaging and sensing applications include synthetic strategies to obtain monodisperse CDs with tunable structural, electronic, and optical properties in order to achieve high-resolution deep-tissue imaging, intracellular detection, and sensing of metal ions with high sensitivity down to nanomolar levels. Herein, we report a novel strategy to synthesize and develop a multifunctional nitrogen-doped CDs probe of different sizes using a new combination of carbon and nitrogen sources. Our results show that the structural characteristics (i.e., the surface density of emissive traps and bandgaps levels) depend on the size of the CDs, which ultimately influences their optical properties. This work also demonstrates the development of a two-photon dual-emissive fluorescent multifunctional probes (3-FCDs) by conjugating fluorescein isothiocyanate on the surface of nitrogen-doped CDs. 3-FCDs show excellent near-infrared two-photon excitation ability, single-wavelength excitation, high photostability, biocompatibility, low cytotoxicity, and good cell permeability. Using two-photon fluorescence imaging, our multifunctional probe shows excellent deep-tissue high-resolution imaging capabilities with penetration depth up to 3000 and 280 μm in hydrogel scaffold and pigskin tissue, respectively. The designed probe exhibits ultrasensitivity and specificity toward Fe3+ ions with a remarkable detection limit of 2.21 nM using two-photon excitation. In addition, we also demonstrate the use of multifunctional CDs probe for ultrasensitive exogenous and real-time endogenous sensing of Fe3+ ions and imaging in live fibroblasts with rapid response times for intracellular ferric ion detection.

Sensing Performance Investigations on Two-Photon Fluorescent Probes for Detecting β-Amyloid in Alzheimer's Disease.

Alzheimer’s disease (AD) is one of the most common forms of senile disease. In recent years, the incidence of AD has been increasing significantly with the acceleration of the aging process of the global population. However, current clinical drugs can only alleviate the symptoms of AD patients without healing the disease fundamentally. Therefore, it is of great significance to develop an effective small molecule diagnostic reagent for the early diagnosis of AD. In this paper, we employ an integrated approach, including molecular docking simulation and quantum mechanics/molecular mechanics calculation, to investigate the sensing performance of a series of donor–acceptor structural probes for the marker protein of AD (β-amyloid). Results show that the probes display evident fluorescence enhancement when bound to the β-amyloid, suggesting the effect of the environment on the molecular properties. Especially, the two-photon absorption cross-section of the probes increase drastically in the β-amyloid compared to that in vacuum, which results from the larger electron delocalization and dipole moment in the fibrillary-like environment. Thus, one can propose that the studied probes are capable of application in two-photon fluorescent imaging, particularly those containing naphthalene rings as the donor or with a longer spacer group. Our calculations elucidate the experimental measurements reasonably, and further establish possible structure–property relationships that can be used to design novel biocompatible two-photon fluorescent probes for the diagnosis of Alzheimer’s.

Nanoscale Metal-Organic Frameworks and Metal-Organic Layers with Two-Photon-Excited Fluorescence.

Metal-organic frameworks (MOFs) based on 9,10-diphenylanthracene-derived ligands had been reported to exhibit upconverted fluorescence through triplet-triplet annihilation. We found that zirconium MOFs based on 9,10-diphenylanthracene can also give upconverted fluorescence via two-photon absorption without adding a triplet photosensitizer when a femtosecond pulsed laser is used as the excitation source. By tuning the synthetic condition, we obtained nanoscale MOFs of UiO structure in both octahedral and hexagonal nanoplate shapes, as well as a hexagonal nanoplate of MOFs of hcp-UiO structure and two-dimensional metal-organic layers. All of them, as well as a homogeneous solution of the 9,10-diphenylanthracene ligand, exhibit upconverted fluorescence upon excitation using a laser pulse of 60 fs with a pulse energy of ∼1.1 × 106 nJ/cm2 (unfocused). Moreover, we observed different emission spectra by two-photon excitation compared to those by one-photon excitation, which indicates access to a unique initial excited state via two-photon excitation. This phenomenon is not observed for a homogeneous solution of the ligand. These nanoscale MOFs may find application in two-photon fluorescence imaging.

