Two-photon Fluorescence Research Articles

Intraoperative microscopic autofluorescence detection and characterization in brain tumors using stimulated Raman histology and two-photon fluorescence.

The intrinsic autofluorescence of biological tissues interferes with the detection of fluorophores administered for fluorescence guidance, an emerging auxiliary technique in oncological surgery. Yet, autofluorescence of the human brain and its neoplasia is sparsely examined. This study aims to assess autofluorescence of the brain and its neoplasia on a microscopic level by stimulated Raman histology (SRH) combined with two-photon fluorescence. With this experimentally established label-free microscopy technique unprocessed tissue can be imaged and analyzed within minutes and the process is easily incorporated in the surgical workflow. In a prospective observational study, we analyzed 397 SRH and corresponding autofluorescence images of 162 samples from 81 consecutive patients that underwent brain tumor surgery. Small tissue samples were squashed on a slide for imaging. SRH and fluorescence images were acquired with a dual wavelength laser (790 nm and 1020 nm) for excitation. In these images tumor and non-tumor regions were identified by a convolutional neural network that reliably differentiates between tumor, healthy brain tissue and low quality SRH images. The identified areas were used to define regions.of- interests (ROIs) and the mean fluorescence intensity was measured. In healthy brain tissue, we found an increased mean autofluorescence signal in the gray (11.86, SD 2.61, n=29) compared to the white matter (5.99, SD 5.14, n=11, p<0.01) and in the cerebrum (11.83, SD 3.29, n=33) versus the cerebellum (2.82, SD 0.93, n=7, p<0.001), respectively. The signal of carcinoma metastases, meningiomas, gliomas and pituitary adenomas was significantly lower (each p<0.05) compared to the autofluorescence in the cerebrum and dura, and significantly higher (each p<0.05) compared to the cerebellum. Melanoma metastases were found to have a higher fluorescent signal (p<0.01) compared to cerebrum and cerebellum. In conclusion we found that autofluorescence in the brain varies depending on the tissue type and localization and differs significantly among various brain tumors. This needs to be considered for interpreting photon signal during fluorescence-guided brain tumor surgery.

Morpholinyl silicon phthalocyanine nanoparticles with lysosome cell death and two-photon imaging functions for in vitro photodynamic therapy of cancer cells.

The lysosome is an important target for realizing antitumor therapy. Lysosomal cell death exerts significant therapeutic effects on apoptosis and drug-resistance. The development of lysosome-targeting nanoparticles to obtain efficient cancer treatment is challenging. In this article, nanoparticles composed of DSPE@M-SiPc and possessing bright two-photon fluorescence, lysosome targeting ability, and photodynamic therapy multifunctionalities are prepared by encapsulating morpholinyl-substituted silicon phthalocyanine (M-SiPc) with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE). Two photon fluorescence bioimaging showed that M-SiPc and DSPE@M-SiPc mainly locate in lysosomes after cellular internalization. Upon irradiation, DSPE@M-SiPc effectively generates reactive oxygen species and damages the function of lysosome, subsequently leading to lysosomal cell death. DSPE@M-SiPc is a promising photosensitizer for cancer treatment.

Carbonized Polymer Dot Probe for Two-Photon Fluorescence Imaging of Lipid Droplets in Living Cells and Tissues.

As a dynamic and multifunctional organelle, lipid droplets (LDs) are essential in maintaining lipid balance and transducing biological signals. LD accumulation and catabolism are closely associated with energy metabolism and cell signaling. In order to easily trace LDs in living cells, a novel carbonized polymer dot (CPD)-based fluorescent nanoprobe is reported to serve the needs of LD-targeting imaging. This probe exhibits the advantages of excellent biocompatibility, simple preparation, good lipophilicity, and high compatibility with commercial dyes. Transient absorption spectroscopy was employed to discuss the luminescence mechanism of CPDs, and the results indicate that the excellent fluorescence property and the environment-responsive feature of our CPDs are derived from the intramolecular charge transfer (ICT) characteristics and the D-π-A structure that possibly formed in CPD. This nanoprobe is available for one-photon fluorescence (OPF) and two-photon fluorescence (TPF) imaging and is also practicable for staining LDs in living/fixed cells and lipids in tissue sections. The staining process is completed within several seconds, with no washing step. The intracellular LDs involving the intranuclear LDs (nLDs) can be selectively lit up. This probe is feasible for visualizing dynamic interactions among LDs, which suggests its great potential in revealing the secret of LD metabolism. The in situ TPF spectra were analyzed to determine surrounding microenvironment according to the polarity-responsive feature of our CPDs. This work expands the applications of CPDs in biological imaging, helps design new LD-selective fluorescent probes, and has implications for studying LD-related metabolism and diseases.

