• 2023

    Navigating Chimeric Antigen Receptor-Engineered Natural Killer Cells as Drug Carriers via Three-Dimensional Mapping of the Tumor Microenvironment

    Journal of Controlled Release 362, 524-535 (2023)

    Shigao Huang, Fuqiang Xing, Yeneng Dai, Zhiming Zhang, Guangyu Zhou, Shuo Yang, Yu-Cheng Liu, Zhen Yuan, Kathy Qian Luo, Tianlei Ying, Dafeng Chu, Tzu-Ming Liu, Chu-Xia Deng, Qi Zhao

    Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are recognized as promising immunotherapeutic agents for cancer treatment. However, the efficacy and trafficking of CAR-NK cells in solid tumors are hindered by the complex barriers present in the tumor microenvironment (TME). We have developed a novel strategy that utilizes living CAR-NK cells as carriers to deliver anticancer drugs specifically to the tumor site. We also introduce a time-lapse method for evaluating the efficacy and tumor specificity of CAR-NK cells using a two-photon microscope in live mouse models and three-dimensional (3D) tissue slide cultures. Our results demonstrate that CAR-NK cells exhibit enhanced antitumor immunity when combined with photosensitive chemicals in both in vitro and in vivo tumor models. Additionally, we have successfully visualized the trafficking, infiltration, and accumulation of drug-loaded CAR-NK cells in deeply situated TME using non-invasive intravital two-photon microscopy. Our findings highlight that tumor infiltration of CAR-NK cells can be intravitally monitored through the two-photon microscope approach. In conclusion, our study demonstrates the successful integration of CAR-NK cells as drug carriers and paves the way for combined cellular and small-molecule therapies in cancer treatment. Furthermore, our 3D platform offers a valuable tool for assessing the behavior of CAR cells within solid tumors, facilitating the development and optimization of immunotherapeutic strategies with clinical imaging approaches. 

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  • Water-Soluble Aggregation-Induced Emission Luminogens with Near-Infrared Emission for Advanced Phototheranostics

    Small Science 2023, 2300052 (2023)

    Hanchen Shen, Changhuo Xu, Ruquan Ye, Tzu-Ming Liu, Jianyu Zhang, Ryan T. K. Kwok, Jacky W. Y. Lam, Zhihong Guo, Jianwei Sun, and Ben Zhong Tang

    The development of water-soluble aggregation-induced emission luminogens (AIEgens) emitting in the near-infrared (NIR) window holds promise for efficient biomedical applications. Nevertheless, synthesizing water-soluble counterparts of NIR AIEgens presents difficulties due to their intrinsic hydrophobic properties. To address this issue, researchers have developed various molecular design strategies to improve the water solubility of NIR AIEgens. The integration of hydrophilic groups and targeting moieties is a crucial aspect of achieving precise phototheranostics. Here, diverse approaches to attain water-soluble NIR AIEgens for biomedical applications are presented, and three commonly used strategies that involve decorating NIR AIEgens with positively or negatively charged groups, hydrophilic chains, and bioactive moieties are elaborated. These rational design strategies are believed to provide solutions for enhancing the water solubility and biological performance of NIR AIEgens in a single action. The remaining challenges and opportunities in this field are also discussed. The aim is to provide new insights into the design of water-soluble NIR AIEgens and inspire more researchers to make significant contributions to this promising research area.

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  • Quantitative multiphoton imaging of cell metabolism, stromal fibers, and keratinization enables label-free discrimination of esophageal squamous cell carcinoma

    Biomedical Optics Express 14, 4137-4155 (2023)

    Feng Xiang, Jia Yu, Danling Jiang, Weiwang Hu, Rongli Zhang, Chenming Huang, Ting Wu, Yufeng Gao, Aiping Zheng, Tzu-Ming Liu, Wei Zheng, Xi Li, and Hui Li

    Esophageal squamous cell carcinoma (ESCC) features atypical clinical manifestations and a low 5-year survival rate (< 5% in many developing countries where most of the disease occurs). Precise ESCC detection and grading toward timely and effective intervention are therefore crucial. In this study, we propose a multidimensional, slicing-free, and label-free histopathological evaluation method based on multispectral multiphoton fluorescence lifetime imaging microscopy (MM-FLIM) for precise ESCC identification. To assess the feasibility of this method, comparative imaging on fresh human biopsy specimens of different ESCC grades is performed. By constructing fluorescence spectrum- and lifetime-coded images, ESCC-induced morphological variations are unveiled. Further quantification of cell metabolism and stromal fibers
    reveals potential indicators for ESCC detection and grading. The specific identification of keratin pearls provides additional support for the early detection of ESCC. These findings demonstrate the viability of using MM-FLIM and the series of derived indicators for histopathological evaluation of ESCC. As there is an increasing interest in developing multiphoton endoscopes and multiphoton FLIM systems for clinical use, the proposed method would probably allow noninvasive, label-free, and multidimensional histological detection and grading of ESCC in the

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  • Label-free optical metabolic imaging of adipose tissues for prediabetes diagnosis

    Theranostics 13, 3550-3567 (2023)

    Liping Chen, Guihui Qin, Yuhong Liu, Moxin Li, Yue Li, Lun-Zhang Guo, Lidong Du, Weiming Zheng, Pei-Chun Wu, Yueh-Hsun Chuang, Xiaoyan Wang, Tzung-Dau Wang, Ja-An Annie Ho, Tzu-Ming Liu

    Rationale: Prediabetes can be reversed through lifestyle intervention, but its main pathologic hallmark, insulin resistance (IR), cannot be detected as conveniently as blood glucose testing. In consequence, the diagnosis of prediabetes is often delayed until patients have hyperglycemia. Therefore, developing a less invasive diagnostic method for rapid IR evaluation will contribute to the prognosis of prediabetes.
    Adipose tissue is an endocrine organ that plays a crucial role in the development and progression of prediabetes. Label-free visualizing the prediabetic microenvironment of adipose tissues provides a less invasive alternative for the characterization of IR and inflammatory pathology.
    Methods: Here, we successfully identified the differentiable features of prediabetic adipose tissues by employing the metabolic imaging of three endogenous fluorophores NAD(P)H, FAD, and lipofuscin-like pigments.
    Results: We discovered that 1040-nm excited lipofuscin-like autofluorescence could mark the location of macrophages. This unique feature helps separate the metabolic fluorescence signals of macrophages from those of adipocytes. In prediabetes fat tissues with IR, we found only adipocytes exhibited a low redox ratio of metabolic fluorescence and high free NAD(P)H fraction a1. This differential signature disappears for mice who quit the high-fat diet or high-fat-high-sucrose diet and recover from IR. When mice have diabetic hyperglycemia and inflamed fat tissues, both adipocytes and macrophages possess this kind of metabolic change. As confirmed with RNA-seq analysis and histopathology evidence, the change in adipocyte’s metabolic fluorescence could be an indicator or risk factor of prediabetic IR.
    Conclusion: Our study provides an innovative approach to diagnosing prediabetes, which sheds light on the strategy for diabetes prevention.

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  • Restriction of molecular motion to a higher level: Towards bright AIE dots for biomedical applications

    iScience 26, 106568 (2023)

    Changhuo Xu, Hanchen Shen, Tzu-Ming Liu, Ryan T.K. Kwok, Jacky W.Y. Lam, and Ben Zhong Tang

    In the late 19th century, scientists began to study the photophysical differences between chromophores in the solution and aggregate states, which breed the recognition of the prototypical processes of aggregation-caused quenching and aggregation-induced emission (AIE). In particular, the conceptual discovery of the AIE phenomenon has spawned the innovation of luminogenic materials with high emission in the aggregate state based on their unique working principle termed the restriction of intramolecular motion. As AIE luminogens have been
    practically fabricated into AIE dots for bioimaging, further improvement of their brightness is needed although this is technically challenging. In this review, we surveyed the recent advances in strategic molecular engineering of highly emissive AIE dots, including nanoscale crystallization and matrix-assisted rigidification. We hope that this timely summary can deepen the understanding about the root cause of the high emission of AIE dots and provide inspiration to the rational design of functional aggregates.

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  • Development of a sensitive luminescent probe to uncover new BRD4 inhibitors in living cells

    Chemical Engineering Journal 463, 142356 (2023)

    Ying-Qi Song, Ke-Jia Wu, Zhiming Zhang, Tzu-Ming Liu, Chung-Nga Ko, Wei-Guo Zhu, Dik-Lung Ma, Wanhe Wang, Chung-Hang Leung

    Bromodomain-containing protein 4 (BRD4) is a reader of acetylated histones that regulates the invasion, metastasis and proliferation of cancer. Inhibition of BRD4 activity is an emerging strategy for anticancer. In this study, an iridium based-luminescent probe for screening BRD4 inhibitors in cellulo was developed. The K293 probe was designed by linking a BRD4 nuclear translocation inhibitor as a “binding unit” to an iridium complex as a luminescent “signal unit”. K293 selectively binds to BRD4 in living cancer cells. Candidate compounds compete with K293 for BRD4, resulting in a decrease in luminescence signal. The long luminescence lifetime of K293 allows its luminescence signal to be distinguished from interfering fluorescence using time-resolved techniques in order to improve sensitivity. Hence, even potential BRD4 inhibitors that are undetectable by conventional fluorescence methods can be identified. As a proof-of-concept, Azelastine, an FDA-approved histamine 1 (H1) antagonist was identified to be a potential BRD4 inhibitor using the K293 probe. Azelastine binds to BRD4 protein in the cytoplasm to prevent its translocation into the nucleus, and suppresses cancer cell growth and migration. The results demonstrated that the probe could be useful for the future drug repurposing. 

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  • Plasma riboflavin fluorescence as a diagnostic marker of mesenteric ischemia-reperfusion injury in rats

    Thrombosis Research 223, 146-154 (2023)

    Xueqin Wu, Lun-Zhang Guo, Yi-Hung Liu, Yu-Cheng Liu, Po-Lun Yang, Yun-Shiuan Leung, Hwan-Ching Tai, Tzung-Dau Wang, Jesse Chih-Wei Lin, Chao-Lun Lai, Yueh-Hsun Chuang, Chih-Hsueh Lin, Pi-Tai Chou, I-Rue Lai, and Tzu-Ming Liu

    Due to the delayed and vague symptoms, it is difficult to early diagnose mesenteric ischemia injuries in the dynamics of acute illness, leading to a 60–80 % mortality rate. Here, we found plasma fluorescence spectra can rapidly assess the severity of mesenteric ischemia injury in animal models. Ischemia-reperfusion damage of the intestine leads to multiple times increase in NADH, flavins, and porphyrin auto-fluorescence of blood. The fluorescence intensity ratio between blue-fluorophores and flavins can reflect the occurrence of shock. Using liquid chromatography and mass spectroscopy, we confirm that riboflavin is primarily responsible for the increased flavin fluorescence. Since humans absorb riboflavin from the intestine, its increase in plasma may indicate intestinal mucosa injury. Our work suggests a self-calibrated and reagent-free approach to identifying the emergence of fatal mesenteric ischemia in emergency departments or intensive care units.

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  • Fluorescence intensity and lifetime imaging of lipofuscin-like autofluorescence for label-free predicting clinical drug response in cancer

    Redox Biology 59, 102578 (2023)

    Yinghan Yan, Fuqiang Xing, Jiayue Cao, Yiling Hu, Ling Li, Zhengyin Gao, Hao Jia, Kai Miao, Fangyuan Shao, Chu-Xia Deng, Kathy Qian Luo, Leo Tsz on Lee, and Tzu-Ming Liu

    Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like autofluorescence acutely accumulated in the cell death process. Its unique red autofluorescence could report the apoptosis without labeling and continuously monitor the treatment responses in 3D tumor-culture models. Lifetime imaging of lipofuscin-like red autofluorescence could further distinguish necrosis from apoptosis of cells. Moreover, this endogenous fluorescent marker could visualize the apoptosis in live zebrafish embryos during development. Overall, this study validates that lipofuscin-like autofluorophore is a generic cell death marker. Its characteristic autofluorescence could label-free predict the efficacy of anti-cancer drugs in organoids or animal models. 

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  • 2022

    Boundary-Preserved Deep Denoising of Stochastic Resonance Enhanced Multiphoton Images

    IEEE Journal of Translational Engineering in Health and Medicine 10, 1-12 (2022)

    Sheng-Yong Niu, Lun-Zhang Guo, Yue Li, Zhiming Zhang, Tzung-Dau Wang, Kai-Chun Liu, You-Jin Li, Yu Tsao, and Tzu-Ming Liu

    Objective: With the rapid growth of high-speed deep-tissue imaging in biomedical research, there is an urgent need to develop a robust and effective denoising method to retain morphological features for further texture analysis and segmentation. Conventional denoising filters and models can easily suppress the perturbative noise in high-contrast images; however, for low photon budget multiphoton images, a high detector gain will not only boost the signals but also bring significant background noise. In such a stochastic resonance imaging regime, subthreshold signals may be detectable with the help of noise, meaning that a denoising filter capable of removing noise without sacrificing important cellular features, such as cell boundaries, is desirable. Method: We propose a convolutional neural network-based denoising autoencoder method — a fully convolutional deep denoising autoencoder (DDAE) — to improve the quality of three-photon fluorescence (3PF) and third-harmonic generation (THG) micros- copy images. Results: The average of 200 acquired images of a given location served as the low-noise answer for the DDAE training. Compared with other conventional denoising methods, our DDAE model shows a better signal-to-noise ratio (28.86 and 21.66 for 3PF and THG, respectively), structural similarity (0.89 and 0.70 for 3PF and THG, respectively), and preservation of the nuclear or cellular boundaries (F1-score of 0.662 and 0.736 for 3PF and THG, respectively). It shows that DDAE is a better trade- off approach between structural similarity and preserving signal regions. Conclusions: The results of this study validate the effec- tiveness of the DDAE system in boundary-preserved image denoising. Clinical Impact: The proposed deep denoising system can enhance the quality of microscopic images and effectively support clinical evaluation and assessment.