Local Resting Ca2+ Controls the Scale of Astroglial Ca2+ Signals

SummaryAstroglia regulate neurovascular coupling while engaging in signal exchange with neurons. The underlying cellular machinery is thought to rely on astrocytic Ca2+ signals, but what controls their amplitude and waveform is poorly understood. Here, we employ time-resolved two-photon excitation fluorescence imaging in acute hippocampal slices and in cortex in vivo to find that resting [Ca2+] predicts the scale (amplitude) and the maximum (peak) of astroglial Ca2+ elevations. We bidirectionally manipulate resting [Ca2+] by uncaging intracellular Ca2+ or Ca2+ buffers and use ratiometric imaging of a genetically encoded Ca2+ indicator to establish that alterations in resting [Ca2+] change co-directionally the peak level and anti-directionally the amplitude of local Ca2+ transients. This relationship holds for spontaneous and for induced (for instance by locomotion) Ca2+ signals. Our findings uncover a basic generic rule of Ca2+ signal formation in astrocytes, thus also associating the resting Ca2+ level with the physiological “excitability” state of astroglia.

Multiplex Stimulated Raman Scattering Imaging Cytometry Reveals Lipid-Rich Protrusions in Cancer Cells under Stress Condition.

SummaryIn situ measurement of cellular metabolites is still a challenge in biology. Conventional methods, such as mass spectrometry or fluorescence microscopy, would either destroy the sample or introduce strong perturbations to target molecules. Here, we present multiplex stimulated Raman scattering (SRS) imaging cytometry as a label-free single-cell analysis platform with chemical specificity and high-throughput capabilities. Using SRS imaging cytometry, we studied the metabolic responses of human pancreatic cancer cells under stress by starvation and chemotherapeutic drug treatments. We unveiled protrusions containing lipid droplets as a metabolic marker for stress-resistant cancer cells. Furthermore, by spectroscopic SRS mapping, we unveiled that triglyceride in lipid droplets are used for local energy production through lipolysis, autophagy, and β-oxidation. Our findings demonstrate the potential of targeting lipid metabolism for selective treatment of stress-resistant cancers. Collectively, these results highlight SRS imaging cytometry as a powerful label-free tool for biological discoveries with a high-throughput, high-content capacity.

Intensity distortion in dual-axis galvo mirror scanning TPF imaging system

Two-photon fluorescence (TPF) imaging system with dual-axis galvo mirror as scanning unit is built. Based on the optical path of imaging system built, mechanism of how intensity distortion generating in dual-axis galvo mirror scanning TPF imaging system is theoretically analyzed. In experiment, TPF imaging is realized with Rhodamine B sample through detecting TPF signal generated from Rhodamine B molecule at excitation of broadband femtosecond pulses. The imaging result verifies the existence of intensity distortion with distribution agreeing well with the theoretical analyzation. To eliminate the intensity distortion in TPF imaging result, a mathematical method is proposed in which a calibration matrix is calculated, As the calibration matrix is applied to the distorted imaging result of Rhodamine B sample, intensity distortion is relieved effectively and precise imaging result is achieved.

Two-photon imaging of aptamer-functionalized Copolymer/TPdye fluorescent organic dots targeted to cancer cells