Ammonia thermal decomposition on quartz and stainless steel walls

Ammonia has been proposed as a promising solution for hydrogen carriers as well as clean fuels. However, the reaction kinetics is still not robust in many engineering applications, where the deviation is mainly caused by the surface reactions of ammonia on the wall. To examine the ammonia surface reaction on engineering materials, in the present study, the thermal decomposition of ammonia is systematically investigated in uniformly heated quartz/SUS304 tubular flow reactors with the prescribed heating length. A decrease in the inner diameter of the quartz tubular reactor leads to an increase in the ammonia thermal decomposition rate, showing that the thermal decomposition reaction occurs even on quartz surfaces, which is usually believed to be inert. The thermal decomposition on the SUS304 reactor surface starts from as low as 700 K, indicating it is much more reactive than the quartz surface. One-step surface reaction models of ammonia for quartz/SUS304 surfaces are proposed, with which the reaction rates are estimated based on the experimental data. The inner surface of the SUS304 tubular reactor after the thermal decomposition experiments is examined by X-ray photoelectron spectroscopy. The formation of iron nitride on SUS304, which in turn facilitates ammonia thermal decomposition, reveals the positive feedback between ammonia decomposition and nitriding. Moreover, the present one-step surface reaction on stainless steel has been validated by measuring ammonia distributions in the preheat zone of NH3/O2/N2 premixed flame impinging on a stainless steel plate through a two-photon absorption laser-induced fluorescence (TALIF) technique.

Mesoporous Silica-Encapsulated Gold Nanorods for Drug Delivery/Release and Two-Photon Excitation Fluorescence Imaging to Guide Synergistic Phototherapy and Chemotherapy.

Photothermal therapy is a promising light-based medical treatment that relies on light absorption agents converting light irradiation into localized heat to destroy cancer cells or other diseased tissues. It is critical to enhance the therapeutic effects of cancer cell ablation for their practical applications. This study reports a high-performance combinational therapy for ablating cancer cells, including both photothermal therapy and chemotherapy to improve therapeutic efficiency. The prepared AuNR@mSiO2 loading molecular Doxorubicin (Dox) assemblies were highlighted by merits of facile acquisition, great stability, easy endocytosis, and rapid drug release in addition to improved anticancer capability upon irradiation with a femtosecond pulsed near-infrared (NIR) laser, where AuNR@mSiO2 nanoparticles afforded a high photothermal conversion efficiency of 31.7%. Two-photon excitation fluorescence imaging was introduced into confocal laser scanning microscope multichannel imaging to track the drug location and cell position in real time for monitoring the process of drug delivery in killing human cervical cancer HeLa cells and then to realize imaging-guiding cancer treatment. These nanoparticles exhibit widespread potential in photoresponsive utilizations including photothermal therapy, chemotherapy, one- and two-photon excited fluorescence imaging, and 3D fluorescence imaging and cancer treatment.

O3 full photo-fragmentation TALIF spectroscopy for quantitative diagnostics of non-thermal O2-mixed plasmas

In this study, we explore the potential of using laser-induced photo-fragmentation of O3 by UV radiation as a quantitative diagnostic tool in non-thermal O2-mixed plasmas. We analyze the optical processes of O3 using a comprehensive kinetic model with a 226 nm laser, which is typically used in the two-photon absorption laser-induced fluorescence (TALIF) measurement of O atoms. Our model demonstrates that the fluorescence intensity from atomic O fragments produced by the same laser is directly proportional to the population of precursor O3. This makes various diagnostic purposes achievable through the proposed O3 full photo-fragmentation (FPF) TALIF spectroscopy, including calibration of TALIF signals of O atoms and quantification of both O and O3 in O2-mixed plasmas. We present detailed theoretical principles, technical requirements, and successful examples of implementation for different diagnostic aims using the proposed O3 FPF-TALIF spectroscopy. However, we also specify the limitations of the developed diagnostic methods, particularly under low E/N conditions (&amp;lt;30 Td), where other interferential species such as the vibrationally excited ground-state O 2 ( X 3 Σ g − , v ≠ 0 ) are abundantly produced.

