Real-time Thermal Imaging of Microwave Accelerated Metal-Enhanced Fluorescence (MAMEF) Based Assays on Sapphire Plates

In this paper, we describe an optical geometry that facilitates our further characterization of the temperature changes above silver island films (SiFs) on sapphire plates, when exposed to microwave radiation. Since sapphire transmits IR, we designed an optical scheme to capture real-time temperature images of a thin water film on sapphire plates with and without SiFs during the application of a short microwave pulse. Using this optical scheme, we can accurately determine the temperature profile of solvents in proximity to metal structures when exposed to microwave irradiation. We believe that this optical scheme will provide us with a basis for further studies in designing metal structures to further improve plasmonic-fluorescence clinical sensing applications, such as those used in microwave accelerated metal-enhanced fluorescence (MAMEF).     

A UV–Visible–NIR fluorescence lifetime imaging microscope for laser-based biological sensing with picosecond resolution

This article describes the design and characterization of a wide-field, time-domain fluorescence lifetime imaging microscopy (FLIM) system developed for picosecond time-resolved biological imaging. The system consists of a nitrogen-pumped dye laser for UV–visible–NIR excitation (337.1–960 nm), an epi-illuminated microscope with UV compatible optics, and a time-gated intensified CCD camera with an adjustable gate width (200 ps-10^-3 s) for temporally resolved, single-photon detection of fluorescence decays with 9.6-bit intensity resolution and 1.4-μm spatial resolution. Intensity measurements used for fluorescence decay calculations are reproducible to within 2%, achieved by synchronizing the ICCD gate delay to the excitation laser pulse via a constant fraction optical discriminator and picosecond delay card. A self-consistent FLIM system response model is presented, allowing for fluorescence lifetimes (0.6 ns) significantly smaller than the FLIM system response (1.14 ns) to be determined to 3% of independently determined values. The FLIM system was able to discriminate fluorescence lifetime differences of at least 50 ps. The spectral tunability and large temporal dynamic range of the system are demonstrated by imaging in living human cells: UV-excited endogenous fluorescence from metabolic cofactors (lifetime ∼1.4 ns); and 460-nm excited fluorescence from an exogenous oxygen-quenched ruthenium dye (lifetime ∼400 ns). Received: 23 February 2003 / Published online: 22 May 2003 RID="*" ID="*"Corresponding author. Fax: +1-734/9361-905, E-mail: mycek@umich.edu     

Characterization and performance of the femtosecond fluorescence up-conversion microscope

The characterization and performance of the femtosecond fluorescence up-conversion microscope is reported in this paper. This new fluorescence microscope is a combination of the frequency up-conversion technique and a confocal optical configuration, which simultaneously achieves femtosecond time and nanometer space resolution. The femtosecond time resolution was evaluated by measuring the rise up of time-resolved fluorescence from a dye molecule, and it was 520 fs and 460 fs with 100× (N.A.=1.3) and 40× (N.A.=0.75) objective lenses, respectively. The best transverse (XY) resolution was 0.34 m with the 100× objective lens for 400 nm excitation. An axial (Z) resolution as high as 1.1 m was obtained for 600 nm fluorescence detection with a 50 m pinhole and a 100× objective lens. The axial resolution was remarkably improved compared with ordinary confocal microscopes owing to the up-conversion process, which requires spatial overlap between the tightly focused gate and the fluorescence beams. Femtosecond time-resolved fluorescence measurements were performed for micro-meter sized particles in liquids, fluorescent beads and C519/toluene micro droplets, by using the laser trapping technique. The high potential of the fluorescence up-conversion microscope was demonstrated. PACS 78.47.+p; 87.64.-t; 82.53.-k     

Portable in situ fluorescence cytometry of microscale cell-based assays

A portable fluorescence cytometric system has been developed for characterizing chemical concentration and cellular status in microscale cell culture analog (microCCA) devices. Based on discrete optical components, the system provides a modular platform for real-time image measurements applicable to a variety of cell-based microassays. As a feasibility study, we investigated the real-time dynamics of daunorubicin uptake with cultured mouse L-cells in a microCCA compartment. Time course results measured by the portable fluorescence cytometric system confirmed that in the microCCA devices daunorubicin accumulation is proportional to the liquid turnover rate.     

Measurement of subpicosecond optical waveforms using a resonator-based phase modulator

We propose a method for the measurement of periodic optical waveforms based on the use of an electrooptic phase modulator placed in an optical Fabry-Perot or ring resonator. Significant broadening of the modulation spectrum extends the recently developed method of periodic modulation for pulse characterization into femtosecond scales. We numerically demonstrate the characterization of a 300-fs optical pulse. We also present a technique based on the temporal fractional Talbot effect for restoration of the pulse phase profile. After fast linear processing, subpicosecond pulses will be observed on the screen of a real-time oscilloscope. This complete characterization of optical pulses is entirely linear and therefore highly sensitive and simple in implementation.     