Fluorescent organic dots (O-dots) recently have emerged as a new class of promising contrast reagents for two-photon fluorescence (TPF) imaging. However, most of these developed two-photon absorption (TPA) O-dots have no tumor-targeting group, which hampers their wide application for targeted tumor imaging. Herein, we fabricated Sgc8c aptamer-mediated TPA O-dots as a proof-of-concept of the sensing platform for targeted imaging in live cells or deep tissues. The O-dots composed of trans-4-[p-(N, N-diethylamino)styryl]-4’-(dimethyl amino) stilbene (DEAS) emerged as TPA organic emissive cores and encapsulation by using poly (methyl methacrylate-co-methacrylic acid) (PMMA-co-MAA) as polymeric encapsulating matrix to form DEAS/PMMA-co-MAA O-dots via a co-precipitation strategy. The obtained O-dots enabled an extremely high TPA absorption cross-section, bright two-photon fluorescence (excitation at 720 nm; emission at 412 nm and 434 nm), excellent cell-permeability and high penetration depth. Sgc8c aptamer, as a protein tyrosine kinase-7 (PTK7) receptor-targetable ligand, was further anchored on the surface of O-dots to obtain DEAS/PMMA-co-MAA@Sgc8c nanoprobes by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-mediated coupling reaction. Guided by Sgc8c aptamer, DEAS/PMMA-co-MAA@Sgc8c nanoprobes could be rapidly internalized into target acute lymphoblastic leukemia cells (CEM) cells with high specificity and great efficiency. It was also performed that two-photon images of TPA nanoprobes exhibited high two-photon brightness not only in target CEM cells, but also in mouse liver tissue slices even a depth of up to 210 μm. In our perception, it is highly promising that this nanoprobe provides a valuable tool for in vivo targeted imaging.

Simultaneous laser-driven x-ray and two-photon fluorescence imaging of atomizing sprays

In this Letter, we report for the first time, to the best of our knowledge, the possibility of visualizing an atomizing spray by simultaneously recording x-ray absorption and two-photon laser-induced fluorescence imaging. This unique illumination/detection scheme is made possible due to the use of soft x rays emitted from a laser-driven x-ray source. An 800 mJ laser pulse of 38 fs duration is used to generate an x-ray beam with up to 4 × 10 8 photons ranging from 1 to 10 keV, allowing projection radiography of water jets generated by an automotive port fuel injector. In addition, a fraction of the laser pulse ( ∼ 10 m J ) is employed to form a light sheet and to induce two-photon fluorescence in a dye added to the water. The resulting high-contrast fluorescence images provide fine details of the spray structure, with reduced blur from multiple light scattering, while the integrated liquid mass is extracted from the x-ray radiography. In this proof of principle, we show that the combination of these two highly complementary techniques, in both the visible and soft x-ray regimes, is very promising for future characterization of challenging spray, as well as for further understanding of the physics of liquid atomization.

Factors associated with obesity alter matrix remodeling in breast cancer tissues.

.Obesity is associated with a higher risk of developing breast cancer and with worse disease outcomes for women of all ages. The composition, density, and organization of the breast tissue stroma are also known to play an important role in the development and progression of the disease. However, the connections between obesity and stromal remodeling are not well understood. We sought to characterize detailed organization features of the collagen matrix within healthy and cancerous breast tissues acquired from mice exposed to either a normal or high fat (obesity inducing) diet. We performed second-harmonic generation and spectral two-photon excited fluorescence imaging, and we extracted the level of collagen-associated fluorescence (CAF) along with metrics of collagen content, three-dimensional, and two-dimensional organization. There were significant differences in the CAF intensity and overall collagen organization between normal and tumor tissues; however, obesity-enhanced changes in these metrics, especially when three-dimensional organization metrics were considered. Thus, our studies indicate that obesity impacts significantly collagen organization and structure and the related pathways of communication may be important future therapeutic targets.

Quantum Mechanics/Molecular Mechanics Density Functional Theory Simulations of the Optical Properties Fingerprinting the Ligand-Binding of Pentameric Formyl Thiophene Acetic Acid in Amyloid-β(1-42).