Molecular and Aggregate Synergistic Engineering of Aggregation-Induced Emission Luminogens to Manipulate Optical/Electronic Properties for Efficient and Diversified Functions.

The optical/electronic properties of organic luminescent materials can be regulated by molecular structure modification, which not only requires sophisticated and time-consuming synthesis but also is unable to accurately afford the optical properties of materials in the aggregate state. Herein, a facile strategy of molecular and aggregate synergistic engineering is proposed to manipulate the optical/electronic properties of a luminogen, ACIK, in the solid state for efficient and diversified functions. ACIK is facilely synthesized and exhibits three polymorphic states (ACIK-Y, ACIK-R, and ACIK-N) with a large emission difference of 102 nm from yellow to near-infrared (NIR). Their structure-property relationships were investigated by crystallographic analyses and computational studies. ACIK-Y, with the most twisted structure, exhibits an intriguing color-tuned fluorescence between yellow and NIR in the solid state in response to multiple stimuli. Shuttle-like ACIK-R microcrystals exhibit an optical waveguide property with a low optical loss coefficient of 19 dB mm-1. ACIK dots display bright NIR-I emission, large Stokes shift, and strong NIR-II two-photon absorption. ACIK dots show specific lipid droplets-targeting capability and can be successfully applied for two-photon fluorescence imaging of mouse brain vasculature with deep penetration and high spatial resolution. This study will inspire more insights in developing advanced optical/electronic materials based on a single chromophore for practical applications.

Growth of single-crystals and microstructures using benzothiadiazole derivatives for efficient optical waveguide and nonlinearity

Organic nanomaterials have been extensively studied for the applications of the electronic and photonic fields. The bottom-up fabrication through self-assembly provides a powerful strategy to construct the nanostructures. In this work, we synthesized a conjugated molecule, namely, 4-(1-phenyl-1H-1,2,3-triazol-4-yl)benzo[c][1,2,5]thiadiazole (BT-SC), based on benzothiadiazole and benzene moieties bridged by a triazole. Then, a simple solvent-vapor annealing technique was presented to drive the self-assembly of BT-SC into highly uniform microrods or microfibers. The morphology of the self-assembly could be controlled by simply changing the solution concentration. The compound also could be assembled into aligned nanowires by wetting into hard templates of anodic aluminum oxide (AAO). The fabricated microrods showed strong emitter and excellent optical waveguide behaviors excited with a UV laser. Moreover, the microrods displayed nonlinear optical responses of two-photon excited fluorescence and could therefore become building blocks for actual photonic devices.

Label-free, multi-parametric assessments of cell metabolism and matrix remodeling within human and early-stage murine osteoarthritic articular cartilage

Osteoarthritis (OA) is characterized by the progressive deterioration of articular cartilage, involving complicated cell-matrix interactions. Systematic investigations of dynamic cellular and matrix changes during OA progression are lacking. In this study, we use label-free two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to assess cellular and extracellular matrix features of murine articular cartilage during several time points at early stages of OA development following destabilization of medial meniscus surgery. We detect significant changes in the organization of collagen fibers and crosslink-associated fluorescence of the superficial zone as early as one week following surgery. Such changes become significant within the deeper transitional and radial zones at later time-points, highlighting the importance of high spatial resolution. Cellular metabolic changes exhibit a highly dynamic behavior, and indicate metabolic reprogramming from enhanced oxidative phosphorylation to enhanced glycolysis or fatty acid oxidation over the ten-week observation period. The optical metabolic and matrix changes detected within this mouse model are consistent with differences identified in excised human cartilage specimens from OA and healthy cartilage specimens. Thus, our studies reveal important cell-matrix interactions at the onset of OA that may enable improved understanding of OA development and identification of new potential treatment targets.

Identifying lipid particle sub-types in live Caenorhabditis elegans with two-photon fluorescence lifetime imaging.