  • Mouse Strain– and Charge-Dependent Vessel Permeability of Nanoparticles at the Lower Size Limit

    Frontiers in Chemistry 10, 944556 (2022)

    Haoran Chen, Yu-Cheng Liu, Zhiming Zhang, Moxin Li, Lidong Du, Pei-Chun Wu, Wai-How Chong, Fuzeng Ren, Weiming Zheng, and Tzu-Ming Liu

    Remarkable advancement has been made in the application of nanoparticles (NPs) for cancer therapy. Although NPs have been favorably delivered into tumors by taking advantage of the enhanced permeation and retention (EPR) effect, several physiological barriers present within tumors tend to restrict the diffusion of NPs. To overcome this, one of the strategies is to design NPs that can reach lower size limits to improve tumor penetration without being rapidly cleared out by the body. Several attempts have been made to achieve this, such as selecting appropriate nanocarriers and modifying surface properties. While many studies focus on the optimal design of NPs, the influence of mouse strains on the effectiveness of NPs remains unknown. Therefore, this study aimed to assess whether the vascular permeability of NPs near the lower size limit differs among mouse strains. We found that the vessel permeability of dextran NPs was size-dependent and dextran NPs with a size below 15 nm exhibited leakage from postcapillary venules in all strains. Most importantly, the leakage rate of 8-nm fluorescein isothiocyanate dextran was significantly higher in the BALB/c mouse strain than in other strains. This strain dependence was not observed in slightly positive TRITC-dextran with comparable sizes. Our results indicate that the influence on mouse strains needs to be taken into account for the evaluation of NPs near the lower size limit.

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  • A universal strategy for the fabrication of single‑photon and multiphoton NIR nanoparticles by loading organic dyes into water‑soluble polymer nanosponges

    Journal of Nanobiotechnology 20, 311 (2022)

    Li‑Xing Yang, Yu‑Cheng Liu, Chang‑Hui Cho, Yi‑Rou Chen, Chan‑Shan Yang, Yin‑Lin Lu, Zhiming Zhang, Yi‑Tseng Tsai, Yu‑Cheng Chin, Jiashing Yu, Hsiu‑Min Pan, Wei‑Rou Jiang, Zi‑Chun Chia, Wei‑Shiang Huang, Yu‑Lin Chiu, Chun‑Kai Sun, Yu‑Ting Huang, Li‑Ming Chen, Ken‑Tsung Wong, Han‑Min Huang, Chih‑Hsin Chen, Yuan Jay Chang, Chih‑Chia Huang, and Tzu‑Ming Liu

    The development of optical organic nanoparticles (NPs) is desirable and widely studied. However, most organic dyes are water-insoluble such that the derivatization and modification of these dyes are difficult. Herein, we demonstrated a simple platform for the fabrication of organic NPs designed with emissive properties by loading ten different organic dyes (molar masses of 479.1–1081.7 g/mol) into water-soluble polymer nanosponges composed of poly(styrene-altmaleic acid) (PSMA). The result showed a substantial improvement over the loading of commercial dyes (3.7–50% loading) while preventing their spontaneous aggregation in aqueous solutions. This packaging strategy includes our newly synthesized organic dyes (> 85% loading) designed for OPVs (242), DSSCs (YI-1, YI-3, YI-8), and OLEDs (ADF-1–3, and DTDPTID) applications. These low-cytotoxicity organic NPs exhibited tunable fluorescence from visible to nearinfrared (NIR) emission for cellular imaging and biological tracking in vivo. Moreover, PSMA NPs loaded with designed NIR-dyes were fabricated, and photodynamic therapy with these dye-loaded PSMA NPs for the photolysis of cancer cells was achieved when coupled with 808 nm laser excitation. Indeed, our work demonstrates a facile approach for increasing the biocompatibility and stability of organic dyes by loading them into water-soluble polymer-based carriers, providing a new perspective of organic optoelectronic materials in biomedical theranostic applications.

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  • Toward Strong Near-Infrared Absorption/Emission from Carbon Dots in Aqueous Media through Solvothermal Fusion of Large Conjugated Perylene Derivatives with Post-Surface Engineering

    Advanced Science 9, 2202283 (2022)

    Yupeng Liu, Josh Haipeng Lei, Gang Wang, Zhiming Zhang, Jun Wu, Bohan Zhang, Huiqi Zhang, Enshan Liu, Liming Wang, Tzu-Ming Liu, Guichuan Xing, Defang Ouyang, Chu-Xia Deng, Zikang Tang, and Songnan Qu

    Carbon dots (CDs) have attracted significant interest as one of the most emerging photoluminescence (PL) nanomaterials. However, the realization of CDs with dominant near-infrared (NIR) absorption/emission peaks in aqueous solution remains a great challenge. Herein, CDs with both main NIR absorption bands at 720 nm and NIR emission bands at 745 nm in an aqueous solution are fabricated for the first time by fusing large conjugated perylene derivatives under solvothermal treatment. With post-surface engineering, the polyethyleneimine modified CDs (PEI-CDs) exhibit enhanced PL quantum yields (PLQY) up to 8.3% and 18.8% in bovine serum albumin aqueous and DMF solutions, which is the highest PLQY of CDs in NIR region under NIR excitation. Density functional theory calculations support the strategy of fusing large conjugated perylene derivatives to achieve NIR emissions from CDs. Compared to the commercial NIR dye Indocyanine green, PEI-CDs exhibit excellent photostability and much lower cost. Furthermore, the obtained PEI-CDs illustrate the advantages of remarkable two-photon NIR angiography and in vivo NIR fluorescence bioimaging. This work demonstrates a promising strategy of fusing large conjugated molecules for preparing CDs with strong NIR absorption/emission to promote their bioimaging applications.

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  • Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo

    Journal of Lipid Research 63, 100207 (2022)

    Yue Li, Yuwei Du, Zhengqing Xu, Yuan He, Ran Yao, Huiran Jiang, Wen Ju, Jianlin Qiao, Kailin Xu, Tzu-Ming Liu, and Lingyu Zeng

    Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.

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  • One Step Synthesis of Efficient Red Emissive Carbon Dots and Their Bovine Serum Albumin Composites with Enhanced Multi-Photon Fluorescence for in vivo Bioimaging

    Light: Science & Applications 11, 113 (2022)

    Huiqi Zhang, Gang Wang, Zhiming Zhang, Josh Haipeng Lei, Tzu-Ming Liu, Guichuan Xing, Chu-Xia Deng, Zikang Tang, and Songnan Qu

    Efficient red emissive carbon dots (CDs) in aqueous solutions are very scarce for high performance bioimaging applications. In this work, we report a one-step solvothermal treatment to synthesize pure red emissive CDs (FA-CDs) from citric acid and urea in formic acid without complicated purification procedures. Photoluminescence quantum yield (PLQY) of 43.4% was observed in their dimethyl sulfoxide solutions. High PLQY up to 21.9% in aqueous solutions was achieved in their bovine serum albumin (BSA) composites (FA-CDs@BSA) with significantly enhanced multiphoton fluorescence. The strong surface electron-withdrawing structure of FA-CDs caused by the high content of C =O groups contributes for their pure red emission. Owing to the significantly enhanced single and multi-photon red fluorescence and enlarged particle sizes after composing with BSA, in vivo tumor imaging and two-photon fluorescence imaging of blood vessels in mouse ear have been realized via intravenous injection of FA-CDs@BSA aqueous solutions.

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  • Noninvasive Assessment of Liver Function Reserve with Fluorescent Dosimetry of Indocyanine Green

    Biomedical Optics Express 13, 1995-2005 (2022)

    Pei-Chun Wu, Lun-Zhang Guo, Shan Yu, Ning Zeng, Yu-Cheng Liu, Jia Yu, Zhiming Zhang, Ke Lu, Liangyu Sun, Chunfei Wang, Yu-Han Chang, Yin-Lin Lu, Yu-Fang Shen, Sheng Tai, Yueh-Hsun Chuang, Ja-An Annie Ho, Kai-Wen Huang, Yao-Ming Wu, and Tzu-Ming Liu

    Using in vivo multiphoton fluorescent dosimetry, we demonstrate that the clearance dynamics of Indocyanine Green (ICG) in the blood can quickly reveal liver function reserve. In normal rats, the ICG retention rate was below 10% at the 15-minute post-administration; While in the rat with severe hepatocellular carcinoma (HCC), the 15-minute retention rate is over 40% due to poor liver metabolism. With a 785 nm CW laser, the fluorescence dosimeter can evaluate the liver function reserve at a 1/10 clinical dosage of ICG without any blood sampling. In the future, this low-dosage ICG 15-minute retention dosimetry can be applied for the preoperative assessment of hepatectomy or timely perioperative examination.

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  • Nanoprobe-based mass spectrometry and Fourier transform infrared spectroscopy for rapid phospholipid profiling

    Journal of the Chinese Chemical Society 69, 94-106 (2022).

    Elias G. Mernie, Mei-Chun Tseng, Wen-Ti Wu, a Tzu-Ming Liu, and Yu-Ju Chen

    Phospholipids are the major components of cellular membranes and possess important biological roles. Despite their significance in diagnosis and therapeutic application in various diseases, systematic profiling of phospholipids remains challenging due to time-consuming sample preparation and their inherent structure complexity. Taking advantages of complementary, simple, and fast structural analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Fourier transform infrared spectroscopy (FTIR), we reported a nanoprobe-based dual detection strategy for large-scale phospholipid profiling. Based on electrostatic interaction between nitrilotriacetic acid (NTA) chelated iron ion and phosphate head on phospholipids, we developed an NTA-decorated magnetic nanoparticle (MNP)-based strategy for enrichment of phospholipids from lung cancer cell lines. Compared to the conventional liquid–liquid extraction, NTA@MNP nanoprobe enrichment demonstrated 2.6-fold more identified phospholipids. By direct on-particle analysis, FTIR confirmed the P-O-C and phosphate stretching characteristic of the phospholipid head group, and MALDI-TOF MS identified a total of 59 phospholipids from PC9 and A549 lung cancer cells. Comparing the variation of phospholipids between two cell lines, we identified significantly more phospholipids in the PC9 cells (49) compared to 35 phospholipids in the A549 cells. The comparison also revealed 24 and 10 cell line-specific phospholipids in the PC9 and A549 cells, respectively. Given the demonstrated rapid enrichment and unambiguous identification of phospholipids at cellular levels, the developed approach can provide systematic profiling to study the under-explored phospholipid species.

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  • 2021

    Plasmon Resonant Two‐Photon Luminescence Inducing Photosensitization and Nonlinear Optical Microscopy In Vivo by Near‐Infrared Excitation of Au Nanopeanuts

    Applied Sciences 11, 10875 (2021)

    Lun‐Zhang Guo, Cheng‐Ham Wu, Ming‐Fong Tsai, Fong‐Yu Cheng, Vijayakumar Shanmugam, Zen‐Uong Tsai, Zhiming Zhang, Tzu‐Ming Liu, and Chen‐Sheng Yeh

    Photodynamic therapy (PDT) provides a potential therapeutic approach for killing malignant cell/solid tumors, but currently approved photosensitizers (PSs) are generally excited by visible light, limiting the penetration depth in tissues. It is necessary to develop a near‐infrared (NIR) responsive photodynamic platform, providing maximum tissue penetration. Here, we present a gold nanopeanut platform exhibiting dual functions of NIR PDT and two‐photon luminescence imaging. The nanopeanut with a size less than 100 nm exhibits two distinct NIR surface plasmon absorption bands at approximately 1110 and 1300 nm. To perform PDT, we conjugated commercial toluidine blue O (TBO) PS on the surface of the nanopeanuts. With spectral overlap, the 1230‐nm femtosecond Cr: forsterite laser can excite the surface plasmons of nanopeanuts, transfer energy to TBO, and generate singlet oxygen to kill cells. Moreover, the plasmon resonance‐enhanced twophoton luminescence of nanopeanuts can be used to map their delivery in vivo. These results demonstrate that the PS‐conjugated gold nanopeanut is an effective theranostic system for NIR PDT.

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  • Novel zebrafish polycystic kidney disease (PKD) models reveal functions of the Hippo pathway in renal cystogenesis

    Disease Models & Mechanisms 14, dmm049027 (2021)

    Zhiqin Ren, Zhiwei Zhang, Tzu-Ming Liu, and Wei Ge

    Hippo signaling pathway is a kinase cascade which plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated Hippo pathway in kidney development and kidney diseases. To further illustrate its roles in vertebrate kidney, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes. The stk3−/- mutant exhibited edema, formation of glomerular cysts and pronephric tubule dilation during larval stage. Interestingly, disruption of wwtr1 but not yap1 significantly alleviated the renal phenotypes of the stk3−/- mutant, and overexpression of Wwtr1 with CMV promoter also induced pronephric phenotypes during larval stage, similar to those of the stk3−/- mutant. Notably, adult fish with Wwtr1 overexpression developed phenotypes similar to those of human polycystic kidney disease (PKD). Overall, our studies revealed roles of Stk3 and Wwtr1 in renal cyst formation. Using pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development

  • Accelerating precision anti-cancer therapy by time-lapse and label-free 3D tumor slice culture platform

    Theranostics 11, 9415-9430 (2021)

    Fuqiang Xing, Yu-Cheng Liu, Shigao Huang, Xueying Lyu, Sek Man Su, Un In Chan, Pei-Chun Wu, Yinghan Yan, Nana Ai, Jianjie Li, Ming Zhao, Barani Kumar Rajendran, Jianlin Liu, Fangyuan Shao, Heng Sun, Tak Kan Choi, Wenli Zhu, Guanghui Luo, Shuiming Liu, De Li Xu, Kin Long Chan, Qi Zhao, Kai Miao, Kathy Qian Luo, Wei Ge, Xiaoling Xu, Guanyu Wang, Tzu-Ming Liu, Chu-Xia Deng

    The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded.

    Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis.

    Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy.

    Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

  • Functionalizing collagen with vessel-penetrating two-photon phosphorescence probes: A new in vivo strategy to map oxygen concentration in tumor microenvironment and tissue ischemia

    Advanced Science 8, 2102788 (2021)

    Cheng-Ham Wu, Kristina S. Kisel, Muthu Kumar Thangavel, Yi-Ting Chen, Kai-Hsin Chang, Ming-Rung Tsai, Chia-Yu Chu, Yu-Fang Shen, Pei-Chun Wu, Zhiming Zhang, Tzu-Ming Liu, Janne Jänis, Elena V. Grachova, Julia R. Shakirova, Sergey P. Tunik, Igor O. Koshevoy, Pi-Tai Chou

    The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio-probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI-diimine complex already exhibits excellent emission yield (34%, 𝝀em = 583 nm) and large two-photon absorption cross-sections (𝝈= 300 GM @800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen-bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 μs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post-extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial-empowered phosphorescence sensing and imaging

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  • Remodeling of Tumor Microenvironment by Tumor-Targeting Nanozymes Enhances Immune Activation of CAR T Cells for Combination Therapy

    Small 12, 2102624 (2021)

    Lipeng Zhu, Jie Liu, Guangyu Zhou, Tzu-Ming Liu, Yunlu Dai, Guangjun Nie, Qi Zhao

    Targeting B7-H3 chimeric antigen receptor (CAR) T cells has antitumor potential for therapy of non-small cell lung cancer (NSCLC) in preclinical studies. However, CAR T cell therapy remains a formidable challenge for the treatment of solid tumors due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Nanozymes exhibit merits modulating the immunosuppression of the tumor milieu. Here, a synergetic strategy by combination of nanozymes and CAR T cells in solid tumors is described. This nanozyme with dual photothermal-nanocatalytic properties is endowed to remodel TME by destroying its compact structure. It is found that the B7-H3 CAR T cells infused in mice engrafted with the NSCLC cells have superior antitumor activity after nanozyme ablation of the tumor. Importantly, it is found that the changes altered immune-hostile cancer environment, resulting in enhanced activation and infiltration of B7-H3 CAR T cells. The first evidence that the process of combination nanozyme therapy effectively improves the therapeutic index of CAR T cells is presented. Thus, this study clearly supports that the TME-immunomodulated nanozyme is a promising tool to improve the therapeutic obstacles of CAR T cells against solid tumors.