The binding pocket proposed by König [ Chem. Commun. 2018 , 54 , 3030 - 3033 ] for the biomarker pentameric formyl thiophene acetic acid (p-FTAA) in the fibrillar structure of amyloid-β(1-42) has been put to the test by the comparison of theoretical and experimental optical absorption and fluorescence spectra obtained in a water environment and inside the protein scaffold. The optical absorption/emission properties of this luminescent conjugated oligothiophene were studied by means of classical force field molecular dynamics simulations to account for the sampling of configuration space in conjunction with polarizable embedding time-dependent density functional theory calculations of spectra. The nuclear motions of residues in the β-sheet were found to be modest, and the time dependence of embedding parameters was shown to be negligible so that a time-independent parameter set could be derived and used for all 300 snapshots considered in the spectrum averaging. In regard to linear absorption spectra, the calculated red shift due to protein binding for the dominant S1 ← S0 transition in p-FTAA was found to be equal to 23 nm (0.17 eV), which is in excellent agreement with the corresponding experimental result of 18 nm and taken as corroborating evidence for having correctly identified the binding pocket of p-FTAA in the amyloid. The underlying mechanisms for the calculated red shift were disentangled, and it is shown that some 20 nm (0.15 eV) of the total 23 nm (0.17 eV) is associated with increased planarity of p-FTAA in the binding pocket, whereas a mere 3 nm (0.02 eV) is associated with changes in the environment. In regard to emission spectra, we demonstrate that intersystem crossing from the excited S1 state to the triplet manifold of states is a less likely event for p-FTAA in the binding pocket as compared to in the aqueous solution, and we thereby partly explain the much higher quantum yield of fluorescence for the more rigid p-FTAA in the binding pocket. Two-photon absorption in p-FTAA is shown to predominantly occur to an overall symmetric excited state and be more than twice as strong for the biomarker in the binding pocket as compared to in water. The corresponding red shift, on the other hand, is very small. Earlier experimental two-photon fluorescence imaging using p-FTAA is shown not to target the dominant two-photon state, and ways to reach a higher image quality (lower signal-to-noise ratio) are proposed in terms of tuning the laser wavelength toward the region of 600 nm or the synthesis of asymmetric ligands with S1 states that are both one- and two-photon allowed.

Efficient dendrimers based on naphthalene indenofluorene for two-photon fluorescent imaging in living cells and tissues

A dendrimer based on naphthalene indenofluorene with local excited state-dominant characteristics exhibited better two-photon fluorescent imaging in living cells and tissues.

Recent progress in efficient organic two-photon dyes for fluorescence imaging and photodynamic therapy

The enhancement of two-photon absorption cross-section, fluorescence quantum yield and singlet oxygen generation efficiency for organic dyes and their applications in two-photon excited fluorescence imaging and photodynamic therapy are summarized.

Stimulating Ca&lt;sup&gt;2+&lt;/sup&gt; photoactivation of nerve cells by near-infrared light

Calcium ions (Ca&lt;sup&gt;2+&lt;/sup&gt;) play a key role of the nerve cells generating universal intracellular signals and controlling important functions. Ca&lt;sup&gt;2+&lt;/sup&gt; activation is of great significance for explaining the subcellular-level biological process. Light stimulated nerve cells to control intracellular signals and membrane activities has become a main method in neuroscience, and the photoactivation is one of the main ways to study intracellular Ca&lt;sup&gt;2+&lt;/sup&gt; transmission. Nerve cells can be directly stimulated by light to produce action potentials, but such techniques are inaccurate in the delivered light energy. To improve this, here in this work we show that gold nanorods (GNRs) can be conjugated to ligands to bound to human neuroblast cells (SH-SY5Y), and introduce an optical method of stimulating and monitoring Ca&lt;sup&gt;2+&lt;/sup&gt; signal in nerve cells in which the plasmonic excitation of GNRs is used. In this paper, we use confocal microscopy to display the 488 nm continuous wave laser irradiating SH-SY5Y cells with Ca&lt;sup&gt;2+&lt;/sup&gt; indicator (Fluo-4, AM) to check fluorescence. Near-infrared pulsed light at the plasmon resonance absorption peak of GNRs is used to stimulate Ca&lt;sup&gt;2+&lt;/sup&gt; signal transduction in SH-SY5Y labeled with GNRs, and Fluo-4, AM is used for two-photon excited fluorescence imaging. In addition, we use the pulsed laser with power of 0.5 mW and a wavelength of 800 nm. The Ca&lt;sup&gt;2+&lt;/sup&gt; activation can be achieved in 10 s on average. The release rate of Ca&lt;sup&gt;2+&lt;/sup&gt; from SH-SY5Y cells labeled with GNRs is 6 times that without GNRs. Next, in order to determine the source of changes in Ca&lt;sup&gt;2+&lt;/sup&gt;, we use the BPATA to deplete the intracellular Ca&lt;sup&gt;2+&lt;/sup&gt;, after 5 min, 200 μmol/L Ca&lt;sup&gt;2+&lt;/sup&gt; solution is added, and its Δ&lt;i&gt;F&lt;/i&gt;/&lt;i&gt;F&lt;/i&gt; is found to be more than 1.5 times that without GNRs. Thus, we believe that GNRs could enhance photoactivation through local surface plasmon resonance induced membrane depolarization and generate an action potential. The results prove the feasibility of using GNRs to enhance the activation of Ca&lt;sup&gt;2+&lt;/sup&gt; in nerve cells, and provide an optical means of lower photodamage and more precise for studying nerve cell ion channels. Our study demonstrates that enhancing photoactivation by GNRs could provide an outlook of basic research in neuroscience.

