利用自体荧光成像量化分析牙菌斑

牙菌斑是造成龋病及牙周病的主要因素之一,通过检测牙齿表面牙菌斑含量多少能在一定程度上了解牙齿健康程度,对口腔健康的维护有重要意义。利用牙菌斑与牙齿组织自体荧光光谱的差异性,本文设计了一套便携式的基于自体荧光成像的检测系统。(1)该系统以中心波长为405 nm的LED作为激发光源,辅以中心波长520 nm的长波通滤光片提高系统信噪比,CCD作为感光器件;系统采集荧光图像后,根据成像结果分析牙面上牙菌斑含量。(2)设计了验证实验采集受试者牙齿图像,并以牙菌斑百分比作为量化牙菌斑多少的指标。实验结果表明荧光图像菌斑百分比与Turesky改良的Quigley-Hein菌斑指数的Spearman等级相关系数为0.944,牙齿荧光图像牙菌斑百分比与染色图像牙菌斑百分比的Pearson相关系数为0.875。由此可见,文中的检测系统具有较好的准确性,而且因为采用光学检测方法,具有非侵入性且可重复性,所以它有广阔的临床应用前景,有望成为家用口腔健康的检查手段。     

不同激发方式对能量色散X射线荧光法测铀的影响

针对能量色散X射线荧光法测铀过程中存在自激发效应对测量结果产生干扰的问题及以往测铀仅使用放射性同位素源作为激发源的测量限制,利用微型X射线对铀矿样品进行自激发效应测量,并分别将109Cd,241Am,微型X光管三种不同激发源测量铀矿样品的结果进行比较分析。结果表明,自激发效应产生的特征X射线峰面积计数仅为有源条件的0.01%以下,属统计涨落范畴,对测量结果的干扰可忽略不计;109Cd源由于其特征射线能量22.11和24.95 keV均在L_α吸收限能量21.75 keV附近,激发光电截面最高,相应的荧光产额也高,故109Cd源相比于241Am源对铀元素的激发效率更高;241Am源测量误差明显大于109Cd源的测量误差,原因是铀的L系能量特征峰与241Am源特征射线26.35 keV的散射峰能量区叠加,造成实测谱线本底偏高;X光管作激发源的铀矿样品中铀含量与化学分析结果之间的误差在10%以内,仅为同位素源激发X射线荧光分析误差的一半,且X光管激发谱峰面积计数值明显大于源激发条件下的峰面积计数,说明X光管作激发源的测铀质量优于源激发模式。     

Chemical Imaging of Phase-Separated Polymer Blends by Fluorescence Microscopy

Blends of poly(vinylacetate) (PVAc) and poly(cyclohexylmethacrylate) (PCHMA) labeled by copolymerization with 4-methacryloylamine-4-nitrostilbene (Sb), with (1-pyrenylmethyl)methacrylate (Py), or with 3-(methacryloylamine)propyl-N-carbazole (Cbz) were prepared by casting dilute solutions in tetrahydrofurane (THF) or chloroform. Films about 10 m thick were formed. Phase separation in two types of domains is observed by transmission optical microscopy (TOM) and epifluorescence microscopy (EFM): small craters of 1 to 10 m placed at the polymer-air interface and larger domains, on the scale of 100 m. The morphology of samples depends on the composition of the polymer blend and on solvent. The green fluorescence of Sb, the violet of Py, or the blue of Cbz provides imaging of the distribution of PCHMA in the different domains and in the matrix. It is thus observed that (i) superficial craters and large domains are formed mainly by PCHMA and (ii) the matrix is composed of PVAc in films cast from THF and it is a blend of the two polymers, homogeneous at the submicrometric scale, for chloroform. The emission intensity of Py, recorded by microfluorescence spectroscopy (MFS), yields a mapping similar to imaging detection. It is remarkable that in films cast from chloroform, the smaller domains are distributed with a 2D hexatic order disrupted by dislocations and disclinations, whereas in films cast from THF, a larger heterogeneity is found, denoting different mechanisms of solvent evaporation.     

Investigations of RhB Langmuir monolayer by Fluorescence Imaging Microscopy

This communication reports the surface pressure vs area per molecule isotherm and Fluorescence Imaging Microscopic studies of the formation of domain structure in the mixed Langmuir monolayer of RhB and Stearic acid (SA) at the air-water interface. Strong repulsive interaction between the unlike components leads to the phase separation and formation of microcrystalline domains at the air-water interface of the Langmuir monolayer. These domains can be directly visualized using fluorescence imaging microscope.     

Measurement of Cell Volume Changes by Fluorescence Self-Quenching

At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (M) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.     