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  • Expansion of Rare Cancer Cells into Tumoroids for Therapeutic Regimen and Cancer Therapy

    Advanced Therapeutics 4, 2100017 (2021)

    Myagmartsend Enkhbat, Yung-Chiang Liu, Jua Kim, Yanshan Xu, Zongyi Yin, Tzu-Ming Liu, Chu-Xia Deng, Chang Zou, Xi Xie, Xiaowu Li, and Peng-Yuan Wang

    Rare cancer cells, such as circulating tumor cells (CTCs) and cancer stem cells (CSCs), are small cell population found in cancer patients. CTCs have been recognized as tumor avatars for real-time cancer monitoring, while CSCs are the most malignant tumor cells that play a dominant role in drug resistance and metastasis. Interestingly, these two types of cells share the same surface markers, such as EpCAM, CD44, and CD133. While capturing these rare cells is available, the expansion of these cells is still challenging due to the limited cell number. These cells are susceptible to the microenvironment and lose the capability to grow in vitro, especially after an intense capturing process. A technology called patient derived tumor organoids (PDOs) or tumoroids is a rising start in cancer modeling but the applicability is still questionable. Recently, assembloids containing multiple tumor-related cells have been developed which is one step closer to the real tumor. In this review, strategies for in vitro expansion of tumoroids are summarized implying that artificial tumor niche composed of optimized biophysical and biological cues is vital in the tumoroid generation. Tumoroids containing rare cancer cells is believed to be beneficial in the diagnosis, therapeutic regimen, and drug discovery for personalized therapy.

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  • 2020

    Low-toxicity FePt nanoparticles for the targeted and enhanced diagnosis of breast tumors using few centimeters deep whole-body photoacoustic imaging

    Photoacoustics 19, 100179 (2020)

    Yubin Liu, Pei-Chun Wu, Sen Guo, Pi-Tai Chou, Chuxia Deng, Shang-Wei Chou, Zhen Yuan, Tzu-Ming Liu

    A considerable amount of early breast tumors grown at a depth over 2 cm in breast tissues. With high near-infrared absorption of iron-platinum (FePt) nanoparticles, we achieved few centimeters deep photoacoustic (PA) imaging for the diagnosis of breast tumors. The imaging depth can extend over 5 cm in chicken breast tissues at the low laser energy density of 20 mJ/cm2 ( ≤ ANSI safety limit). After anti-VEGFR conjugation and the tail-vein injection, we validated their targeting on tumor sites by the confocal microscopy and PA imaging. Using a home-made whole-body in vivo PA imaging, we found that the nanoparticles were rapidly cleared away from the site of the tumor and majorly metabolized through the liver. These results validated the clinical potential of the FePt nanoparticles in the low-toxicity PA theragnosis of early breast cancer and showed the capacity of our whole-body PA imaging technique on monitoring the dynamic biodistribution of nanoparticles in the living body.

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  • Imaging of macrophage mitochondria dynamics in vivo reveals cellular activation phenotype for diagnosis

    Theranostics 10, 2897-2917 (2020)

    Yue Li, Yuan He, Kai Miao, Ying Zheng, Chuxia Deng, Tzu-Ming Liu

    Highly plastic macrophages are pivotal players in the body’s homeostasis and pathogenesis. Grasping the molecular or cellular factors that drive and support the macrophage activation will help to develop diagnostics and manipulate their functions in these contexts. However, the lack of in vivo characterization methods to reveal the dynamic activation of macrophages impedes these studies in various disease contexts.

    Methods: Here, in vitro bone marrow-derived macrophages (BMDMs) and in vivo Matrigel plug were used to evaluate how mitochondria dynamics supports cellular activation and functions. We conducted macrophage repolarization in vitro to track mitochondria dynamics during the shift of activation status. For in vivo diagnosis, a novel MitoTracker-loaded liposome was first developed to label macrophage mitochondria in mice before/after inflammatory stimulation.

    Results: Based on the typical activation of in vitro BMDMs, we found glycolysis based macrophages have punctate and discrete mitochondria, while OXPHOS active macrophages have elongated and interconnected mitochondria. M1, M2a, M2b, and M2c activated BMDMs showed clustered and differentiable features in mitochondrial morphology. These features also hold for Matrigel plug-recruited macrophages in mice. Furthermore, with the interventions on M2a macrophages in vitro, we demonstrated that mitochondria morphology could be a metabolic index to evaluate macrophage activation status under drug manipulation. Using the MitoTracker-loaded liposomes, we further achieved subcellular imaging of macrophage mitochondria in vivo. Their organization dynamics revealed the dynamic change from anti-inflammatory macrophages to inflammatory ones in vivo under the lipopolysaccharide (LPS) challenge.

    Conclusion: These results reveal that subcellular imaging of mitochondria organization can characterize the activation status of macrophage in vitro and in vivo at a single-cell level, which is critical for the studies of noninvasive diagnosis and therapeutic drug monitoring.

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  • 2019

    New Templated Ostwald Ripening Process of Mesostructured FeOOH for Third‐Harmonic Generation Bioimaging

    Small 15, 1805086 (2019). Back cover

    Chien‐Wei Lee, Pei‐Chun Wu, I‐Ling Hsu, Tzu‐Ming Liu, Wai‐How Chong, Cheng‐Ham Wu, Tsung‐Yuan Hsieh, Lun‐Zhang Guo, Yu Tsao, Po‐Ting Wu, Jiashing Yu, Pei‐Jane Tsai, Huei‐Sheng Huang, Yu‐Chun Chuang, and Chih‐Chia Huang

    Emerging advances in iron oxide nanoparticles exploit their high magnetization for various applications, such as bioseparation, hyperthermia, and magnetic resonance imaging. In contrast to their excellent magnetic performance, the harmonic generation and luminescence properties of iron oxide nanoparticles have not been thoroughly explored, thus limiting their development as a tool in photomedicine. In this work, a seed/growth‐inspired synthesis is developed combined with primary mineralization and a ligand‐assisted secondary growth strategy to prepare mesostructured α‐FeOOH nanorods (NRs). The sub‐wavelength heterogeneity of the refractive index leads to enhanced third‐harmonic generation (THG) signals under near‐infrared excited wavelengths at 1230 nm. The as‐prepared NRs exhibit an 11‐fold stronger THG intensity compared to bare α‐FeOOH NRs. Using these unique nonlinear optical properties, it is demonstrated that mesostructured α‐FeOOH NRs can serve as biocompatible and nonbleaching contrast agents in THG microscopy for long‐term labeling of cells as well as in angiography in vivo by modifying lectin to enhance the binding efficiency to the glycocalyx layers on the wall of blood vessels. These results provide a new insight into Fe‐based nanoplatforms capable of emitting coherent light as molecular probes in optical microscopy, thus establishing a complementary microscopic imaging method for macroscopic magnetic imaging systems.

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  • 2018

    Discovering Macrophage Functions Using In Vivo Optical Imaging Techniques

    Frontiers in Immunology 9, 502 (2018)

    Yue Li and Tzu-Ming Liu

    Macrophages are an important component of host defense and inflammation and play a pivotal role in immune regulation, tissue remodeling, and metabolic regulation. Since macrophages are ubiquitous in human bodies and have versatile physiological functions, they are involved in virtually every disease, including cancer, diabetes, multiple sclerosis, and atherosclerosis. Molecular biological and histological methods have provided critical information on macrophage biology. However, many in vivo dynamic behaviors of macrophages are poorly understood and yet to be discovered. A better understanding of macrophage functions and dynamics in pathogenesis will open new opportunities for better diagnosis, prognostic assessment, and therapeutic intervention. In this article, we will review the advances in macrophage tracking and analysis with in vivo optical imaging in the context of different diseases. Moreover, this review will cover the challenges and solutions for optical imaging techniques during macrophage intravital imaging.

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  • Harmonic Generation Microscopy of Bone Microenvironment in vivo

    Optics Communications 422, 52-55 (2018)

    Pei-Chun Wu, Yu-Fang Shen, Chi-Kuang Sun, Charles P. Lin and Tzu-Ming Liu

    Here we report and review the investigation of bone microenvironment in vivo with harmonic generation microscopy. Excited by an infrared femtosecond laser, the second harmonic generation images can reveal the bone structures and boundaries. The third harmonic generation images can reveal the osteocytes, connecting canaliculi, and granular bone marrow cells. These imaging features can be used to observe and analyze the histology of the bone microenvironment in the future.

  • 2017

    Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy

    PLoS ONE12, e0186846 (2017)

    Danielle Tokarz, Richard Cisek, Marc N. Wein, Raphaël Turcotte, Christa Haase, Shu-Chi A. Yeh, Srinidhi Bharadwaj, Anthony P. Raphael, Hari Paudel, Clemens Alt, Tzu-Ming Liu, Henry M. Kronenberg, Charles P. Lin

    Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

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  • Entropy change of biological dynamics in COPD

    International Journal of COPD 12, 2997-3005 (2017)

    Yu Jin, Chang Chen, Zhixin Cao, Baoqing Sun, Iek Long Lo, Tzu-Ming Liu, Jun Zheng, Shixue Sun, Yan Shi, Xiaohua Douglas Zhang

    In this century, the rapid development of large data storage technologies, mobile network technology, and portable medical devices makes it possible to measure, record, store, and track analysis of large amount of data in human physiological signals. Entropy is a key metric for quantifying the irregularity contained in physiological signals. In this review, we focus on how entropy changes in various physiological signals in COPD. Our review concludes that the entropy change relies on the types of physiological signals under investigation. For major physiological signals related to respiratory diseases, such as airflow, heart rate variability, and gait variability, the entropy of a patient with COPD is lower than that of a healthy person. However, in case of hormone secretion and respiratory sound, the entropy of a patient is higher than that of a healthy person. For mechanomyogram signal, the entropy increases with the increased severity of COPD. This result should give valuable guidance for the use of entropy for physiological signals measured by wearable medical device as well as for further research on entropy in COPD.

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  • Resonant dipolar coupling of microwaves with confined acoustic vibrations in a rod-shaped virus

    Scientific Reports 7, 4611 (2017)

    Chi-Kuang Sun, Yi-Chun Tsai, Yi-Jan E. Chen, Tzu-Ming Liu, Hui-Yuan Chen, Han-Ching Wang, and Chu-Fang Lo

    In this letter, we treat a rod-shaped virus as a free homogenous nanorod and identify its confined acoustic vibration modes that can cause strong resonant microwave absorption through electric dipolar excitation with a core-shell charge distribution. They are found to be the n=4N-2 modes of the longitudinal modes of the nanorods, where N is an integer starting from 1 and n is the mode order quantum number. This study was confirmed by measuring the microwave absorption spectra of white spot syndrome virus (WSSV), which is a rod-shaped virus. This is also the first study to identify the “dipolar-like” mode in a rod-shaped nano-object. Our study is not only an important step to achieve rapid and sensitive detection of rod-shaped viruses based on their microwave spectroscopic features and a non-contact method to measure the Young’s modulus of rod-shaped viruses, but also is critical to formulate an efficient epidemic prevention strategy to deactivate viruses with the structure-resonant microwaves.

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  • Mesoporous silica promoted deposition of bioinspired polydopamine onto contrast agent: A universal strategy to achieve both biocompatibility and multiple scale molecular imaging

    Particle & Particle Systems Characterization 34, 1600415 (2017)

    Yu-Wei Chen, Yung-Kang Peng, Shang-Wei Chou, Yu-Jui Tseng, Pei-Chun Wu, Shin-Kung Wang, Yi-Wei Lee, Jing-Jong Shyue, Jong-Kai Hsiao, Tzu-Ming Liu, Pi-Tai Chou

    Polydopamine (PDA) preserves universal coating and metal-binding ability, and is suitable for application in synthesizing multifunctional agents. Herein, utilizing mesoporous silica assisted deposition to enhance both heterogeneous nucleation and loading amounts of PDA, the magnetic resonance (MR) T1 component (PDA-Fe3+) and MR T2/computed tomography (CT)/multiphoton luminescence (MPL) component (FePt) have been successfully integrated in aqueous solution. This four-in-one (T1, T2, CT, MPL) imaging nanocomposite, FePt@mSiO2 @PDA-polyethylene glycol (PEG), demonstrated its multi-imaging power both in vitro/in vivo. According to our in vitro/in vivo results, FePt@mSiO2@PDA-PEG reveals water-content-dependent property in T1 MR imaging, which suggests the necessity of having dual-modal MR ability in a single particle for the precision diagnosis. Most importantly, this dual (T1,T2)-MRI/CT contrast agent is demonstrated complementary to each other in the in vivo testing. PDA coated mesoporous silica also offers an advantage of delayed degradation that prevents adverse effects caused by silica fragments before excretion. The potential of this nanocomposites in both drug carrier and photothermal agent was further evaluated by using doxorubicin and monitoring solution temperature after irradiating 808 nm continuous-wave, respectively The merits of controlled polymerization, enhanced PDA loading, and biofavorable degradation make this methodology promising to other nanoparticle@mSiO2 for a wide range of bioapplications.