Fast and accurate group delay ripple measurement technique for ultralong chirped fiber Bragg gratings

A simple and very precise group delay ripple (GDR) measurement technique for linearly chirped fiber Bragg gratings (CFBGs) is proposed. It is based on real-time optical Fourier transformation of an ultrashort pulse directly induced by the CFBG dispersion. We have experimentally demonstrated highly accurate characterization of the GDR profile of a commercial 10-m-long CFBG with a dispersion of +2000 ps/nm, having achieved a remarkably small standard deviation in our measurements of about 4 ps over a bandwidth of 28 nm. The proposed method has the unique potential to provide real-time GDR monitoring (in the MHz range) by use of commercially available high-speed sampling electronics.     

Endogenous synchronous fluorescence spectroscopy (SFS) of basal cell carcinoma-initial study

The human skin is a complex, multilayered and inhomogeneous organ with spatially varying optical properties. Analysis of cutaneous fluorescence spectra could be a very complicated task; therefore researchers apply complex mathematical tools for data evaluation, or try to find some specific approaches, that would simplify the spectral analysis. Synchronous fluorescence spectroscopy (SFS) allows improving the spectral resolution, which could be useful for the biological tissue fluorescence characterization and could increase the tumour detection diagnostic accuracy.     

CdSe/ZnS量子点在倏逝波光纤pH传感中的应用

将新型量子点荧光传感技术与光纤倏逝波传感技术相结合,发展了一种基于量子点荧光效应并结合倏逝波传导进行溶液酸碱度检测的新型传感技术,具有灵敏度高、检测速度快、便于微环境检测、可实现远程探测、实时监测和原位分析等特点。详细介绍了用于倏逝波传感的锥柱组合型光纤荧光探头的制备方法,量子点在光纤探头表面的修饰流程,光谱与强度两种光纤pH传感平台的建立,并分别从响应范围、线性度、重复性和稳定性等方面对CdSe/ZnS量子点应用于光纤pH传感进行了评价。结果表明,在pH值为2~12的范围内,CdSe/ZnS量子点的荧光光谱信号的峰位在强酸和强碱的情况下都会产生红移,且红移量随pH值的变化呈线性关系,其量子点荧光强度信号随pH值的减小呈线性降低关系,通过在强酸和强碱下交替测试的实验表明其具有较好的重复性,利用荧光强度传感平台进行实时监测的实验表明其具有较好的稳定性。因此,将CdSe/ZnS量子点用于倏逝波光纤pH传感具有可行性,在生物化学、环境监测、医学临床、食品安全等领域的pH值测量方面有着广泛的应用前景。     

Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy

We report the first simultaneous measurement of surface-confined and solution fluorescence correlation spectroscopy (FCS). We use an optical configuration for tightly focused excitation and separate detection of light emitted below (undercritical angle fluorescence, UAF) and above (supercritical angle fluorescence, SAF) the critical angle of total internal reflection of the coverslip/sample interface. This creates two laterally coincident detection volumes which differ in their axial extent. While detection of far-field UAF emission producesa standard confocal volume, near-field-mediated SAF produces a highly surface-confined detection volume at the coverslip/sample interface which extends only ~200 nm into the sample. A characterization of the two detection volumes by FCS of free diffusion is presented and compared with analytical models and simulations. The presented FCS technique allows to determine bulk solution concentrations and surface-near concentrations at the same time.     

Scanning near-field optical/atomic force microscopy for fluorescence imaging and spectroscopy of biomaterials in air and liquid: Observation of recombinantEscherichia coli with gene coding to green fluorescent protein

We have developed a system of scanning near-field optical/atomic force microscopy (SNOM/AFM) for fluorescence imaging and spectroscopy of biomaterials in air and liquid. SNOM/AFM uses a bent optical fiber simultaneously as a dynamic force AFM cantilever and a SNOM probe. Optical resolution of SNOM images shows about 50 nm in an illumination mode for a standard sample of a patterned chromium layer of 20 nm thickness on a quartz glass plate. The SNOM/AFM system contains a photon counting system and polychrometer/ICCD (intensified charge coupled device) system for observation of the fluorescence image and spectrograph of micro areas, respectively. The gene coding to green fluorescence protein (GFP) was cloned in recombinantEscherichia coli (E. coli). Topography, fluorescence image and spectrograph of recombinantE. coli by SNOM/AFM showed a difference in fluorescence in individualE. coli. Fluorescence activity of GFP can thus be used as a convenient indicator of transformation. SNOM/AFM is also applicable to observe immobilizedE. coli on a glass plate in water with a liquid chamber and may allow the viewing of observation of floating organisms.     