Fat metabolism is an important modifier of aging and longevity in Caenorhabditis elegans. Given the anatomy and hermaphroditic nature of C. elegans, a major challenge is to distinguish fats that serve the energetic needs of the parent from those that are allocated to the progeny. Broadband coherent anti-Stokes Raman scattering (BCARS) microscopy has revealed that the composition and dynamics of lipid particles are heterogeneous both within and between different tissues of this organism. Using BCARS, we have previously succeeded in distinguishing lipid-rich particles that serve as energetic reservoirs of the parent from those that are destined for the progeny. While BCARS microscopy produces high-resolution images with very high information content, it is not yet a widely available platform. Here we report a new approach combining the lipophilic vital dye Nile Red and two-photon fluorescence lifetime imaging microscopy (2p-FLIM) for the in vivo discrimination of lipid particle sub-types. While it is widely accepted that Nile Red staining yields unreliable results for detecting lipid structures in live C. elegans due to strong interference of autofluorescence and non-specific staining signals, our results show that simple FLIM phasor analysis can effectively separate those signals and is capable of differentiating the non-polar lipid-dominant (lipid-storage), polar lipid-dominant (yolk lipoprotein) particles, and the intermediates that have been observed using BCARS microscopy. An advantage of this approach is that images can be acquired using common, commercially available 2p-FLIM systems within about 10% of the time required to generate a BCARS image. Our work provides a novel, broadly accessible approach for analyzing lipid-containing structures in a complex, live whole organism context.

Retinal microvascular and neuronal pathologies probed in vivo by adaptive optical two-photon fluorescence microscopy.

The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.

Simultaneous single-shot imaging of H and O atoms in premixed turbulent flames using femtosecond two-photon laser-induced fluorescence.

A method based on femtosecond two-photon excitation has been developed for simultaneous visualization of interference-free fluorescence of H and O atoms in turbulent flames. This work shows pioneering results on single-shot simultaneous imaging of these radicals under non-stationary flame conditions. The fluorescence signal, showing the distribution of H and O radicals in premixed CH4/O2 flames was investigated for equivalence ratios ranging from ϕ = 0.8 to ϕ = 1.3. The images have been quantified through calibration measurements and indicate single-shot detection limits on the order of a few percent. Experimental profiles have also been compared with profiles from flame simulations, showing similar trends.

Abstract 3703: Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma

Abstract Radiation resistance can halt a well-curated treatment plan in its tracks, but it may take weeks to realize this lack of efficacy resulting in lost time and unnecessary side effects. In this study, we sought to elucidate novel biomarkers of radiation resistance to predict treatment response in head and neck squamous cell carcinoma (HNSCC) at much earlier timepoints. We grew tumor xenografts from radiation-resistant UM-SCC-47 (n=25) and -sensitive UM-SCC-22B (n=37) cells in nude athymic mice. Each group was randomly divided into treatment and control groups, which were further subdivided into four timepoints for tumor excision: pre-treatment baseline, 1-, 24-, and 48-hours post-treatment. Prior to excision, we injected pimonidazole to quantify tumor hypoxia. We determined the two-photon excited fluorescence intensity and lifetime of NADH and FAD and quantified the optical redox ratio, ratio of free to bound NADH, and mean NADH lifetime of frozen sections of each tumor. We observed spatial and temporal heterogeneity in these high-resolution optical maps that indicated different metabolic programs in radiation-resistant and -sensitive tumors. A histogram-based heterogeneity analysis revealed time dependent shifts toward glycolysis in the radiation-resistant UM-SCC-47 treatment group, while the sensitive UM-SCC-22B treatment group showed the opposite trend. Proteomics performed on both cell lines identified multiple dysregulated proteins that are often implicated in HNSCC and associated with metabolic reprogramming — specifically, EGFR and SMAD4. We also found elevated levels of hypoxia and HIF-1 in the UM-SCC-47 tumors compared with the UM-SCC-22B tumors prior to and post-radiation. Using spatially co-registered optical imaging and immunohistochemical maps of the same field of view, we will present correlations between our optical markers and EGFR, SMAD4, and HIF-1. These studies will provide evidence of how protein dysregulation causes changes to optical metabolic imaging markers. Citation Format: Jesse D. Ivers, Sina Dadgar, Narasimhan Rajaram. Multimodal metabolic imaging and proteomics of radiation resistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3703.