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  • Smart NIR linear and nonlinear optical nanomaterials for cancer theranostics: Prospects in photomedicine

    Progress in Materials Science 88, 89-135 (2017)

    Tzu-Ming Liu, João Conde, Tomasz Lipiński, Artur Bednarkiewicz, and Chih-Chia Huang

    Light-based diagnostics and therapy have become indispensable tools in the field of cancer nanomedicine. Various optical imaging modalities with tomographic capability have been developed to visualize cellular and organismic distributions of molecules. Microscopic pharmacokinetics and the tumor-targeting efficacy of nanoscale effectors can now be precisely evaluated. Moreover, phototherapy using intense laser light has been widely used for treating cancers. Using light-active nanoscale effectors, photothermal and photodynamic therapies on superficial tumors can be achieved with low-illumination lasers. Consequently, for the next generation of photo-medical techniques, the use of near infrared (NIR) excitation sources on NIR-activatable nanoparticles may offer deeper light penetration owing to less extensive scattering and absorption by endogenous chromophores in the NIR spectral region. Therefore, treatments and biodetection within higher tissue volumes and with less side effects (e.g. overheating) may be successfully implemented. This comprehensive review covers the state-of-the-art technologies on (a) advanced laser light sources appropriate for deep tissue theranostics, (b) types of laser interactions with pure- NIR and NIR-upconverting nanomaterials, (c) current development of NIR and multiphoton nanoparticles, (d) application fields of NIR nanomaterials in cancer theranostics, and (e) nanotoxicology of NIR nanoscale effectors for cancer treatment.

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  • Fiber-based 1150-nm femtosecond laser source for the minimally invasive harmonic generation microscopy

    Journal of Biomedical Optics 22, 036008 (2017)

    Jing-Yu Huang, Lun-Zhang Guo, Jing-Zun Wang, Tse-Chung Li, Hsin-Jung Lee, Po-Kai Chiu, Lung-Han Peng, and Tzu-Ming Liu

    Harmonic generation microscopy (HGM) has become one unique tool of optical virtual biopsy for the diagnosis of cancer and the in vivo cytometry of leukocytes. Without labeling, HGM can reveal the submicron features of tissues and cells in vivo. For deep imaging depth and minimal invasiveness, people commonly adopt 1100- to 1300-nm femtosecond laser sources. However, those lasers are typically based on bulky oscillators whose performances are sensitive to environmental conditions. We demonstrate a fiber-based 1150-nm femtosecond laser source, with 6.5-nJ pulse energy, 86-fs pulse width, and 11.25-MHz pulse repetition rate. It was obtained by a bismuth borate or magnesium-doped periodically poled lithium niobate (MgO:PPLN) mediated frequency doubling of the 2300-nm solitons, generated from an excitation of 1550-nm femtosecond pulses on a large mode area photonic crystal fiber. Combined with a home-built laser scanned microscope and a tailor-made frame grabber, we achieve a pulse-per-pixel HGM imaging in vivo at a 30-Hz frame rate. This integrated solution has the potential to be developed as a stable HGM system for routine clinical use.

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  • 2016

    Imaging cytometry of human leukocytes with third harmonic generation microscopy

    Scientific Reports 6, 37210 (2016)

    C.-H. Wu, T.-D. Wang, C.-H. Hsieh, S.-H. Huang, J.-W. Lin, S.-C. Hsu, H.-T. Wu, Y.-M. Wu, and T.-M. Liu

    Based on third-harmonic-generation (THG) microscopy and a k-means clustering algorithm, we developed a label-free imaging cytometry method to differentiate and determine the types of human leukocytes. According to the size and average intensity of cells in THG images, in a two-dimensional scatter plot, the neutrophils, monocytes, and lymphocytes in peripheral blood samples from healthy volunteers were clustered into three differentiable groups. Using these features in THG images, we could count the number of each of the three leukocyte types both in vitro and in vivo. The THG imaging-based counting results agreed well with conventional blood count results. In the future, we believe that the combination of this THG microscopy-based imaging cytometry approach with advanced texture analysis of sub-cellular features can differentiate and count more types of blood cells with smaller quantities of blood.

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  • Revisiting the classification of NIR-absorbing/emitting nanomaterials for in vivo bioapplications

    NPG Asia Materials 8, e295 (2016)

    T.-M. Liu, J. Conde, T. Lipiński, A. Bednarkiewicz and C.-C. Huang

    With the development of nonlinear optics and new imaging methods, near-infrared (NIR) light can excite contrast agents to probe biological specimens both functionally and structurally with a deeper imaging depth and a higher spatial resolution than linear optical approaches. There is considerable and growing interest in how biological specimens respond to NIR light. Moreover, the visible absorption band of most functional nanomaterials becomes NIR-excitable through multiphoton processes, thus allowing multifunctional imaging and combined therapy with noble metal and magnetic nanoparticles both in vitro and in vivo. A groundbreaking example is the use of different laser techniques to excite single-type NIR-absorbing/emitting nanomaterials to produce multiphoton emission by femtosecond lasers using either a remote control system for photodynamic therapy or photo-induced chemical bond dissociation. These techniques provided superior anatomical resolution and detection sensitivity for in vivo tumor-targeted imaging than those offered by conventional methods. Here we summarize the most recent progress in the development of smart NIR-absorbing/emitting nanomaterials for in vivo bioapplications.

  • Infrared-active quadruple contrast FePt nanoparticles for multiple scale molecular imaging

    Biomaterials 85, 54-64 (2016)

    S.-W. Chou, C.-L. Liu, T.-M. Li.u, Y.-F Shen, L.-C. Kuo, C.-H. Wu, T.-Y. Hsieh, P.-C. Wu, M.-R. Tsai, C.-C. Yang, K.-Y. Chang, M.-H. Lu, P.-C. Li, S.-P. Cheng, Y.-H. Wang, C.-W. Lu, Y.-A. Chen, C.-C. Huang, C.-R. C. Wang, J.-K. Hsiao, M.-L. Li, P.-T. Chou

    A single nanomaterial with multiple imaging contrasts and functions is highly desired for multiscale theragnosis. Herein, we demonstrate single 1–1.9 μm infrared-active FePt alloy nanoparticles (FePt NPs) offering unprecedented four-contrast-in-one molecular imaging – computed tomography (CT), magnetic resonance imaging (MRI), photoacoustic (PA) imaging, and high-order multiphoton luminescence (HOMPL) microscopy. The PA response of FePt NPs outperforms that of infrared-active gold nanorods by 3- to 5.6-fold under identical excitation fluence and particle concentrations. HOMPL (680 nm) of an isolated FePt NP renders spatial full-width-at-half-maximum values of 432 nm and 300 nm beyond the optical diffraction limit for 1230-nm and 920-nm excitation, respectively. The in vivo targeting function was successfully visualized using HOMPL, PA imaging, CT, and MRI, thereby validating FePt as a single nanomaterial system covering up to four types (Optical/PA/CT/MRI) of molecular imaging contrast, ranging from the microscopic level to whole-body scale investigation.

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  • Detection of malformations in sea urchin plutei exposed to mercuric chloride using different fluorescent techniques

    Ecotoxicology and Environmental Safety 123, 72-80 (2016)

    I. Buttino, D. Sartori, J.-S. Hwang, D. Pellegrini, G. Romano, A. Gaion, C.-K. Sun, T.-M. Liu, D. Pellegrini, A. Gaion, D. Sartori

    Embryos of Mediterranean sea urchin Paracentrotus lividus and subtropical Echinometra mathaei were exposed to 5,10, 15 and 20 μg L-1 and to 1, 2, 3 and 4 μg L-1 mercuric chloride (HgCl2), respectively. The effective concentration (EC50) inducing malformation in 50% of 4-arm pluteus stage (P4) was 16.14 μg L-1 for P. lividus and 2.41 μg L-1for E. mathaei. Two-photon (TP), second (SHG) and third harmonic generation (THG) microscopy techniques, TUNEL staining,  propidium iodide (PI) and Hoechst 33342 probes were used to detect light signals or to stain apoptotic and necrotic cells in fixed and alive plutei. Signals were detected differently in the two species: TP fluorescence, commonly associated with apoptotic cells, did not increase with increasing HgCl2 concentrations in P. lividus and in fact, the TUNEL did not reveal induction of apoptosis. PI fluorescence increased in P. lividus in a dose-dependent manner, suggesting a loss of cell permeability. In E. mathaei plutei TP fluorescence increased at increasing HgCl2 concentrations. THG microscopy revealed skeletal rods in both species. Different fluorescent techniques, used in this study, are proposed as early-warning systems to visualize malformations and physiological responses in sea urchin plutei.

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  • 2015

    Efficient structure resonance energy transfer from microwaves to confined acoustic vibrations in viruses

    Scientific Reports 5, 18030 (2015)

    S.-C. Yang, H.-C. Lin, T.-M. Liu, J.-T. Lu, W.-T. Hung, Y.-R. Huang, Y.-C. Tsai, C.-L. Kao, S.-Y. Chen, and C.-K. Sun*

    Virus is known to resonate in the confined-acoustic dipolar mode with microwave of the same frequency. However this effect was not considered in previous virus-microwave interaction studies and microwave-based virus epidemic prevention. Here we show that this structure-resonant energy transfer effect from microwaves to virus can be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public. We demonstrate this effect by measuring the residual viral infectivity of influenza A virus after illuminating microwaves with different frequencies and powers. We also established a theoretical model to estimate the microwaves power threshold for virus inactivation and good agreement with experiments was obtained. Such structure-resonant energy transfer induced inactivation is mainly through physically fracturing the virus structure, which was confirmed by real-time reverse transcription polymerase chain reaction. These results provide a pathway toward establishing a new epidemic prevention strategy in open public for airborne virus.

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  • Controlled Au-polymer nanostructures for multiphoton imaging, prodrug delivery, and chemo-photothermal therapy platforms

    ACS Applied Materials & Interfaces 7, 25259-25269 (2015)

    C-C Huang* and T.-M. Liu

    We have successfully introduced proton-induced control reaction of HAuCl4 and poly(styrene-alt-maleic acid) (PSMA) sodium salt to prepare triangular and multicore Au@polymer nanoparticles (NPs). The inter-particle interactions in the core gave rise to an absorption band at the near-infrared wavelength. The near-infrared optical properties of the resulting Au-polymer nanostructures are highly stabile in physiological environment, which offered strong photo-to-thermal conversion by a moderate continuous-wave 808 nm laser and exhibited multiphoton fluorescence for imaging using a 1230 nm-light excitation (femtosecond laser). Exposure of the carboxylate groups at the polymer shell made the surface structure of the Au multicore @polymer NPs to directly conjugate Pt(II)/Pt(IV)-based drugs, which possessed the elimination of the immediate toxicity over the short time and performed anticancer effect after three days. A synergistic effect of the chemo-photothermal therapy showed a moderate hyperthermia assistance (< 1 W/cm2) and a better anticancer performance over time compared with the individual treatments. We demonstrated that such PSMA-based methodology enables a broad range of chemical material synthesis in the kinetic control to not only form Au nano-octahedrons and nano-triangles using Br/I ions additives, but also could be extended to form Au/Fe3O4@polymer nanocomposites via proton-assisted PSMA self-assembly.

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  • Imaging endogenous bilirubins with two-photon fluorescence of bilirubin dimers

    Analytical Chemistry 87, 7575-7582 (2015)

    Y.-F. Shen, M.-R. Tsai, S.-C. Chen, Y.-S. Leung, C.-T. Hsieh, Y.-S. Chen, F.-L. Huang, R. P. Obena, M. M. L. Zulueta, H.-Y. Huang, W.-J. Lee, K.-C. Tang, C.-T. Kung, M.-H. Chen, D.-B. Shieh, Y.-J. Chen, T.-M. Liu, P.-T. Chou, and C.-K. Sun

    Based on an infrared femtosecond Cr:forsterite laser, we developed a semi-quantitative method to analyze the microscopic distribution of bilirubins. Using 1230 nm femtosecond pulses, we selectively excited the two-photon red fluorescence of bilirubin dimers around 660 nm. Autofluorescences from other endogenous fluorophores were greatly suppressed. Using this distinct fluorescence measure, we found that poorly-differentiated hepatocellular carcinoma (HCC) tissues on-average showed 3.7-times lower concentration of bilirubins than the corresponding non-tumor parts. The corresponding fluorescence lifetime measurements indicated that HCC tissues exhibited a longer lifetime (500 ps) than those of non-tumor parts (300 ps). Similarly, oral cancer cell lines had longer lifetimes (>330 ps) than those of non-tumor ones (250 ps). We anticipate the developed methods of bilirubin molecular imaging to be useful in diagnosing cancers or studying the dynamics of bilirubin metabolisms in live cells.

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  • In vivo quantification of the structural changes of collagens in a melanoma microenvironment with second and third harmonic generation microscopy

    Scientific Reports 5, 8879 (2015)

    Pei-Chun Wu, Tsung-Yuan Hsieh, Zen-Uong Tsai, and Tzu-Ming Liu

    Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). In the early stage of melanoma development, we found that collagen fibers adjacent to a melanoma have increased OI values and SHG intensities. In the late stages, these collagen networks have more directionality and less homogeneity. The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes. In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity. Multiplying OI, VarGLCM, and RTHG/SHG together, the combinational collagen remodeling (CR) index at 4 weeks post melanoma implantation showed a 400-times higher value than normal ones. These results validate that our quantitative indices of SHG and THG microscopies are sensitive enough to diagnose the collagen remodeling in vivo. We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

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  • THz acoustic phonon spectroscopy and nanoscopy by using piezoelectric semiconductor heterostructures

    Ultrasonics 56, 52-65 (2015)

    Pierre-Adrien Mante, Yu-Ru Huang, Szu-Chi Yang, Tzu-Ming Liu, Alexei A. Maznev, Jinn-Kong Sheu, and Chi-Kuang Sun

    Thanks to ultrafast acoustics, a better understanding of acoustic dynamics on a short time scale has been obtained and new characterization methods at the nanoscale have been developed. Among the materials that were studied during the development of ultrafast acoustics, nitride based heterostructures play a particular role due to their piezoelectric properties and the possibility to generate phonons with over-THz frequency and bandwidth. Here, we review some of the work performed using this type of structure,
    with a focus on THz phonon spectroscopy and nanoscopy. First, we present a brief description of the theory of coherent acoustic phonon generation by piezoelectric heterostructure. Then the first experimental observation of coherent acoustic phonon generated by the absorption of ultrashort light pulses in piezoelectric heterostructures is presented. From this starting point, we then present some methods developed to realize customizable phonon generation. Finally we review some more recent applications of these structures, including imaging with a nanometer resolution, broadband attenuation measurements with a frequency up to 1 THz and phononic bandgap characterization.