Furthering Precision in Sentinel Node Navigational Surgery for Oral Cancer: a Novel Triple Targeting System

To describe an innovative sentinel lymph node (SLN) guidance approach using a radionuclide tracer, 3D augmented reality-guided imaging, and near infrared (NIR) fluorescence over-lay imaging with hand-held probes to optimize accuracy, efficiency, and precise navigation for sentinel node (SN) localization in head and neck cancer. In a cT1N0M0 squamous cell carcinoma of the tongue, pre-operative radionuclide lymphoscintigraphy was performed with a sentinel node-specific radiolabeled tracer. Intraoperatively, a 3D hand-held augmented reality (AR) scanning SPECT probe assessed concordance of the SN with pre-operative SPECT-CT images. The real-time optical video was linked to the SPECT-CT images for added precision. Final guidance to the SN was performed using ICG fluorescence imaging. Dynamic and SPECT-CT showed bilateral lymphatic drainage from the tumor. The 3D hand-held AR SPECT probe SN localization was concordant with pre-operative imaging. The optical video successfully demonstrated the lymphatic drainage in real-time through a unique overlay fluorescence image. The ICG localized to the same nodes identified by both the SPECT-CT and hand-held SPECT images. The use of dual radiation and fluorescence tracers improved SN detection, especially for SN close to the injection site. The hand-held probes allowed the surgeon to dissect continuously, without needing to change tools. The combination of augmented reality, nuclear medicine, and over-lay fluorescence imaging allowed greater accuracy for matching the preoperative imaging with intraoperative identification and precisely guiding the dissection. This method uniquely permitted the surgeon to efficiently dissect the SN with accurate visualization and optimal precision.     

Geometrical characterization techniques for microlens made by thermal reflow of photoresist cylinder

Microlens characterization is a prerequisite for improving fabrication process, and for satisfying the end user needs. In this paper we explore techniques to characterize geometrical properties of microlens made by thermal reflow: viz. microlens profile; radius of curvature; microlens height; contact angle and focal length. The geometrical characterization is done using techniques such as contact profilometry, scanning electron microscopy (SEM), optical microscopy, white light confocal microscopy and fluorescence confocal microscopy. All the above techniques are studied and compared, keeping in mind the characterization requirements of polymer microlens made by thermal reflow technique.     

壳聚糖碳点的水热法制备及其对金属离子的选择性研究

以壳聚糖为碳源通过水热法合成碳点,对影响碳点荧光强度的水热温度、水热时间和壳聚糖质量分数进行考察。通过紫外分光光度计、荧光分光光度计、原子力显微、Fourier红外光谱仪、X-ray光电子能谱、X射线衍射仪对壳聚糖碳点的光学性质、化学结构、晶体结构、形貌结构等进行表征分析。结果表明,在水热温度200℃、水热时间9 h、壳聚糖质量分数2%的条件下制备得到的壳聚糖碳点量子产率为32.86%。碳点呈现出主要尺寸为3～10 nm的球形颗粒状,且在波长335 nm激发下,发射峰位于410 nm(蓝)。对金属离子的选择性研究分析表明,Fe~(3+)对碳点溶液的荧光猝灭效应最显著,说明碳点对Fe~(3+)具有较好敏感性和高选择性,且荧光猝灭效率对Fe~(3+)浓度在0～100μmol/L范围内呈现线性响应,因此有望将碳点作为荧光探针应用于Fe~(3+)的检测表征。     

鼻咽癌离体组织拉曼光谱的测量

拉曼光谱分析技术在探测与组织病理学相关的分子变化方面具有特别的潜力和优势,并且使无损、实时、快速的光学诊断成为可能.采用785 nm半导体激光器,透射式的全息光栅,背向感光、深度耗尽的CCD探测器及特殊设计的光纤拉曼探头构建了一台快速拉曼光谱测试装置.所设计的光纤拉曼探头可在减少荧光信号和瑞利散射影响的同时,最大限度地收集生物组织的拉曼信号,同时解决了平面光栅衍射光束的谱线弯曲问题,提高了仪器的信噪比,使装置具有较高的灵敏度并可快速测量获得人体组织的近红外拉曼信号.通过新鲜猪肉的脂肪和肌肉组织的拉曼光谱信号的检测,验证了测试装置的良好性能; 在此基础上,研究了鼻咽癌组织样品存放时间对拉曼光谱的影响,并在1～5 s时间内快速测量获得了人离体鼻咽癌组织的近红外拉曼光谱.     

