Raman microspectroscopy, an alliance of Raman spectroscopy and confocal optical microscopy, is now widely used for label-free and molecular-level investigations of living cells. Here I review recent technical advances in our research group of the hardware as well as the software of Raman microspectroscopy of living cells. The advances in the hardware includes the developments of a multi-focus confocal Raman microspectrometer and a fast spiral raster scanning confocal Raman microspectrometer.These new techniques enable fast Raman imaging of living cells in several to several tens of second, an order of magnitude faster than existing commercial Raman microspectrometers. The new software utilizes the multivariate curve resolution-alternating least squares (MCR-ALS) method to obtain molecular component distribution imaging of living cells. Under appropriate model constraints, such as non-negativity and norm regularization for spectral profiles and their intensities, this new software provides physically interpretable spectral components without a priori knowledge on existing chemical components. These new software/hardware have been successfully applied to fast, automatic and label-free discrimination of living cells such as white blood cells and cardiac muscle cells.

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We developed an application of femtosecond-laser ablation to create a microscopic injury at selected sites of zebrafish heart. We analyzed the morphological and functional recovery of the zebrafish heart during regeneration. Our results might provide insight into the development of novel regenerative therapy.

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Full-field optical coherence tomography (FF-OCT)is suitable for rapid en-face imaging as it uses a 2D imaging device for planar interference and pixel processing of a sample plane. In this study, a red LED was applied in an FF-OCT system for skin imaging. A three-dimensional derm tissue image was obtained from the system to demonstrate the potential of LED in FF-OCT system. Noninvasive 3D skin imaging contributes to advance dermatology and cosmetology.

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Two-photon fluorescence microscopy (2PFM) features deep penetration and high resolution. However, the near infrared excitation of 2PFM results in inferior resolution to that of confocal imaging. To further enhance the resolution of 2PFM, here we propose super-resolution 2PFM. Resolution improvement is demonstrated in brain neurons imaging.

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We established the FRET fluorophore-labeling Dengue virus particles to visualize viral uncoating in living cells. In acceptor-bleaching imaging and single-particle tracking, the FRET signal decay was colocalized with autophagosomes. In addition, the autophagy inhibitors caused the reduction of viral uncoating process, suggesting that Dengue virus uncoating is correlated with autophagy.

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Formation of perfusable vascular networks is an important topic for tissue engineering. We cocultured human umbilical vein endothelial cells with hepatocellular carcinoma cells as spheroids of various sizes, and used selective plane illumination and confocal microscopy to monitor the structures of the endothelial cells inside a spheroid.

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The fabrication of Ce-doped fibers (CeDFs) employing drawing-tower method with rod-in-tube and powder-in-tube technique is demonstrated. The fluorescence spectrum of CeDFs exhibited a 120-nm broadband emission with 1.45 μm axial resolution. This CeDF may be functioned as a high- resolution light source for optical coherence tomography (OCT) applications.

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A THz near-field transmission type imaging system is proposed and demonstrated to investigate the blood absorption coefficient in-vivo. The in-vivo absorption coefficient of the blood of nude mouse is successfully found to be around 140cm-1 at 340GHz.

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Platelets adhering to circulating tumor cells may play an import role in cancer metastasis. In this study, we reveal that platelets adhesion increases cell viscoelasticity and enhance resistance to cellular damage caused by fluid shear stress, resulting in less apoptotic cells and higher metastasis ability.

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Nonlinear fiber optics and nonlinear light microscopy are two totally distinct research areas. In this work, we demonstrate that by efficient wavelength-conversion mechanisms in a highly nonlinear fibers. And the fiber-delivered femtosecond 1.2-1.35 micron down-converted and 0.6-0.8 micron up-converted light sources were then utilized for nonlinear microscopy for the first time.

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We submitted the design of an IR microbolometer to Chip Implementation Center (CIC) in Taiwan. After we received the chips back from CIC, we ink-jetted Cytochrome c protein onto the aluminum electrode. Finally, we demonstrated the TCR of 28.27%. This value is much higher than most materials used in microbolometers.

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In this paper, we proposed a three-layer structure Au/ITO/Au fiber sensor based on surface plasmon resonance to improve the sensing sensitivity for the index-matching oil measurements with the refractive index ranged from 1.30 to 1.39. The results show the sensitivity can be increased 25.8% significantly.

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In light of the limitation of conventional animal models of thrombosis, we develop an application of a photochemical means to initiate thrombosis in targeted blood vessels of zebrafish larvae. This approach is extensible to induce ischemic stroke in zebrafish and may facilitate the screening of drugs with thrombolytic capability.