Abstract 5616: Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome

Abstract Lung cancer remains the leading cause of cancer deaths in the US and worldwide.​ The five-year survival rate of patients with non-small cell lung carcinoma (NSCLC) remains significantly low given that over half present with locally advanced or metastatic disease at time of diagnosis, and experience tumor recurrence following therapeutic intervention.​Current evaluation techniques to assess treatment response are lacking, given they are implemented several weeks after treatment completion and are solely based on anatomical changes in tumor size, forgoing other criteria such as functional or metabolic changes.​ There is a critical need to identify markers early on following diagnosis that are indicative of patient long-term outcome. Two photon microscopy (TPM) techniques are well-suited to provide non-invasive high-resolution information on cell metabolism within three-dimensional tissue. Specifically, two-photon excited fluorescence imaging of autofluorescent cofactors, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), can be used to metabolically characterize cancerous tissue. The goal of this study is to utilize the optical redox ratio (ORR) of FAD/[NADH+FAD] autofluorescence and NADH fluorescence lifetime decay to identify measurable differences in optical metabolic endpoints of human NSCLC that are indicative of their long-term outcome. Twenty-nine NSCLC specimens were obtained from the Lung Cancer Biospecimen Resource Network. They were resected from the lung prior to the patient receiving therapy. Clinical detail reports were used to evaluate the follow-up data of each patient and classify them into groups that reflected their eventual outcomes: responder (N=21), non-responder (N=18), non-metastatic (N=25), and metastatic (N=14). TPM was used to determine the ORR for each sample. NADH lifetime images were collected and fit to a biexponential model to separate the short (A1) and long (A2) components of free and bound NADH. We observed a significantly higher optical redox ratio for tumors within the metastatic group compared to non-metastatic. ​This is consistent with previous findings from our lab that revealed increases in the ORR with increasing metastatic potential in cells, as well as other studies reporting a decrease in NADH concentration in metastatic cancers.​ Additionally, a significantly shorter mean NADH lifetime in the metastatic tumor group was revealed when compared to non-metastatic. This stands in agreement with other studies highlighting decreases in the average lifetime of NADH in metastatic cells compared to non-metastatic. These results demonstrate the feasibility of using optical imaging of autofluorescence and lifetimes of metabolic cofactors to characterize treatment-naïve primary NSCLC tumors and determine differences in their optical metabolic endpoints that are indicative of their long-term outcome. Citation Format: Paola Monterroso Diaz. Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5616.

Temperature measurements in heated gases and flames using carbon monoxide femtosecond two-photon laser-induced fluorescence

Temperature measurements in heated gases and flames using carbon monoxide femtosecond two-photon laser-induced fluorescence

Design and development of a mitochondrial-targeted photosensitizer for two-photon fluorescence imaging and photodynamic therapy

• A rationally designed mitochondrial-targeted photosensitizer, TTTP, for PDT • TTTP can track living cells in vitro and in vivo by two-photon fluorescence imaging • TTTP shows selective phototoxicity to cancer cells, particularly under irradiation • TTTP stimulates ROS in mitochondria, activating apoptosis to inhibit tumor growth Mitochondria are well-acknowledged as ideal targets for tumor therapy due to their important role in energy supply and cellular signal regulation. Mitochondria-specific photosensitizers have been reported to be critical for inducing cell apoptosis. Two-photon fluorescence imaging provides a new technique for delineating biological structures and activities in deep tissues. Herein, we developed a new aggregation-induced emission (AIE) active photosensitizer by attaching a pyridinium group for mitochondrial targeting. The rationally designed photosensitizer (TTTP) exhibited excellent photophysical properties, good biocompatibility, reactive oxygen species (ROS) stimulation ability, anticancer efficacy, and two-photon imaging properties. TTTP was highly taken up by cells and accumulated specifically in mitochondria but was selectively cytotoxic to cancer cells. Under light irradiation, the generation of ROS was significantly boosted, leading to actively induced apoptosis. The in vivo tumor photodynamic therapeutic efficacy of TTTP showed significant inhibition of tumor growth. Furthermore, the underlying mechanism of TTTP tumor suppression revealed that the apoptosis agonist Bax was markedly up-regulated while the antagonist Bcl-xL was down-regulated. This research provides a potential mitochondrial-targeted phototherapeutic agent for effective therapy and two-photon fluorescence imaging.