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  • Femtosecond laser bone ablation with a high repetition rate fiber laser source

    Biomedical Optics Express 6, 32-42 (2015)

    Luke J. Mortensen, Clemens Alt, Raphaël Turcotte, Marissa Masek, Tzu-Ming Liu, Daniel C. Côté, Chris Xu, Giuseppe Intini and Charles P. Lin

    Femtosecond laser pulses can be used to perform very precise cutting of material, including biological samples from subcellular organelles to large areas of bone, through plasma-mediated ablation. The use of a kilohertz regenerative amplifier is usually needed to obtain the pulse energy required for ablation. This work investigates a 5 megahertz compact fiber laser for near-video rate imaging and ablation in bone. After optimization of ablation efficiency and reduction in autofluorescence, the system is demonstrated for the in vivo study of bone regeneration. Image-guided creation of a bone defect and longitudinal evaluation of cellular injury response in the defect provides insight into the bone regeneration process.

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  • 2014

    Synergistic delivery of gold nanorods using multifunctional microbubbles for enhanced plasmonic photothermal therapy

    Scientific Reports 4, 5685 (2014)

    Yu-Hsin Wang, Shi-Ping Chen, Ai-Ho Liao, Ya-Chuen Yang, Cheng-Ru Lee, Cheng-Han Wu, Pei-Chun Wu, Tzu-Ming Liu, Churng-Ren Chris Wang, and Pai-Chi Li

    Plasmonic photothermal therapy (PPTT) using plasmonic nanoparticles as efficient photoabsorbing agents has been proposed previously. One critical step in PPTT is to effectively deliver gold nanoparticles into the cells. This study demonstrates that the delivery of gold nanorods (AuNRs) can be greatly enhanced by combining the following three mechanisms: AuNRs encapsulated in protein-shell microbubbles (AuMBs), molecular targeting, and sonoporation employing acoustic cavitation of microbubbles (MBs). Both in vitro and in vivo tests were performed. For molecular targeting, the AuMBs were modified with anti-VEGFR2. Once bound to the angiogenesis markers, the MBs were destroyed by ultrasound to release the AuNRs and the release was confirmed by photoacoustic measurements. Additionally, acoustic cavitation was induced during MB destruction for sonoporation (i.e., increase in transient cellular permeability). The measured inertial cavitation dose was positively correlated with the temperature increase at the tumor site. The quantity of AuNRs delivered into the cells was also determined by measuring the mass spectrometry and observed using third-harmonic-generation microscopy and two-photon fluorescence microscopy. A temperature increase of 20°C was achieved in vitro. The PPTT results in vivo also demonstrated that the temperature increase (>45°C) provided a sufficiently high degree of hyperthermia. Therefore, synergistic delivery of AuNRs was demonstrated.

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  • One-step shell polymerization of inorganic nanoparticles and their applications in SERS/nonlinear optical imaging, drug delivery, and catalysis

    Scientific Reports 4, 5593 (2014)

    Tzu-Ming Liu, Jiashing Yu, Allen Chang, Arthur Chiou, Huihua Kenny Chiang, Yu-Chun Chuang, Cheng-Han Wu, Che-Hao Hsu, Po-An Chen, and Chih-Chia Huang

    Surface functionalized nanoparticles have found their applications in several fields including biophotonics, nanobiomedicine, biosensing, drug delivery, and catalysis. Quite often, the nanoparticle surfaces must be post-coated with organic or inorganic layers during the synthesis before use. This work reports a generally one-pot synthesis method for the preparation of various inorganic-organic core-shell nanostructures (Au@polymer, Ag@polymer, Cu@polymer, Fe3O4@polymer, and TiO2@polymer), which led to new optical, magnetic, and catalytic applications. This green synthesis involved reacting inorganic precursors and poly(styrene-alt-maleic acid). The polystyrene blocks separated from the external aqueous environment acting as a hydrophobic depot for aromatic drugs and thus illustrated the integration of functional nanoobjects for drug delivery. Among these nanocomposites, the Au@polymer nanoparticles with good biocompatibility exhibited shell-dependent signal enhancement in the surface plasmon resonance shift, nonlinear fluorescence, and surface-enhanced Raman scattering properties. These unique optical properties were used for dual-modality imaging on the delivery of the aromatic photosensitizer for photodynamic therapy to HeLa cells.

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  • Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

    Scientific Reports 4, 5574 (2014)

    Yuen Yung Hui, Long-Jyun Su, Oliver Yenjyh Chen, Yit-Tsong Chen, Tzu-Ming Liu, and Huan-Cheng Chang

    Through high density ensembles of negatively charged nitrogen-vacancy centers (NV), nanodiamonds are promising fluorescent biomarkers due to their excellent photostability and biocompatibility.  These NV centers have fluorescence lifetimes of up to 20 ns, which is significantly longer than those of cell and tissue autofluorescence (<10 ns), making it possible to achieve background-free detection in vivo by time gating.  Here we demonstrate   the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells. Combined with a nanosecond intensified charge-coupled device (ICCD), this technique allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness, to detect single FND-labeled HeLa cell in blood flowing through a microfluidic device at a frame rate of 23 Hz, and to locate and trace FND-labeled lung cancer cells in the capillary  of a mouse ear.  It opens up a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

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  • Multiphoton imaging to identify grana, stroma thylakoid, and starch inside an intact leaf

    BMC Plant Biology 14, 175 (2014)

    Mei-Yu Chen, Guan-Yu Zhuo, Kuan-Chieh Chen, Pei-Chun Wu, Tsung-Yuan Hsieh, Tzu-Ming Liu, and Shi-Wei Chu

    Background: Grana and starch are major functional structures for photosynthesis and energy storage of plant, respectively. Both exhibit highly ordered molecular structures and appear as micrometer-sized granules inside chloroplasts. In order to distinguish grana and starch, we used multiphoton microscopy, with simultaneous acquisition of two-photon fluorescence (2PF) and second harmonic generation (SHG) signals. SHG is sensitive to crystallized structures while 2PF selectively reveals the distribution of chlorophyll.
    Result: Three distinct microstructures with different contrasts were observed, i.e. “SHG dominates”, “2PF dominates”, and “SHG collocated with 2PF”. It is known that starch and grana both emit SHG due to their highly crystallized structures, and no autofluorescence is emitted from starch, so the “SHG dominates” contrast should correspond to starch. The contrast of “SHG collocated with 2PF” is assigned to be grana, which exhibit crystallized structure with
    autofluorescent chlorophyll. The “2PF dominates” contrast should correspond to stroma thylakoid, which is a non-packed membrane structure with chrolophyll. The contrast assignment is further supported by fluorescence lifetime measurement.
    Conclusion: We have demonstrated a straightforward and noninvasive method to identify the distribution of grana and starch within an intact leaf. By merging the 2PF and SHG images, grana, starch and stroma thylakoid can be visually distinguished. This approach can be extended to the observation of 3D grana distribution and their dynamics in living plants.

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  • 2013

    In vivo metabolic imaging of insulin with multiphoton fluorescence of human insulin-Au nanodots

    Small 9, 2103-2110 (2013)

    Chien-Liang Liu, Tzu-Ming Liu, Tsung-Yuan Hsieh, Han-Wen Liu, Yu-Shing Chen, Cheng-Kun Tsai, Hsieh-Chih Chen, Jong-Wei Lin, Ron-Bin Hsu, Tzung-Dau Wang, Chien-Cheng Chen, Chi-Kuang Sun, and Pi-Tai Chou

    Functional human insulin–Au nanodots (NDs) are synthesized for the in vivo imaging of insulin metabolism. Benefi ting from its effi cient red to near infrared fluorescence, deep tissue subcellular uptake of insulin–Au NDs can be clearly resolved through a least-invasive harmonic generation and two-photon fl uorescence (TPF) microscope. In vivo investigations on mice ear and ex vivo assays on human fat tissues conclude that cells with rich insulin receptors have higher uptake of administrated insulin. Interestingly, the insulin–Au NDs can even permeate into lipid droplets (LDs) of adipocytes. Using this newly discovered metabolic phenomenon of insulin, it is found that enlarged adipocytes in type II diabetes mice have higher adjacent/LD concentration contrast with small-sized ones in wild type mice. For human clinical samples, the epicardial adipocytes of patients with diabetes and coronary artery disease (CAD) also show elevated adjacent/LD concentration contrast. As a result, human insulin–Au nanodots provide a new approach to explore subcellular insulin metabolism in model animals or patients with metabolic or cardiovascular diseases.

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  • Surface states mediated NIR two-photon fluorescence of iron oxides for nonlinear optical microscopy

    Advanced Functional Materials 23, 2044-2051 (2013)

    Mei-Yi Liao, Cheng-Ham Wu, Ping-Shan Lai, Jiashing Yu, Hong-Ping Lin, Tzu-Ming Liu, and Chih-Chia Huang

    The development of fluorescent iron oxide nanomaterials is highly desired for multimodal molecular imaging. Instead of incorporating fluorescent dyes on the surface of iron oxides, a ligand-assisted synthesis approach is developed to allow near-infrared (NIR) fl uorescence in Fe3O4 nanostructures. Using a trimesic acid (TMA)/citrate-mediated synthesis, fabricated Fe3O4 nanostructures can generate a NIR two-photon florescence (TPF) peak around 700 nm under the excitation by a 1230-nm femtosecond laser. By tailoring the absorption of Fe3O4 nanostructures toward NIR band, the NIR-TPF efficiency can be greatly increased. Through internal etching, surface peeling, and ligand replacement, spectroscopic results validated that such resonantly enhanced NIR-TPF is mediated by surface states with strong NIR-IR absorption. This TPF signal evolution can be generalized to other iron oxide nanomaterials like magnetite nanoparticles and α– Fe2O3 nanoplates. Using the developed fluorescent Fe3O4 nanostructures, it is demonstrated that their TPF and third harmonic generation (THG) contrast in the nonlinear optical microscopy of live cells. It is anticipated that the synthesized NIR photofunctional Fe3O4 will serve as a versatile platform for dual-modality magnetic resonance imaging (MRI) as well as a magnet-guided theragnostic agent.

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  • Virtual optical biopsy of human adipocytes with third harmonic generation microscopy

    Biomedical Optics Express 4, 178-186 (2013)

    Cheng-Kun Tsai, Tzung-Dau Wang, Jong-Wei Lin, Ron-Bin Hsu, Lun-Zhang Guo, San-Tai Chen, and Tzu-Ming Liu

    Using the sectioning capability of third harmonic generation (THG) microscopy, we assessed the morphologic features of human adipocytes directly without fixation and labeling. At the plane of the largest cross-sectional area, both area-equivalent circular diameters (AECD) and perimeters of adipocytes were measured, and their statistical distributions were examined. We found, in patients with no cardiovascular risk factors, the average AECD of epicardial adipocytes were 70–90 μm with 11–17 μm standard deviations. In contrast, for patients with coronary artery disease, amounts of small-sized (AECD <40 μm) epicardial adipocytes were observed and the corresponding standard deviations of AECD were increased to 20–29 μm. Our results indicate that the THG tomography platform can be used to explore the histopathological features of adipocytes in clinical scenarios based on its superior resolution for virtual optical biopsy.

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  • 2012

    Imaging morphodynamics of human blood cells in vivo with video-rate third harmonic generation microscopy

    Biomedical Optics Express 3, 2860-2865 (2012)

    Chien-Kuo Chen and Tzu-Ming Liu

    With a video-rate third harmonic generation (THG) microscopy system, we imaged the micro-circulation beneath the human skin without labeling. Not only the speed of circulation but also the morphohydrodynamics of blood cells can be analyzed. Lacking of nuclei, red blood cells (RBCs) shows typical parachute-like and hollow-core morphology under THG microscopy. Quite different from RBCs, every now and then, round and granule rich blood cells with strong THG contrast appear in circulation. The corresponding volume densities in blood, evaluated from their frequencies of appearance and the velocity of circulation, fall within the physiological range of human white blood cell counts.

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  • Imaging granularity of leukocytes with third harmonic generation microscopy

    Biomedical Optics Express 3, 2234-2243 (2012)

    Cheng-Kun Tsai, Yu-Shing Chen, Pei-Chun Wu, Tsung-Yuan Hsieh, Han-Wen Liu, Chiou-Yueh Yeh, Win-Li Lin, Jean-San Chia, and Tzu-Ming Liu

    Using third harmonic generation (THG) microscopy, we demonstrate that granularity differences of leukocytes can be revealed without a label. Excited by a 1230 nm femtosecond laser, THG signals were generated at a significantly higher level in neutrophils than other mononuclear cells, whereas signals in agranular lymphocytes were one order of magnitude smaller. Interestingly, the characteristic THG features can also be observed in vivo to track the newly recruited leukocytes following lipopolysaccharide (LPS) challenge. These results suggest that label-free THG imaging may provide timely tracking of leukocyte movement without disturbing the normal cellular or physiological status.

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  • Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy

    Biomedical Optics Express 3, 1972-1977 (2012)

    Ke Wang, Tzu-Ming Liu, Juwell Wu, Nicholas G. Horton, Charles P. Lin, and Chris Xu

    We demonstrate a fiber-based, three-color femtosecond source for simultaneous imaging of three fluorescent proteins (FPs) using two-photon fluorescence microscopy (2PM). The three excitation wavelengths at 775 nm, 864 nm and 950 nm, are obtained through second harmonic generation (SHG) of the 1550-nm pump laser and the 1728-nm and 1900-nm solitons generated through soliton self-frequency shift (SSFS) in a large-mode-area (LMA) fiber. These energetic pulses are well matched to the two-photon excitation peaks of red, cyan and yellow fluorescent proteins (TagRFPs, TagCFPs, and TagYFPs) for efficient excitation. We demonstrate simultaneous 2PM of human melanoma cells expressing a “rainbow” combination of these three fluorescent proteins.

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  • 2011

    Study of apoptosis induction using fluorescent and higher harmonic generation microscopy techniques in Acartia tonsa nauplii exposed to chronic concentrations of nickel

    Chemistry and Ecology 27, supplement 2, 97-104 (2011)

    Isabella Buttino, David Pellegrini, Giovanna Romano, Jiang-Shiou Hwang, Tzu-Ming Liu, Davide Sartori, Chi- Kuang Sun, Simona Macchia, and Adrianna Ianora

    In this study, we applied different fluorescent techniques to analyse the induction of apoptosis in the copepod Acartia tonsa nauplii exposed to low concentrations of nickel chloride (NiCl2). Newly hatched and later naupliar stages were exposed to increasing concentrations of NiCl2 (0.016, 0.025 and 0.063 mg·L−1) for 7 days and then stained with annexin V–FITC, a vital fluorescent probe commonly used to visualize apoptotic cells in live samples. Nauplii were also stained with TUNEL, a non-vital fluorescent probe used to detect apoptosis in fixed copepods. Moreover, we used for the first time, two-photon fluorescence (2PF) microscopy and higher harmonic generation microscopy (second SHG and third THG harmonic generation) to study apoptosis induction in A. tonsa nauplii without the use of fluorescent probes. 2PF and THG intensity increased in samples exposed to higher Ni concentrations, with respect to the control, whereas SHG signals were similar in all treated samples and visualized muscles. Future perspectives on the use of these new technologies to reveal apoptosis are discussed.