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We propose a new optical fiber sensor with bi-metal layers structure based on surface plasmon resonance to study the sensing sensitivity improvement under an applied external magnetic field. The results show that the magnetic field can enhance the sensing sensitivity and change the resonance absorption dip wavelength obviously.

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We numerically investigate a periodic array of silver-shell nanorod pair structure by using finite element method. Simulation results show that the SPR modes are very sensitive to the relative permittivities change in the surrounding materials, which could be used as highly sensitive sensors.

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Recognizing different types of leukocytes without a label is very helpful for flow cytometry in clinical medicine. Here we show a new method to differentiate the types of human leukocyte by using third harmonic generation and two-photon auto-fluorescence microscopy. Cell identities were confirmed by immunohistochemical fluorescence labels.

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In this study, the spectrum of each picture element of the patient’s skin image was obtained by multi-spectral imaging technology (MSI). Spectra of normal or pathological skin were collected from 30 patients (10 early stage MF, 10 psoriasis vulgaris, and 10 atopic dermatitis). An algorithm combined with multi-spectral imaging and color reproduction technique is applied to find the best enhancement of the difference between normal and abnormal skin regions. Accordingly, an illuminant with specific intensity ratio of red, green, and blue LEDs is proposed, which has optimal color enhancement for MF detection. Compared with the fluorescent lighting commonly in the use now, the color difference between normal and inflamed skin can be improved from 11.8414 to 17.4002 with a 47% increase, 8.7671 to 12.8544 with a 26.3% increase, and 11.0735 to 17.2634 with a 34.3% increase for MF, psoriasis, and atopic dermatitis patients, respectively, thus making medical diagnosis more efficient, so helping patients receive early treatment.

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The first,to our knowledge,very low glucose sensing limit(0.01mg/dL) after immobilized glucose oxidase technique was demonstrated from our surface plasmon resonator sensing with high telecommunication wavelengthresolution. Its sensitivity was showing 1.56 nm/(mg/dL).

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In order to whether psoralen plus UVA (PUVA) could help photochemotherapy, the transmittance and Raman spectra will be done for the detection. The results exhibited the psoralen plus UVA LED lighting source could add the transmittance and the reaction peaks in the Raman spectra.

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We combine surface plasmon resonance and electrochemical techniques to regulate the oxygen-containing functional groups of graphene oxide, and thus induced band gap to enhanced surface plasmon coupling effect.

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High-speed optical imaging is an essential tool for studying fast dynamics in plasma physics, microfluidics, phononics, and photochemistry. Unfortunately, conventional cameras based on CCD and CMOS image sensors are not fast enough to capture such events with high sensitivity. This is due to the fundamental trade-off between sensitivity and speed – at high frame rate, fewer photons are detected during each frame. In this talk, I introduce unconventional approaches to high-speed optical imaging that overcome the trade-off in the traditional image sensors and offer a record high frame rate and shutter speed without sacrificing sensitivity. I also present a new class of biomedical applications enabled by the technology such as high-throughput screening for cancer detection and drug discovery.

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The interaction of light with metal nanostructures gives rise to the collective oscillation of electrons, which can be engineered to resonate at different wavelengths by tuning the size and geometry of the structures. This phenomenon can be used to create high-resolution color prints. While the resolution of inkbased color printers is limited to ~1,000 dots-per-inch (dpi) due to the intermixing and the size of the ink drops, nanostructured metal can achieve prints at ~100,000 dpi1. At this resolution, different color pixels with dimensions of ~ 250 nm can be juxtaposed without registration errors, and color prints can be achieved at the optical diffraction limit.

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We used a spatial light modulator to project optical micropatterns with an intensity gradient on lung cancer cell A549. We observed that the amounts of reactive oxygen species (ROS) in cells were in proportion to the optical intensity, and the optical intensity gradients could drive directional cell migration.

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Human blood coagulation is investigated by instant and temporal THz spectra measurement of 56 ex-vivo human blood samples either with or without adding anticoagulant. Our result indicates a high sensitivity of THz wave to the first-stage platelet aggregation and amplification process, but not the later stage blood clotting process.

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Process of cytolysis observed in laser treated HepG2 was analyzed by three-dimensional time-lapse imaging. Transgenic HepG2 cells expressing EGFP and YFP in cytosol and plasma membrane revealed increase of membrane permeability and generation of vesicles having plasma membrane and cytosol were generated from the laser treated cell.

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In this experiment, we utilized a home-made optical tweezers system to study the effect on collagen Type I viscosity caused by thermal denaturation with varying pH and additives (i.e. glycerol, urea, glucose, and NaCl). Viscosity of collagen samples were determined by measuring the thermal motion of the trapped microsphere by optical tweezers.