Two Dicyanostilbene-Based Two-Photon Fluorescence Environmentally Sensitive Probes with Large Two-Photon Absorption Cross Sections and Two-Photon Triple Fluorescence

The unusually sensitive solvatochromism of 2,5-dicyano-4-methyl-4’-dimethylaminostilbene (1a) whose emission maximum varies from 445 nm in cyclohexane to 641 nm in DMSO are described. 1a with remarkably large two-photon cross sections exhibits very strong polarity-, viscosity-, and temperature-dependence of fluorescence, and can be used to detect polarities, viscosities, and temperature. The successful application of 1a provides a brilliant prototype for the developments of ideal two-photon fluorescence probes.

Two-dimensional laser-induced fluorescence thermometry in a medium- to high-enthalpy CO2 flow

Ground-based experimental facilities are needed to qualify materials subjected to aerothermodynamic loads during an entry into the Martian atmosphere. In the present work, the focus is on quantifying the temperature field in a medium- to high-enthalpy CO_{2} flow (from 4.5 to {10.5}, hbox {MJ}/hbox {kg}) of a plasma wind tunnel using one- and two-dimensional two-photon absorption laser-induced fluorescence (CO-TALIF) thermometry at a CO number density in the range of 1–2 times 10^{17} cm^{-3}. However, the measurement method developed here can also be used to study satellite propulsion systems. Due to absorption by air, a two-photon excitation scheme of the CO molecule at 115 nm (VUV) was used. Experimental and simulated excitation spectra are compared for temperature determination. For spectrum simulation in the B^1varSigma ^+leftarrow X^1varSigma ^+ system, an existing program was extended and a polynomial regression correlation was developed for signals with low signal-to-noise ratios. After the first one-dimensional temperature measurements using CO-TALIF in the free-stream to quantify the radial temperature profile for three conditions in the medium- to high- enthalpy range, respectively, two-dimensional measurements in a steady state test chamber at room temperature were carried out. For the two-dimensional investigations, a light sheet optic was designed to generate a light sheet free of divergence in width and thickness. The results of the one- and two-dimensional temperature measurements performed at medium- to high-enthalpy conditions provided plausible results concerning both the values and the course in radial and axial direction, the latter being, to the authors’ knowledge, the first two-dimensional temperature measurements under such conditions using CO-TALIF.

Water-soluble iridium(III) complexes as multicolor probes for one-photon, two-photon and fluorescence lifetime imaging

Fluorescence microscopy imaging provides an indispensable way to visualize morphological details in tissue with subcellular resolution. Of the various imaging agents currently available, heavy-metal complex based probes offer a powerful tool for live cell imaging, primarily due to their excellent photophysical properties, including evident Stokes shifts, high photoluminescence efficiency and relatively long emission lifetime of phosphorescent signals. Herein, we have designed and synthesized seven water soluble cationic iridium(III) solvato complexes (1 − 7) for live cell imaging. The emission colors of these iridium(III) complexes can be tuned from green to red in PBS buffer (phosphate buffered saline). In particular, multicolor phosphorescent imaging for cytoplasm staining of living cells has been realized. More importantly, two-photon excited imaging and fluorescence lifetime imaging microscopy have also successfully been applied for complex 7.

Endoplasmic Reticulum Peroxynitrite Fluctuations in Hypoxia-Induced Endothelial Injury and Sepsis with a Two-Photon Fluorescence Probe.

Sepsis is a serious systemic inflammatory disease that frequently results in death. Early diagnosis and timely targeted interventions could improve the therapeutic effect. Recent work has revealed that the reactive oxygen species (ROS) in the endoplasmic reticulum (ER) and hypoxia-induced endothelial injury play significant roles in sepsis. However, the relationship between the levels of peroxynitrite (ONOO-) and hypoxia-induced endothelial injury as well as different states of sepsis remain unexplored. Herein, we developed a unique two-photon fluorescent probe (ER-ONOO-) for detecting ONOO- in aqueous solution that has high sensitivity, high selectivity, and ultrafast response time. In addition, ER-ONOO- was successfully used to evaluate the levels of ONOO- at the ER with three kinds of methods in a hypoxia-induced endothelial injury model. Furthermore, ER-ONOO- is capable of monitoring the changes in organ fluorescence through ONOO- variation in different stages of a cecum ligation and puncture (CLP) mouse model. Moreover, we also confirmed that the endoplasmic reticulum stress and oxidative stress participated in the CLP model. Consequently, this research can provide a reliable tool for studying ONOO- fluctuation in sepsis and provide new insights into the pathogenic and therapeutic mechanisms involved.