Fluorescence polarization imaging for delineating nonmelanoma skin cancers

We present a method for detecting nonmelanoma skin cancers using exogenous fluorescence polarization. We built an automated system that permits exogenous fluorescence polarization imaging. It includes a tunable linearly polarized monochromatic light source and a CCD camera equipped with a rotating linear polarizer and a filter to reject excitation light. Two fluorophores that are retained in tumors, toluidine blue and methylene blue, are employed. We demonstrate that fluorescence polarization imaging can be used for accurate delineation of nonmelanoma cancers. The results suggest that this optical technique may be suitable for real-time noninvasive demarcation of epithelial cancers.     

Large scale synthesis of cadmium selenide nanowires using template synthesis technique and their characterization

We report the fabrication, and structural and optical characterization of CdSe nanowires. Large scale uniform nanowires with length 40 micron and diameter 100 nm were grown using the simple chemical reaction technique. Morphological study of CdSe nanowires was done using scanning electron microscopy (SEM). X-ray diffraction (XRD), and Raman studies show the crystalline structure of CdSe nanowires. Energy dispersive X-ray fluorescence (EDXRF) technique was used to study the composition of CdSe nanowires. UV–Vis absorption studies show a blue shift of 0.26 eV in the optical band gap of CdSe nanowires.     

秸秆还田对旱田黄土可溶性有机质三维荧光光谱的影响

以西北旱田黄土为研究对象,分析玉米秸秆还田前后黄土可溶性有机质(DOM)的三维荧光光谱差异,探讨黄土腐殖化程度的变化情况及对Pb(Ⅱ)赋存形态的影响。实验结果表明：黄土DOM的荧光峰主要分布在λ_ex/em=240～270/280～340区域和λ_ex/em=325/450附近,分别归属为紫外区类富里酸以及可见光区类富里酸和腐殖酸类物质的荧光峰。秸秆还田60 d后,紫外区类富里酸荧光峰值增加,在λ_ex/em=250/440附近和λ_ex/em=320～350/350～400区域出现新的腐殖酸荧光峰。黄土腐殖化程度随秸秆还田时间的延长而增加,Pb(Ⅱ)的生物有效性随腐殖化程度的增加而降低。三维荧光光谱可以有效表征秸秆还田前后黄土DOM的变化特性。     

The Synthesis of Magnetic and Fluorescent Bi-functional Silica Composite Nanoparticles via Reverse Microemulsion Method

In this paper, a simple synthesis method of small-size( about 50 nm in diameter), high magnetic and fluorescent bi-functional silica composite nanoparticles were developed, in which water-soluble Fe₃O₄ magnetic nanoparticlels (MNs) and CdTe quantum dots (QDs) were directly incorporated into a silica shell by reverse microemulsion method. The high luminescent QDs can be used as luminescent marker, while the high magnetic MNs allow the manipulation of the bi-functional silica composite nanoparticles by external magnetic field. Poly (dimethyldiallyl ammonium chloride) was used to balance the electrostatic repulsion between CdTe QDs and silica intermediates to enhance the fluorescence intensity of MNs-QDs/SiO₂ composite nanoparticles. The optical property, magnetic property, size characterization of the bi-functional composite nanoparticles were studied by UV-Vis and PL emission spectra, VSM, TEM, SEM. The stabilities toward time, pH and ionic strength and the effect of MNs on the fluorescence properties of bi-functional silica composite nanoparticles were also studied in detail. By modifying the surface of MNs-QDs/SiO₂ composite nanoparticles with amino and methylphosphonate groups, biologically functionalized and monodisperse MNs-QDs/SiO₂composite nanoparticles can be obtained. In this work, bi-functional composite nanoparticles were conjugated with FITC labeled goat anti-rabbit IgG, to generate novel fluorescent-magnetic-biotargeting tri-functional composite nanoparticles, which can be used in a number of biomedical application.     

六通道光纤传感药物溶出度监测仪的研制及应用

药物溶出度试验是药品检验的重要项目,在药物质量评价方面起着非常重要的作用。利用光纤传感技术可以实现药物溶出度的自动化、过程化监测。以氙灯、氘灯或卤钨灯作为荧光、紫外光及可见光的光源,以Y型分支光纤作为光路传输介质,紫外-可见吸收探头或荧光分子探头作为光响应器件,CCD作为检测器,通过自编软件实现紫外-可见吸收及荧光猝灭两种模式的检测。光纤传感药物溶出度监测仪不但解决了目前离线取样分析方法耗时、耗力的缺点,而且提供了药物溶出过程的实时信息,为药物质量控制提供了更好的评价手段。