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  • Observation of spontaneous polarization misalignments in periodically poled crystals using second-harmonic generation microscopy

    Optics Express 19, 11106-11113 (2011)

    Yu-Yi Tzeng, Zong-Yan Zhuo, Ming-Yin Lee, Chien-Sheng Liao, Pei-Chun Wu, Chin-Jie Huang, Ming-Che Chan, Tzu-Ming Liu, Yen-Yin Lin, and Shi-Wei Chu

    Periodically poled crystal (PPC) is a key component for nonlinear optical applications. Its poling quality relies largely on successful domain inversion and the alignment of spontaneous polarization (SP) vectors in each domain. Here we report the unexpected observation of bulk second harmonic generation (SHG) in PPC when excitation propagating along its optical axis. Based on its tensorial nature, SHG is highly sensitive to the orientation of SP, and therefore the misalignment of SP in each domain of PPC can be revealed noninvasively by SHG microscopy. This nonlinear imaging modality provides optical sectioning capability with 3D sub-micrometer resolution, so it will be useful for in situ investigation of poling quality in PPC.

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  • Graphite-shelled Si nanoparticles and their Au/Si heterodimers: Preparation, photoluminescence, and second harmonic generation

    Journal of Physical Chemistry C 115, 9952-9960 (2011)

    Chih-Chia Huang, Kuei-Yi Chuang, Chin-Jie Huang, Tzu-Ming Liu, and Chen-Sheng Yeh

    New structured graphite-shelled Si nanocrystals were prepared by laser ablation in various solvents including H2O, ethanol, 2-propanol, hexane, octane, octadecene, trioctylamine, toluene, trioctylamine/toluene, and oleylamine/toluene. With the aromatic debris, containing hexagonal carbon rings, from organic media seems to facilitate graphitization process to build up graphitic carbon layers on Si. The luminescent feature was strongly related to the presence of graphite shells. Excited by infrared emtosecond lasers, the graphiteshelled Si nanocrystals showed extraordinarily strong second harmonic generation (SHG). Both fluorescence and SHG signals were growing with the aging days. The single photonexcited fluorescence and quantum yield were increased from as-prepared graphite-shelled Si quantum dots (QDs) (8%) to 30-day aged samples (13.0%). Because of oxidation upon aging, it is suggested that the possible increase of the SiO bonds on Si surface could have diminished nonradiative centers resulting in enhancing fluorescence. The SHG signal was also up to 32 times higher than that of as-prepared graphite-shelled Si QDs after the aging process. Such an oxidative aging process likely created strain at the Si/SiO2 interface, breaking the central symmetry, and allowed the SHG. Additionally, the poling of SiO2 on the surface of graphiteshelled Si QDs and the transition states of quantum-confined excitons have been addressed for the SHG observation as well. The enhancements of fluorescence and SHG signals have opened an avenue for the applications of graphite-shelled Si QDs as cell biolabeling agents and contrast agents in deep tissue microscopy and tomography. The blue emission of graphite-shelled Si QDs integrated with Rhodamine 610 dye has allowed us to tune luminous spectra giving white light luminescence. The first synthesis of the Au/Si heterodimer nanostructures were formed through replacement reaction between graphite-shelled Si nanocrystals and tetraoctylammonium-Au3+ complexes in organic solvent.

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  • Apoptosis to predict copepod mortality: state of the art and future perspectives

    Hydrobiologia 666, 257-264

    I. Buttino, J.-S. Hwang, C.-K. Sun, C.-T. Hsieh, T.-M. Liu, D. Pellegrini, A. Ianora, D. Sartori, G. Romano, S.-H. Cheng, A. Miralto

    Apoptosis or programmed cell death is the result of complex biochemical and genetic signals triggered by normal physiological events or by exposure to toxic compounds. Apoptosis can be revealed early in cells and tissues due to morphological and biochemical alterations of the cell. Here, we review recent progress in the study of the induction and detection of apoptosis in zooplankton copepods after their exposure to different apoptotic-inducer compounds, and we propose methods to predict copepod mortality even when animals appear normal. We also discuss the use of higher harmonic generation microscopy and multiphoton fluorescence microscopy techniques as possible tools to visualize apoptosis in vivo copepods without using any fluorescent probes.

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  • Roles of dislocation density to the scattering of nano-acoustic waves in GaN

    Chinese Journal of Physics 49, 171-175 (2011)

    Tzu-Ming Liu, Shih-Ze Sun, Chieh-Feng Chang, Guan-Ting Chen, Chang-Chi Pan, Jen-Inn Chyi, and Chi-Kuang Sun

    We have investigated how the density of threading dislocations in GaN affects the decay of the intensity of nano-acoustic waves. We carried out measurements using a reflection-type femtosecond pump probe, and thus, we determined the local dislocation density from the lifetime of nano-acoustic waves. We found that for a dislocation density of 108 cm−2, defect scattering will surmount other scattering mechanisms and dominate the attenuation of 100GHz acoustic phonons.

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  • 2010

    A sub-100fs self-starting Cr:forsterite laser generating 1.4W output power

    Optics Express 18, 24085- 24091 (2010)

    Shih-Hsuan Chia, Tzu-Ming Liu, Anatoly A. Ivanov, Andrey B. Fedotov, Aleksey M. Zheltikov, Ming-Rung Tsai, Ming-Che Chan, Che-Hang Yu, and Chi-Kuang Sun

    Without cavity dumping or external amplification, we report a femtosecond Cr:forsterite laser with a 1.4W output power and 2W in continuous wave (CW) operated with a crystal temperature of 267K. In the femtosecond regime, the oscillator generates Kerr-lens-mode-locked 84fs pulses with a repetition rate of 85MHz, corresponding to a high 16.5nJ pulse energy directly from a single Cr:forsterite resonator. This intense femtosecond Cr:forsterite laser is ideal to pump varieties of high power fiber light sources and could be thus ideal for many biological and spectroscopy applications.

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  • Miniaturized video-rate epi-third-harmonic-generation fiber-microscope

    Optics Express 18, 17382-17391 (2010)

    Shih-Hsuan Chia, Che-Hang Yu, Chih-Han Lin, Nai-Chia Cheng, Tzu-Ming Liu, Ming-Che Chan, I-Hsiu Chen, and Chi-Kuang Sun

    With a micro-electro-mechanical system (MEMS) mirror, we successfully developed a miniaturized epi-third-harmonic-generation (epi-THG) fiber-microscope with a video frame rate (31Hz), which was designed for in vivo optical biopsy of human skin. With a large-mode-area (LMA) photonic crystal fiber (PCF) and a regular microscopic objective, the nonlinear distortion of the ultrafast pulses delivery could be much reduced while still achieving a 0.4μm lateral resolution for epi-THG signals. In vivo real time virtual biopsy of the Asian skin with a video rate (31Hz) and a sub-micron resolution was obtained. The result indicates that this miniaturized system was compact enough for the least invasive hand-held clinical use.

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  • Quantitative and qualitative investigation into the impact of focused ultrasound with microbubbles on the triggered release of nanoparticles from vasculature in mouse tumors

    Journal of Controlled Release 146, 291-298 (2010)

    Chung-Yin Lin, Tzu-Ming Liu, Chao-Yu Chen, Yen-Lin Huang, Wei-Kai Huang, Chi-Kuang Sun, Fu-Hsiung Chang, Win-Li Lin

    Ultrasound-mediated microbubble destruction may enhance the release of nanoparticles from vasculature to tumor tissues. In this study, we used four different sizes of lipid-coated CdSe quantum dot (LQD) nanoparticles ranging from 30 to 180 nm, 1.0-MHz pulsed focused ultrasound (FUS) with a peak acoustic pressure of 1.2-MPa, and an ultrasound contrast agent (UCA; SonoVue®) at a dose of 30 μL/kg to investigate any enhancement of targeted delivery. Tumor-bearing male Balb/c mice were first injected with UCA intravenously, were then sonicated at the tumors with FUS, and were finally injected with 50 μL of the LQD solution after the sonication. The mice were sacrificed about 24 h after the sonication, and then we quantitatively and qualitatively evaluated the deposition of LQDs in the tumors by using graphite furnace atomic absorption spectrometry (GF-AAS), photoluminescence spectrometry (PL), and harmonic generation microscopy (HGM). Further, immunoblotting analysis served to identify the biochemical markers reflecting the vascular rupture. The experimental results show that the amount of LQDs deposited in tumor tissues was greater in cases of FUS/UCA application, especially for smaller LQDs, being 4.47, 2.27, 0.99, and 0.82 (μg Cd)/ (g tumor) for 30, 80, 130, and 180 nm of LQDs, respectively; compared to 1.12, 0.75, 0.26, and 0.34 (μg Cd)/ (g tumor) in absence of FUS/UCA. The immunoblotting analysis further indicates that FUS-induced UCA oscillation/destruction results in rupture areas in blood vessels increasing the vascular permeability and thus justifying for the higher quantity of nanoparticles deposited in tumors.

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  • 2009

    Effects of hydration levels on the bandwidth of microwave resonant absorption induced by confined acoustic vibrations

    Applied Physics Letters 95, 173702 (2009)

    Tzu-Ming Liu, Hung-Pin Chen, Shih-Chia Yeh, Chih-Yu Wu, Chung-Hsiung Wang, Tang-Nian Luo, Yi-Jan Chen, Shen-Iuan Liu, and Chi-Kuang Sun

    We found the hydration levels on the capsid surface of viruses can affect the bandwidth of microwave resonant absorption (MRA) induced by the confined acoustic vibrations (CAV). By decreasing the pH value of solution down to 5.2 or inactivating the capsid proteins, we enhanced the surface hydrophilicity and increased the magnitude of surface potentials. Both of these surface manipulations raised the surface affinity to water molecules and narrowed the bandwidths of CAV-induced MRA. Our results validate the viscoelastic transition of hydration shells.

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  • FoxD5 mediates anterior-posterior polarity through upstream modulator Fgf signaling during zebrafish somitogenesis

    Developmental Biology 336, 232-245 (2009)

    Hung-Chieh Lee, Wei-An Tseng, Fang-Yi Lo, Tzu-Ming Liu, Huai-Jen Tsai

    The transcription factor FoxD5 is expressed in the paraxial mesoderm of zebrafish. However, the roles of FoxD5 in anterior pre-somitic mesoderm (PSM) during somitogenesis are unknown. We knocked down FoxD5 in embryos, which resulted in defects of the newly formed somites, including loss of the striped patterns of anterior–posterior polarity genes deltaC, notch2, notch3 and EphB2a, as well as the absence of mespa expression in S-I. Also, the expression of mespb exhibited a ‘salt and pepper’ pattern, indicating that FoxD5 is necessary for somite patterning in anterior PSM. Embryos were treated with SU5402, an Fgf receptor (FGFR) inhibitor, resulting in reduction of FoxD5 expression. This finding was consistent with results obtained from Tg(hsp70l:dnfgfr1-EGFP)pd1 embryos, whose dominant-negative form of FGFR1 was produced by heat-induction. Loss of FoxD5 expression was observed in the embryos injected with fgf3-/fgf8-doublemorpholinos (MOs). Excessive FoxD5 mRNA could rescue the defective expression levels of mespa and mespb in fgf3-/fgf8-double morphants, suggesting that Fgf signaling acts as an upstream modulator of FoxD5 during somitogenesis. We concluded that FoxD5 is required for maintaining anterior–posterior polarity within a somite and that the striped pattern of FoxD5 in anterior PSM is mainly regulated by Fgf. An Fgf-FoxD5-Mesps signaling network is therefore proposed.

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  • Efficient near-IR hyperthermia and intense nonlinear optical imaging contrast on the gold nanorod-in-shell nanostructures

    Journal of the American Chemical Society 131, 14186-14187 (2009)

    Kuo-Wei Hu, Tzu-Ming Liu, Kuei-Yi Chung, Keng-Shiang Huang, Chien-Tai Hsieh, Chi-Kuang Sun, and Chen-Sheng Yeh

    New gold nanorod (Au NR)-in-shell nanostructures were developed to be more efficacious than Au NRs in near-IR (NIR) hyperthermia and nonlinear optical imaging contrast. Au NR-in-shell nanostructures are composed of an intact Au NR in a Au/Ag nanoshell. These nanostructures have a broad, intense absorption band that extends from 400 nm to 900 nm in the NIR. They are more efficient and efficacious than Au NRs with respect to in vitro hypothermia performance. Au NR-in-shell-labeled cancer cells were destroyed using continuous-wave NIR radiation with 50% less laser power than needed for Au NRs. Noticeably, the area of the destroyed cells was significantly larger than the size of the laser irradiation beam; in contrast, the destroyed area was usually restricted to the size of the laser beam spot when Au NRs were used. With their extraordinarily broad and strong surface plasmon resonance band, Au NR-in-shell nanostructures efficiently augmented several multiphoton nonlinear processes as well. The multiphoton emission spectrum covered almost the entire visible wavelength. The yield of the multiphoton signals of Au NR-in-shell nanostructures was on average 55 times larger than that of Au NRs. In vitro images of cancer cells targeted by Au NR-in-shell nanostructures revealed a stronger multiphoton contrast than those targeted by Au NRs.

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  • Selective imaging in second-harmonic-generation microscopy with anisotropic radiation

    Journal of Biomedical Optics 14, 010504 (2009)

    Shi-Wei Chu, Shih-Peng Tai, Tzu-Ming Liu, Chi-Kuang Sun, and Chi-Hung Lin

    As a novel modality of optical microscopy, second-harmonic generation SHG provides attractive features including intrinsic optical sectioning, noninvasiveness, high specificity, and high penetrability. For a biomedical application, the epicollection of backward propagating SHG is necessary. But due to phase-matching constraint, SHG from thick tissues is preferentially forward propagation. Myosin and collagen are two of the most abundant fibrous proteins in vertebrates, and both exhibit a strong second-harmonic response. We find that the radiation patterns of myosin-based muscle fibers and collagen fibrils are distinct due to coherence effects. Based on these asymmetric radiation patterns, we demonstrate selective imaging between intertwining muscle fibers and type I collagen fibrils with forward and backward SHG modalities, respectively. Thick muscle fibers dominate the forward signal, while collagen fibril distribution is preferentially resolved in the backward channel without strong interference from muscle. Moreover, we find that wellformed collagen fibrils are highlighted by forward SHG, while loosely arranged collagen matrix is outlined by backward signal.