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In this study,we will use hydrogen peroxide catalyzed by peroxidase, and then with 4-aminophenazone, phenol reacts to specific wavelengths of light product has absorption characteristics; take advantage of this feature spectral analysis of the relationship between the concentration of hydrogen peroxide.

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Silicon wire biosensors’ detection sensitivities with TE and TM modes for typical test solutions have been investigated. It was found that the detection sensitivity of TM mode is about 7 times of that of a TE mode. Silicon wires’ detection sensitivities are a few thousand times of that of SOI rib waveguides’.

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The ion-sensitive field-effect-transistor-based urea biosensors were fabricated using the AlGaN/GaN heterostructure. To enhance the sensing performance, the photoelectrochemical etching and oxidation method were applied to recess the gate region and directly grow the sensing membrane, respectively. The sensing sensitivity achieved 17.5 mA/decade within the urea concentration from 0.015M to 50M.

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We proposed an in-line fiber sensor to measure H2O2 concentration with multimode-fiber interferometer. The novel fiber sensor with a no-core fiber (NCF) immobilized peroxidase enzyme (POD) on surface. POD will catalyze H2O2 and lead interference wavelength shifting. The unknown H2O2 concentration sample can be obtained by the wavelength shifting signal.

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Previous study usually believes that dependency between THG spectra and dispersion is negligible. In this letter, we show that dispersions play roles in changing the THG spectral shapes, and this effect should not be neglected for deep tissue THG spectroscopy and microscopy.

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We developed a frequency-domain nanosecond-resolved fluorescence anisotropy detection system, which utilized homodyning method to record the fast dynamic fluorescence signals. We applied this technique to study immunoglobulin G (IgG) aggregation level. With our system, we were able to identify difference size of IgG aggregate from time-resolved anisotropy data.

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The study aims to illustrate that both absorption and scattering optical coefficients images of breast can be reconstructed by only continuous-wave data reliably within a certain extent. Reconstructed images for phantoms with designated absorption / scattering properties of inclusion and background are presented to demonstrate this perspective.

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Electric field distributions of a pair of silver nanorod on a substrate are performed by the finite element method. We find the incident waves are reflected back by the substrate and the superposition of the electric fields of the incident wave and the reflection wave.

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Stimulated Emission (SE) has distinctive characteristics different from fluorescence lifetime image microscopy (FLIM) in that the coherence nature of SE signals enables long distance observation. In SE microscopy, the coincidences of pump and probe beams in space and time are essential. Previously the coincidence in time was accomplished by translational stage and delay cables, now we can accomplish this by a fully digital system, in which the relative delay between pump and probe beam is controlled by a function generator and delay generator, and delay can be adjusted as short as 30ps.

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To fabricate complex 3D structure with nature proteins and overcome the nature limitation of fibronectin (FN) stacking in 3D conditions, bovine serum albumin (BSA) protein can easily form complex 3D scaffold via multiphoton excited photochemistry, then FN can be fabricated at specific location on BSA scaffold to help and guide cell growing three-dimensionally

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We developed two laser based methods – photochemical thrombosis and femtosecond laser ablation – to induce ischemic and hemorrhagic stroke at selected cerebral blood vessels of zebrafish larvae. Our novel zebrafish models of stroke combined with fluorescent imaging facilitate visualization of neovascularization and rerouting of cerebral vessels after stroke.

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We measured the membrane roughness of neuroblastoma cells under the treatments of gold nanoparticles (AuNPs) and paclitaxel. The positive charges on AuNPs reduced the roughness of cell membranes. The stabilization of intracellular microtubules by paclitaxel led to similar results.

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Transdermal drug delivery systems (TDDS) have been an attracting field in drug delivery because of its benefit over parenteral and oral administration. Recent studies illustrate that microneedle (MN) can penetrate through stratum corneum (SC) barrier without damaging nerves. In this study, we used optical coherence tomography (OCT) to investigate the temporal effects on skin induced by MN for transdermal drug delivery. With OCT, the morphological changes in skin due to MN can be identified from 2D or 3D OCT image. Moreover, the penetration depth and damaged area can be quantitatively evaluated. In addition, OCT was used to reconstruct micro-angiography of mouse skin and to observe the changes in micro-angiography induced by MN. Finally, OCT is implemented for dynamic evaluation of skin recovery after using MN array for drug delivery.

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Conventional Photoacoustic microscopy (PAM) enhances ultrasonically determined resolution at the expense of penetration depth. Also, high resolution PAM usually uses time consuming mechanical scanning, which lacks of practicality. In this paper, we propose a high frame rate laser scanning PAM, which features an optically determined submicron resolution.

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We experimentally observed the change of imaging property of confocal microscopy during observation of a gold nanoparticle with different illumination intensities. Modification of the point-spread function through the nonlinear nature of plasmonic scattering, which has been reported previously, was confirmed. We extracted the nonlinear scattering components for high spatial-resolution imaging.