Highly efficient singlet oxygen generation, two-photon photodynamic therapy and melanoma ablation by rationally designed mitochondria-specific near-infrared AIEgens.

Photosensitizers (PSs) with multiple characteristics, including efficient singlet oxygen (1O2) generation, cancer cell-selective accumulation and subsequent mitochondrial localization as well as near-infrared (NIR) excitation and bright NIR emission, are promising candidates for imaging-guided photodynamic therapy (PDT) but rarely concerned. Herein, a simple rational strategy, namely modulation of donor–acceptor (D–A) strength, for molecular engineering of mitochondria-targeting aggregation-induced emission (AIE) PSs with desirable characteristics including highly improved 1O2 generation efficiency, NIR emission (736 nm), high specificity to mitochondria, good biocompatibility, high brightness and superior photostability is demonstrated. Impressively, upon light irradiation, the optimal NIR AIE PS (DCQu) can generate 1O2 with efficiency much higher than those of commercially available PSs. The excellent two-photon absorption properties of DCQu allow two-photon fluorescence imaging of mitochondria and subsequent two-photon excited PDT. DCQu can selectively differentiate cancer cells from normal cells without the aid of extra targeting ligands. Upon ultralow-power light irradiation at 4.2 mW cm−2, in situ mitochondrial photodynamic activation to specifically damage cancer cells and efficient in vivo melanoma ablation are demonstrated, suggesting superior potency of the AIE PS in imaging-guided PDT with minimal side effects, which is promising for future precision medicine.

Poly(fluorenone-co-thiophene)-based nanoparticles for two-photon fluorescence imaging in living cells and tissues.

Conjugate polymer nanoparticles (CPNs) were constructed based on poly(fluorenone-co-thiophenes) (PFOTs) synthesized through a direct arylation polymerization (DArP) approach. Results demonstrate that the developed novel CPNs have potential applications in two-photon fluorescence imaging of both cells and tissues.

Two-photon excited fluorescent silica nanoparticles loaded with iron(II) as a probe for determination and imaging of hydrogen peroxide in living cells.

A method is described for determination and optical imaging of hydrogen peroxide (H2O2) by using the two-photon (TP) excited fluorescence of silica (SiO2) nanoparticles containing Fe(II) ions. In the presence of H2O2, hydroxyl radicals (•OH) are produced via the Fenton reaction. This leads to quenching of the green fluorescence of a TP-excitable organic dye loaded into the SiO2NPs. Fluorescence is excited at 370nm and has an emission peaking at 447nm. The degree of quenching increases linearly in the 2.5 to 100μM H2O2 concentration range. The nanoprobe is highly selective and sensitive, with a detection limit of 336nM. The nanoprobe is biocompatible and was successfully used to image changes in the H2O2 concentration in HeLa cells via TP fluorescence imaging. Graphical abstractSchematic rpresentation of the detection of H2O2 by using the two-photon excited fluorescence of silica nanoparticles (TP-SiO2NPs) containing Fe2+. H2O2 triggers the Fenton reaction to produce hydroxyl radicals (•OH), which quench the green fluorescence of theSiO2NPs.