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  • Microwave resonant absorption of viruses through dipolar coupling with confined acoustic vibrations

    Applied Physics Letters 94, 043902 (2009)

    Tzu-Ming Liu, Hung-Ping Chen, Li-Tzu Wang, Jen-Ren Wang, Tang-Nian Luo, Yi-Jan Chen, Shen-Iuan Liu, and Chi-Kuang Sun

    In this letter, with an electric double layer on the surface of spherical viruses, we confirm that one of the microwave resonant absorption (MRA) mechanisms of viruses is through dipolar coupling with confined acoustic vibrations. By treating spherical virions as free homogeneous nanoparticles, we found that the MRA frequencies agree well with that of l=1 dipolar modes predicted by the elastic continuum theory. The magnitude of MRA was also found to change with the amount of adsorbed charges on the surface of virions. Our results provide a method to observe three-dimensionally confined acoustic vibrations in biological systems.

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  • 2008

    In vivo long term continuous observation of gene expression in zebrafish embryos’ nerve system by using harmonic generation microscopy and morphant technology

    Journal of Biomedical Optics 13, 064041

    Cho-Shuen Hsieh, Ching-Yi Ko, Szu-Yu Chen, Tzu-Ming Liu, Jian-Shiung Wu, Chin-Hwa Hu, and Chi-Kuang Sun

    Gene expression plays an important role in embryo development and organ function. Previous studies have shown that harmonic generation microscopy HGM can be used as a fluorescence signal-independent, minimally invasive method with a subcellular 3-D resolution and a penetration depth in the order of millimeters for long-term continuous imaging of vertebrate embryos. We show that it is ideal to combine in vivo HGM with the morphant technology for minimally invasive, long-term continuous observation of gene expression in the nervous system of vertebrate embryos. Since second- and third-harmonic generations SHG, THG are virtual-state-transitionbased systems that depend only on the structure of the organisms, they are not temporally limited by the expression of the fluorescence proteins. We successfully identified the expression of the zarnt2a and the hif-1α, , and  genes in the nervous system of zebrafish embryos with specific knockdown genes by microscopically observing the embryos from the early stages of embryogenesis. The results from a combination of the two different modalities, i.e., SHG microscopy and THG microscopy, successfully revealed the weak cell adhesion, cell apoptosis, nerve formation reduction, and neural tube distortion in the morphant zebrafish embryos.

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  • Miniaturized multiphoton microscope with a 24Hz frame-rate

    Optics Express 16, 10501-10506

    Tzu-Ming Liu, Ming-Che Chan, I-Hsiu Chen, Shih-Hsuan Chia, and Chi-Kuang Sun

    With miniaturized tube lenses and a micro-electro-mechanical system (MEMS) mirror, we constructed a miniaturized multiphoton microscope system. Through a two-dimensional asynchronous scanning of the MEMS mirror, 24Hz frame rate can be realized. With a high numerical aperture objective, sub-micron resolution can also be achieved at the same time.

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  • 1.2~2.2-μm tunable raman soliton source based on a Cr:forsterite-laser and a photonic-crystal fiber

    IEEE Photonics Technology Letters 20, 900-902

    Ming-Che Chan, Shih-Hsuan Chia, Tzu-Ming Liu, Tsung-Han Tsai, Min-Chen Ho, Anatoly A. Ivanov, Aleksei M. Zheltikov, Jiun-Yi Liu, Hsiang-Lin Liu, and Chi-Kuang Sun

    A 1.2- to 2.2-μm tunable femtosecond light source based on the soliton-self-frequency-shift effect of high-power Cr : forsterite laser pulses propagating inside a highly nonlinear photonic crystal fiber is reported. The demonstrated soliton self-frequency shift is higher than 42% of the pump laser frequency, corresponding to a record 910-nm wavelength tuning range. Due to the advantages of simplicity, easy tunability, high-temperature stability, and low cost of this new femtosecond light source, it accordingly, could be widely applicable for many applications.

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  • Imaging polyhedral inclusion bodies of nuclear polyhedrosis viruses with second harmonic generation microscopy

    Optics Express 16, 5602-5608

    Tzu-Ming Liu, Yen-Wei Lee, Chieh-Feng Chang, Shih-Chia Yeh, Chung-Hsiung Wang, Shi-Wei Chu, and Chi-Kuang Sun

    We studied the polarization anisotropy of second harmonic generation (SHG) in polyhedral inclusion bodies (PIBs) of nuclear polyhedrosis viruses (NPV). Due to a body-centered-cubic arrangement of polyhedrin trimers, a characteristic SHG polarization property with a mixture of I23 and I3 symmetry was measured from PIBs. With this characteristic SHG anisotropy, it provides an intrinsic nonlinear signal for virus infection studies in living cells. With multimodal harmonic generation microscopy, we also demonstrated 3D imaging on PIBs of NPV in living cells. The distribution and the number of PIBs in intact infected cells can be revealed without the help of fluorescent labeling.

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  • Resonance-enhanced dipolar interaction between terahertz photons and confined acoustic phonons in nanocrystals

    Applied Physics Letters 92, 093122

    Tzu-Ming Liu, Ja-Yu Lu, Hung-Ping Chen, Chung-Chiu Kuo, Meng-Ju Yang, Chih-Wei Lai, Pi-Tai Chou, Ming-Hao Chang, Hsiang-Lin Liu, Yu-Tai Li, Ci-Ling Pan, Shih-Hung Lin, Chieh-Hsiung Kuan, and Chi-Kuang Sun

    Taking advantage of the specific core-shell charge separation structure in the CdSe/CdTe coreshell type-II nanocrystals, we experimentally observed and verified the existence of the resonance-enhanced dipolar interaction between terahertz photons and their corresponding confined acoustic phonons. From the measured terahertz transmission spectra, we found that the photon frequency of the terahertz resonant absorption is inversely proportional to the diameter D of the nanocrystals and agrees with that of dipolar active l=1, n=0 confined acoustic modes. The corresponding absorption cross section shows a D4 dependence, supporting a charged simple-harmonic-oscillator model. These facts verify the occurrence of dipolar interaction between terahertz photons and confined terahertz acoustic phonons.

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  • Piezoelectricity-induced terahertz photon absorption by confined acoustic phonons in wurtzite CdSe nanocrystals

    Physical Review B 77, 085428

    Tzu-Ming Liu, Meng-Ju Yang, Chih-Wei Lai, Pi-Tai Chou, Ming-Hao Chang, Hsiang-Lin Liu, and Chi-Kuang Sun

    We report here the observation of piezoelectricity-induced terahertz photon absorption related to confined acoustic phonons in nanoparticles. Terahertz photon inactive l=0 breathing modes become terahertz photon active in wurtzite CdSe nanocrystals due to the piezoelectric coupling. Obvious absorption peaks related to l=0 modes appeared in the terahertz absorption spectra and the frequency of the peaks were inversely proportional to the size of the CdSe nanocrystals. This piezoelectrically coupled electric vibration provides a mechanism for low-dimensional systems to convert a terahertz photon into a phonon of the same frequency.

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  • 2007

    Molecular imaging of cancer cells using plamon-resonant-enhanced third-harmonic-generation in silver nanoparticles

    Advanced Materials 19. 4520-4523

    Shih-Peng Tai, Yana Wu, Dar-Bin Shieh, Lung-Jin Chen, Kuan-Jiuh Lin, Che-Hang Yu, Shi-Wei Chu, Chien-Huei Chang, Xuan-Yu Shi, Yu-Chieh Wen, Kung-Hsuan Lin, Tzu-Ming Liu, and Chi-Kuang Sun

    We demonstrate molecule-specific third-harmonic-generation (THG) microscopy by using silver nanoparticles as THG contrast agents. Through matching surface plasmon wavelength to THG wavelength, strong contrast can be provided by silver nanoparticles under THG microscopy. By conjugating anti-Her2 antibodies with silver nanoparticles, the expression of the Her2/neu oncogene in the cancer cell membranes is successfully imaged under THG modality for the first time.

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  • Narrow-band detection of propagating coherent acoustic phonons in piezoelectric InGaN/GaN multiple-quantum wells

    Applied Physics Letters 91, 133101

    Cheng-Ying Chen, Yu-Chieh Wen, Hung-Ping Chen, Tzu-Ming Liu, Chang-Chi Pan, Jen-Inn Chyi, and Chi-Kuang Sun

    The authors demonstrated that the piezoelectric superlattice, can serve as narrow-band detectors for propagating coherent longitudinal acoustic phonons at multiple frequencies corresponding to the spatial frequency of the superlattice and its higher harmonics, and its detection bandwidth is determined by the total structure width. By optically exciting a broadband propagating longitudinal acoustic pulse from a thin Ni film, the authors studied the acoustic spectral sensitivity function of a ten-period In0.12Ga0.88N/GaN multiple quantum well. Because the barriers 19 nm and wells 3.6 nm are of different widths, the second detection band, corresponding to the second harmonic of the fundamental frequency, can be resolved.

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  • Anharmonic decay of sub-THz coherent acoustic phonons in GaN

    Applied Physics Letters 90, 041902

    Tzu-Ming Liu, Shih-Ze Sun, Chieh-Feng Chang, Chang-Chi Pan, Guan-Ting Chen, Jen-Inn Chyi, Vitalyi Gusev, and Chi-Kuang Sun

    This letter examines the anharmonic decay in GaN of subthermal (hω<< kBT) coherent longitudinal acoustic (LA) phonons with a frequency in the subterahertz range. In a collisionless regime (ωτ>1), the anharmonic decay rate of subterahertz coherent LA phonons in GaN shows square dependence on the phonon frequency and cubic dependence on the crystal temperature. As confirmed by the authors experiments, this behavior agrees with a model based on Herring scattering, indicating its importance for the estimation of acoustic attenuation in the sub-terahertz range.

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  • 2006

    Measuring plasmon-resonance enhanced third-harmonic χ(3) of Ag nanoparticles

    Applied Physics Letters 89, 043122

    Tzu-Ming Liu, Shih-Peng Tai, Che-Hang Yu, Yu-Chieh Wen, Shi-Wei Chu, Lung-Jin Chen, Muppa Ramakrishna Prasad, Kuan-Jiuh Lin, and Chi-Kuang Sun

    By coinciding the plasmon frequency with the third-harmonic frequency of the excitation light, the authors determined the plasmon-resonance enhanced optical third-harmonic-generation THG susceptibility of a polyvinylpyrrolidone-coated Ag nanoparticle with a 5–7 nm diameter. With dispersed Ag nanoparticles on a quartz surface and through measuring the frequency dependent THG intensities, interface THG showed evident enhancement when the third harmonic of excitation matched the Ag-nanoparticle’s plasmon-resonant frequency. According to the effective medium theory and by analyzing the interface THG under focused Gaussian beams, the ensemble-averaged χ(3) of a Ag nanoparticle can be estimated to be on the order of  2×10-11 esu.

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  • 2005

    Compact fiber-delivered Cr:forsterite laser for nonlinear light microscopy

    Journal of Biomedical Optics 10, 054006

    Ming-Che Chan, Tzu-Ming Liu, Shih-Peng Tai, and Chi-Kuang Sun

    We demonstrate a compact and self-starting fiber-delivered femtosecond Cr:forsterite laser for nonlinear light microscopy. A semiconductor saturable absorber mirror provides the self-starting mechanism and maintains long-term stability in the laser cavity. Four double-chirped mirrors are employed to reduce the size of the cavity and to compensate for group velocity dispersion. Delivered by a large-mode-area photonic crystal fiber, the generated laser pulses can be compressed down to be with a nearly transform-limited pulse width with 2.2-nJ fiber-output pulse energy. Based on this fiber delivered Cr:forsterite laser source, a compact and reliable two-photon fluorescence microscopy system can thus be realized.

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  • Optical piezoelectric transducer for nano-ultrasonics

    IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control 52, 1404-1414

    Kung-Hsuan Lin, Gia-Wei Chern, Cheng-Ta Yu, Tzu-Ming Liu, Chang-Chi Pan, Guan-Ting Chen, Jen-Inn Chyi, Sheng-Wen Huang, Pai-Chi Li, and Chi-Kuang Sun

    Piezoelectric semiconductor strained layers can be treated as piezoelectric transducers to generate nanometer-wavelength and THz-frequency acoustic waves. The mechanism of nano-acoustic wave (NAW) generation in strained piezoelectric layers, induced by femtosecond optical pulses, can be modeled by a macroscopic elastic continuum theory. The optical absorption change of the strained layers modulated by NAW through quantum-confined Franz-Keldysh (QCFK) effects allows optical detection of the propagating NAW. Based on these piezoelectric-based optical principles, we have designed an optical piezoelectric transducer (OPT) to generate NAW. The optically generated NAW is then applied to one-dimensional (1-D) ultrasonic scan for thickness measurement, which is the first step toward multidimensional nano-ultrasonic imaging. By launching a NAW pulse and resolving the returned acoustic echo signal with femtosecond optical pulses, the thickness of the studied layer can be measured with < 1 nm resolution. This nano-structured OPT technique will provide the key toward the realization of nano-ultrasonics, which is analogous to the typical ultrasonic techniques but in a nanometer scale.

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  • Multiplying the repetition rate of passive mode-locked femtosecond lasers by an intracavity flat surface with low reflectivity

    Optics Letters 30, 439-441

    Tzu-Ming Liu, Franz X. Kärtner, James G. Fujimoto, and Chi-Kuang Sun

    By inserting a low-reflectivity flat surface inside the oscillator cavity, we demonstrate a flexible and phase insensitive method for multiplying the repetition rate of a femtosecond passive mode-locked solid-state laser. Without mode matching and feedback control, we successfully multiplied the repetition rate of a passively mode-locked Cr:forsterite laser from 124 MHz to 1.24 GHz. High-repetition-rate femtosecond optical pulses with average power of .100 mW can be obtained with the demonstrated method.

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  • 2-GHz repetition-rate femtosecond blue source

    Applied Physics Letters 86, 061112

    Tzu-Ming Liu, Cheng-Ta Yu, and Chi-Kuang Sun

    We report a 2 GHz repetition-rate, all-solid-state femtosecond blue source. Pumped by a 740 mW femtosecond Ti:sapphire laser with the same repetition rate, 150 mW femtosecond pulses at 409 nm can be efficiently generated from the external resonant cavity with a lithium triborate crystal.