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Deoxyribonucleic acid (DNA) biopolymer material has been attracting great interest for years and became a promising material for many optoelectronic applications. In this study, we reported the fabrication of the DNA biopolymer with silver nanoparticles using a photochemical method and demonstrated a device performing a write-read-erase memory characteristic.

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Epithelial–mesenchymal transition (EMT), a crucial process in tumor metastasis, is often associated with the reorganization and restructuring of actin cytoskeletons, resulting in a change in intracellular stiffness. We measured the intracellular stiffness at different stages of EMT for potential application in the evaluation of metastatic potential of cancer cells.

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We developed a portable LED induced autofluorescence (LIAF) imager through 365nm and 472nm wavelength as excitation light sources to capture ex-vivo oral tissue autofluorescence image. The device achieves a sensitivity of 87.26% and specificity of 62.90% when using normalized blue-to-green ratio analysis method on autofluorescence image with 365nm excitation light source.

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To ensure proper energy delivery without damaging tissue structures beneath the nail plate, optical coherence tomography (OCT) was implemented for quantitative evaluation of induced microscopic ablation zone (MAZ). To further study the feasibility of drug delivery, normal saline was dripped on the exposure area of fingernail and the speckle variance in OCT signal was used to observe water diffusion through the ablative channels into the nail plate.

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The heart of larval-zebrafish was ablated with a femtosecond laser in vivo under asystole. Fluorescence image of transgenic fish expressing EGFP-myosin and mCherry-histone revealed that a targeted cardiomyocyte was ablated. Vesicles that contained cytosol and were enclosed by plasma membrane were produced immediately after ablation, which was followed by cytolysis.

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We applied two photon fluorescence lifetime imaging microscopy (TP-FLIM) to determine the fluorescence lifetime as well as the amounts of endogenous fluorophores, NADH and FAD, in normal and Parkinson’s disease induced PC12 cells. Our results show a significant difference in cellular metabolic state in normal vs. diseased cells.

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We apply pseudodynamic 3D imaging to assess the two-chambered heart of zebrafish larvae. We reveal distinct functional aspects of the heart of larval zebrafish with emphasis on the subtle difference relative to the heart tube of zebrafish embryos and the four-chambered human heart.

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A three-dimensional (3D) single fluorescent particle tracking strategy based on temporal focusing multiphoton excitation microscopy (TFMPEM) combined with astigmatism imaging is proposed for delivering nanoscale-level axial information that reveals 3D trajectories of single fluorospheres in the axially-resolved multiphoton excitation volume without z-axis scanning.

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Ammonia sensors are easily affected by relative humidity (RH) when performing electrical measurement. In this paper, we have found that this undesirable effect can be improved by applying cyclohexane-triphenylene (cchxnTP) as modification layers of nanowire devices. We also discuss the relation between surface modification and morphology in sensing mechanisms.

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A multi-channel Single Plane Illumination Microscopy (SPIM) has been established and applied to study the cell dynamics during the Eye-Antenna Disc Primordium (EADP) formation of Drosophila melanogaster. The contribution of en gene to eyes development in embryonic stage has also been verified by comparing the dual-channel in vivo three-dimensional time-lapsed images of the expression patterns of CD enhancer and en gene.

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Diffuse optical imaging can show the map of chromophore concentration in the medium from multi-wavelength light measurement. In this study, initial simulation of diffuse optical imaging for reconstructing chromophore concentration, such as oxy-hemoglobin, deoxy-hemoglobin, and water, is presented.

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In this article, the mathematical analysis and experiment set up of low-coherence fiber optic Fabry-Perot interference have been demonstrated. Since we want to confirm the ability in determining refractive index of liquid, a series of test with water samples and without sample were conducted under favorable conditions.

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We developed an in vivo non-invasive detection system with two-photon fluorescence to conduct a preoperative assessment of hepatic functions. In our rat models, the residual ICG on 15 minutes post administration (ICG-R15) in rats with hepatocellular carcinoma is above 40%; control group, below 15%. It's as accurate as traditional methods.

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The pseudospectral time-domain (PSTD) technique is employed to obtain numerical solutions of Maxwell’s equations. Outgoing light is recorded and used to generate phase-conjugated light which back-propagates through the scattering medium. By changing the angular span, we can analyze different angular spans and help determine the optimal angular span for back-propagation of light.

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The finite-difference time-domain method (FDTD) can help us to analyze data acquired in the optical coherence tomography (OCT). We reconstruct the structure of melanin, which is the key component of human pigment system. Simulation reconstruction shows structures of the melanin that may be useful for biomedical optics application.

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The light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and structured light, respectively.

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