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  • 2004

    Higher harmonic generation microscopy for developmental biology

    Journal of Structural Biology 147, 19-30

    Chi-Kuang Sun, Shi-Wei Chu, Szu-Yu Chen, Tsung-Han Tsai, Tzu-Ming Liu, Chung-Yung Lin, and Huai-Jen Tsai

    Optical higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to its interacted matters due to an energy-conservation characteristic, providing the ‘‘noninvasiveness’’ nature desirable for biological studies. Combined with its nonlinearity, higher harmonic generation microscopy provides excellent three-dimensional (3D) sectioning capability, offering new insights into the studies of embryonic morphological changes and complex developmental processes. By choosing a laser working in the biological penetration window, here we present a noninvasive in vivo light microscopy with sub-micron 3D resolution and millimeter penetration, utilizing endogenous higher harmonic generation signals in live specimens. Noninvasive imaging was performed in live zebrafish (Danio rerio) embryos. The complex developmental processes within >1-mm-thick zebrafish embryos can be observed in vivo without any treatment. No optical damage was found even with high illumination after long-term observations and the examined embryos all developed normally at least to the larval stage. The excellent 3D resolution of the demonstrated technology allows us to capture the subtle developmental information on the cellular or subcellular levels occurring deep inside the live embryos and larvae. This technique can not only provide in vivo observation of the cytoarchitecture dynamics during embryogenesis with submicron resolution and millimeter penetration depth, but would also make strong impact in developmental and structural biology studies.

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  • Nonlinear behaviors of low-temperature-grown GaAs-based photodetectors around 1.3μm telecommunication wavelength

    IEEE Photonics Technology Letters 16, 242-244

    Jin-Wei Shi, Yen-Hung Chen, Kian-Giap Gan, Yi-Jen Chiu, John E. Bowers, Ming-Chun Tien, Tzu-Ming Liu, and Chi-Kuang Sun

    We observed distinct bandwidth degradation behaviors in low-temperature-grown GaAs (LTG-GaAs)-based traveling-wave photodetectors (PDs) under 1300-nm telecommunication wavelength operation. Compared with the bandwidth degradation behaviors of different excitation wavelengths ( 800 and 1550 nm) in LTG-GaAs-based PDs, the saturation behaviors at the studied wavelength are more serious and can be attributed to “hot electron” effect of photogenerated carriers. The disclosed unique material properties of LTG-GaAs are important for its applications in ultrafast optoelectronics and understanding its carrier dynamics with the defect states.

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  • 2003

    In vivo developmental biology study using noninvasive multi-harmonic generation microscopy

    Optics Express 11, 3093-3099

    Shi-Wei Chu, Szu-Yu Chen, Tsung-Han Tsai, Tzu-Ming Liu, Cheng-Yung Lin, Huai-Jen Tsai, and Chi-Kuang Sun

    Morphological changes and complex developmental processes inside vertebrate embryos are difficult to observe noninvasively with millimeter-penetration and sub-micrometer-resolution at the same time. By using higher harmonic generation, including second and third harmonics, as the microscopic contrast mechanism, optical noninvasiveness can be achieved due to the virtual-level-transition characteristic. The intrinsic nonlinearity of harmonic generations provides optical sectioning capability while the selected 1230-nm near-infrared light source provides the deeppenetration ability. The complicated development within a ~1.5-mm thick zebrafish (Danio rerio) embryo from initial cell proliferation, gastrulation, to tissue formation can all be observed clearly in vivo without any treatment on the live specimen.

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  • Real-time second-harmonic-generation microscopy based on a 2-GHz repetition rate Ti: sapphire laser

    Optics Express 11, 933-938

    Shi-Wei Chu, Tzu-Ming Liu, and Chi-Kuang Sun

    The problem of weak harmonic generation signal intensity limited by photodamage probability in optical microscopy and spectroscopy could be resolved by increasing the repetition rate of the excitation light source. Here we demonstrate the first photomultiplier-based real-time second-harmonic-generation microscopy taking advantage of the strongly enhanced nonlinear signal from a high-repetition-rate Ti:sapphire laser. We also demonstrate that the photodamage possibility in common biological tissues can be efficiently reduced with this high repetition rate laser at a much higher average power level compared to the commonly used ~80-MHz repetition rate lasers.

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  • 2002

    Nonlinear bio-photonic crystal effects revealed with multi-modal nonlinear microscopy

    Journal of Microscopy 208, 190-200

    S.-W. Chu, I.-H. Chen, T.-M. Liu, C.-K. Sun, S.-P. Lee, B.-L. Lin, P.-C. Cheng, M.-X. Kuo, D. - J. Lin, and H.-L. Liu

    Highly optically active nonlinear bio-photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through secondharmonic generation (SHG) interactions, behaving similarly to man-made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third-harmonic generation, and multiphoton-induced fluorescence. With no energy release during harmonic generation processes, the nonlinear-photonic-crystal-like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.

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  • Characterization of ultrashort optical pulses with third-harmonic- generation based triple autocorrelation

    IEEE Journal of Quantum Electronics 38, 1529-1535

    Tzu-Ming Liu, Yin-Chieh Huang, Gia-Wei Chern, Kung-Hsuan Lin, Yu-Chueh Hung, Chih-Jie Lee, and Chi-Kuang Sun

    We present a method to obtain complete information of femtosecond pulses. By measuring triple-optical autocorrelation
    directly with third-harmonic generation, without spectral information, a temporal pulse shape can be obtained by analytical calculation without direction-of-time ambiguity. Combining the resulting optical pulse shape with its corresponding optical spectrum, the exact phase and color variations in time can all be recovered with a Gerchberg–Saxton algorithm through an iterative calculation with an O(n) complexity.

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  • Triple-optical autocorrelation for direct optical pulse-shape measurement

    Applied Physics Letters 81, 1402-1404

    Tzu-Ming Liu, Yin-Chieh Huang, Gia-Wei Chern, Kung-Hsuan Lin, Chih-Jie Lee, Yu-Chueh Hung, and Chi-Kuang Sun

    Triple optical autocorrelation of femtosecond optical pulses was realized simply with third-harmonic-generation technique. This optical technique provides complete knowledge of transient pulse intensity variation directly in time domain. Only analytic calculation is needed to obtain the pulse-shape from data without direction-of-time ambiguity. Combined with a spectral measurement and the Gerchberg–Saxton algorithm, except for pulses with complete temporal and spectral symmetry that will cause a twofold ambiguity, exact phase variation in time can also be retrieved through an iterative calculation with an O(n) complexity.

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  • Locked multichannel generation and management by use of a Fabry–Perot etalon in a mode-locked Cr:forsterite laser cavity

    IEEE Journal of Quantum Electronics 38, 458-463

    Tzu-Ming Liu, Hsu-Hao Chang, Shi-Wei Chu, and Chi-Kuang Sun

    We demonstrate a method to manage the locked multichannel output from a mode-locked Cr:forsterite laser cavity, which was achieved by inserting a Fabry–Perot etalon into the oscillator. It is found that the thickness of the etalon determines the channel spacing and its surface reflectivity affects channel linewidth and channel pulsewidth. Following Fabry–Perot theory, we can use these facts to control the characteristics of these locked multichannels.

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  • 2001

    Multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser

    Optics Letters 26, 1909-1911

    Shi-Wei Chu, I-Hsiu Chen, Tzu-Ming Liu, Ping Chin Chen, and Chi-Kuang Sun, Bai-Ling Lin

    We demonstrate a novel multimodal nonlinear spectral microscopy based on a femtosecond Cr:forsterite laser at 1230 nm. By acquiring the whole nonlinear spectrum in the visible and near-NIR region, this novel technique allows a combination of different imaging modalities, including second-harmonic generation, third-harmonic generation, and multiple-photon f luorescence. Combined with the selected excitation wavelength, which is located in the IR transparency window, this microscopic technique can provide high penetration depth with reduced damage and is ideal for studying living cells.

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  • Multi-photon confocal microscopy using a Femtosecond Cr:forsterite laser

    Scanning 23, 249-254

    Tzu-Ming Liu, Shi-Wei Chu, Chi-Kuang Sun, Bai-Ling Lin, Ping-Chin Cheng, and I. Johnson

    With its output wavelength covering the infrared penetrating window of most biological tissues at 1200–1250 nm, the femtosecond Cr:forsterite laser shows high potential to serve as an excellent excitation source for the multiphoton fluorescence microscope. Its high output power, short optical pulse width, high stability, and low dispersion in fibers make it a perfect replacement for the currently widely used Ti:sapphire laser. In this paper, we study the capability of using a femtosecond Cr:forsterite laser in multiphoton scanning microscopy. We have performed the multiphoton excited photoluminescence spectrum measurement on several commonly used bioprobes using the 1230 nm femtosecond pulses from a Cr:forsterite laser. Efficient fluorescence can be easily observed in these bioprobes through two-photon or threephoton excitation processes. These results will assist in the selection of dichroic beam splitter and band pass filters in a multiphoton microscopic system. We have also performed the autofluorescence spectrum measurement from chlorophylls in live leaves of the plant Arabidopsis thaliana excited by 1230 nm femtosecond pulses from the Cr:forsterite laser. Bright luminescence from chlorophyll, centered at 673 and 728 nm, respectively, can be easily observed. Taking advantage of the bright two-photon photoluminescence from chlorophyll, we demonstrated the two-photon scanning paradermal and cross-sectional images of palisade mesophyll cells in live leaves of Arabidopsis thaliana.

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  • Simultaneous multiwavelength generation from a mode-locked all-solid-state Cr:forsterite laser

    Optics Letters 26, 834-836

    Tzu-Ming Liu, Shi-Peng Tai, Hsu-Hao Chang, and Chi-Kuang Sun

    We demonstrate a multiple-channel, mode-locked, all-solid-state Cr:forsterite laser. By inserting an etalon into the laser cavity, we have generated 12 phase-locked channels with 9–19-ps pulse width near 1230 nm with 280-mW average output power from a single laser oscillator. By tuning the etalon bandwidth we can shorten the pulse width in a specific channel to 1.8 ps.

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  • Intracavity frequency-doubled femtosecond Cr4+:forsterite laser

    Applied Optics 40, 1957-1960

    Tzu-Ming Liu, Shi-Peng Tai, and Chi-Kuang Sun

    The generation of femtosecond optical pulses centered at 620 nm directly from an all-solid-state laser oscillator is reported. Red pulses with pulse widths of the order of 170 fs were obtained with 24-mW average power at an 81-MHz repetition rate. They were achieved by intracavity frequency doubling of a mode-locked Cr4+:forsterite laser with a 1-mm-thick β-BaB2O4 crystal. The process of laser mode locking was modified by surface coating the doubling crystal.

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  • 2000

    Multi-photon spectroscopy of plant tissues

    Scanning 22, 193-194

    Fu-Jen Kao, Ping-Chin Cheng, Chi-Kuang Sun, Bai-Ling Lin, Yi-Min Wang, Jian-Cheng Chen, Yung-Sheng Wang, Tzu-Ming Liu, and Mao-Kuo Huang

    Considering its non-linear nature, two-photon excitation may generate very different spectral response in samples when compared with single-photon excitation. It is thus necessary to measure the two-photon spectra of samples, so that the fluorescence images can be properly interpreted. However, fluorescence spectra obtained from bulk samples may not provide adequate information for microscopy. For instance, due to the relatively small contribution to the total fluorescence intensity, a small number of fluorescent particles in a generally fluorescing specimen may escape detection when the spectrum of the specimen as a whole is obtained. In addition, signals resulted from second harmonic generation (SHG) may be mixed with lowlevel broadband background auto-fluorescence commonly found in biological specimen. Therefore, measuring fluorescence spectrum from a micro-focused volume is essential to properly interpret multi-photon fluorescence images. In this study, leaf protoplasts and stem slices of maize were used as samples to address this issue in a micro-spectroscopic set-up.


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  • Multi-photon excited fluorescence spectra of common bio-probes

    Scanning 22, 187-188

    Ping-Chin Cheng, Fu-Jen Kao, Chi-Kuang Sun, Bai-Ling Lin, Tzu-Ming Liu, Yung-Sheng Wang, MAao-Kuo Huang, Yi-Min Wang, Jian-Cheng Chen, and I. Johnson

    Fluorescent probes are commonly used in biological fluorescence microscopy for tracking specific structures and subcellular compartments, and for indicating cellular ionic conditions. Recent development in multi-photon fluorescence microscopy has greatly expanded the usage of fluorescent probes in biomedical research. Considering its non-linear nature, two-photon excitation may generate very different fluorescence spectral response in the sample when compared with single photon excitation. It is thus necessary to measure the two-photon spectra of various fluorescent probes, so that two-photon fluorescence microscopy may be performed effectively and the images properly interpreted. This report represents the second installment of a continued effort in characterizing the multiphoton fluorescence spectra of commonly used bioprobe.

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  • High intensity scanning microscopy with a femtosecond Cr:forsterite laser

    Scanning 22, 95-96

    Chi-Kuang Sun, Shi-Wei Chu, Tzu-Ming Liu, and Ping Chin Cheng

    Accompanied by the improvements in ultrafast laser technology, there is a rapid advance in recent developments in high-intensity scanning microscopy. The primary advantage of this technique over laser scanning confocal microscopy derives from the confined excitation volume by using high-intensity-light induced multi-photon excitation fluorescence or high-order harmonic generation. This technique will not only provide a better z-axis resolution but also eliminate the need for a confocal pinhole1. The use of infrared excitation wavelength not only provides a deeper penetration depth but also avoids unnecessary out-of-focus photo-bleaching and photo-damages. However, the full potential of high-intensity scanning microscopy has not been realized due to the limited wavelength available in the market with a primary focus on Ti:sapphire lasers. Although this femtosecond source provides excellent images, comparative studies have shown that the Ti:sapphire wavelength is not optimized for penetrating thick biological tissues. An optimally penetrating optical source will take advantage of the diminishing scattering cross-section with wavelength, while avoiding the resonant molecular absorption of common tissue constituents such as water. In this paper we present a high-intensity microscopy technique by using a femtosecond Cr:forsterite laser source with a center wavelength of 1220–1240nm, which corresponds to the penetrating window in most biological tissues like human skin2 and maize stem3. Other advantages of using the Cr:forsterite laser include that its two-photon fluorescence/second-harmonic-generation (SHG) falls in the red region and its three-photon fluorescence/third-harmonic-generation (THG) falls in the blue-to-green regions, which are visible and can be easily separated from the infrared pump-wavelength.

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