Photo-Degradation Study of CdTe Nanocrystals by Fluorescence Measurement

The photo-degradation of green-, yellow-, orange- and red-emitting CdTe nanocrystals (NCs) in sol–gel SiO₂ films was investigated quantitatively by measuring the PL efficiency as a function of the irradiation intensity. The degradation behaviors of the NCs depended strongly on the particle size and the surface state. Green- and yellow-emitting CdTe NCs exhibited a red-shifted PL peak wavelength and decreased PL efficiency after irradiation. In contrast, the PL peak wavelength of red-emitting CdTe NCs remained unchange and their PL efficiency increased. Furthermore, the degraded degree of green-emitting NCs depended linearly on the irradiation intensity ( \textrate \textconstant k₁ = ( 1.10±0.04 ) ×10 ^{- 6} \textphoton ) \left( {{\text{rate}}\,{\text{constant}}\,{k_{{1}}} = \left( {{1}.{1}0\pm 0.0{4}} \right) \times {1}{0^{{ - {6}}}}\,{\text{photon}}} \right) , whereas hat of red-emitting NCs showed a quadratic dependence ( \textrate \textconstant k₂ = ( 2.26±0.1 ) ×10 ^{- 26}( \textc\textm² \texts )/\textphoton ) \left( {{\text{rate}}\,{\text{constant}}\,{k_{{2}}} = \left( {{2}.{26}\pm 0.{1}} \right) \times {1}{0^{{ - {26}}}}\left( {{\text{c}}{{\text{m}}^{{2}}}\,{\text{s}}} \right)/{\text{photon}}} \right) at room temperature. This is ascribed to the different surface state of green- and red-emitting CdTe NCs.     

Effect of Scattering on Spectral Imaging of Exposed Cortical Tissue for Brain Function Measurement

Optical imaging of an exposed cortex for brain function measurement is an attractive method for both clinical and physiological investigations. Spectral imaging of the exposed cortical tissue enables measurement of the activity-dependent changes in oxy- and deoxy-haemoglobin independently. Since the diffusely reflected light from the exposed cortex is highly scattered in the cortical tissue, the wavelength dependence of the optical properties of the tissue is likely to affect the images of oxy- and deoxy-haemoglobin changes obtained from the spectral images. In this study, the spectral images of the model of exposed cortical tissue are calculated by Monte Carlo simulation to investigate the influence of blurring by tissue scattering on the brain function measurement by the imaging. The images of the change in oxy- and deoxy-haemoglobin obtained from the spectral images at near infrared wavelengths are broadened and blurred due to the scattering in the cortical tissue. The influence of cross talk is more significant in the image calculated from the spectral images at the visible wavelengths. Inappropriate choice of the spectral range of images might increase cross talk and error in the images of oxy- and deoxy-haemoglobin changes.     

DNA纳米传感器荧光成像技术

把基于量子点的DNA纳米传感器模型应用到ICCD荧光显微成像系统中,利用量子点的量子产率高、荧光寿命长、激发谱宽而发射谱窄、发射波长可由材料尺寸调谐等特性,以量子点为供体,Cy5(一种小分子荧光染料)为受体,结合ICCD系统的全内反射荧光成像功能、实时成像功能和双通道成像功能,证实了DNA纳米传感器可以在溶液中检测到含30个碱基的单链目标DNA片段。实时拍摄了溶液中链霉亲和素包被的量子点对两头分别连着Cy5和生物素的单链DNA片段(Cy5-ssDNA-Biotin)的捕获过程。并在活的中国仓鼠卵巢细胞样品中加入链霉亲和素包被的量子点和Cy5-ssDNA-Biotin进行实时荧光成像,拍摄到链霉亲和素包被的量子点和Cy5-ssDNA-Biotin进入细胞中并发生FRET的过程,初步表明了DNA纳米传感器在活细胞内进行DNA(或RNA)片段检测的可行性。     

荧光成像及手性特异性生物识别

癌症是威胁人类健康的重大疾病之一,实现早查早治是降低癌症死亡率的重要手段。目前在癌症的众多识别方式中,荧光检测凭借无创伤、检测快和可视化等优点受到了持续关注。文章对荧光探针靶向识别肿瘤的研究新进展进行了综述;对赋予荧光探针靶向性的叶酸(FA)和叶酸受体(FR)介导研究进展进行了介绍和深入分析。叶酸受体是癌细胞表面过量表达的特有物质,利用叶酸和叶酸受体特异性结合的特点,叶酸对荧光探针分子进行修饰可赋予荧光探针识别癌细胞的靶向性。叶酸受体有4种亚型（FRα,FRβ,FRγ和FRδ）,前两者FRα和FRβ因分别在癌细胞和炎症巨噬细胞表面过量表达而备受关注,FRα和FRβ约有70%的同源性,两者均具有能与叶酸结合的特性,造成了荧光探针分子在生物识别过程中难以区分癌细胞和炎症巨噬细胞的弱势;针对叶酸修饰荧光探针难以区分叶酸受体亚型造成癌细胞和炎症巨噬细胞混淆问题的结构性缺陷,分析了两种叶酸受体亚型结构具有的手性区别特征：FRα和FRβ主要区别位于三个末端未翻译区,有三种不同手性特征的氨基酸形成一个用来包结叶酸分子的三角空腔,分别固定在氨基酸堆积体节点上的三种氨基酸形成了不同手性特性的区域性“手性空间”。FRα和FRβ本身的结构对不同的配体表现出立体差异性。讨论了基于叶酸受体亚型存在的“手性空间”差异性,构建叶酸受体亚型荧光及手性识别探针的可能性;有望借助光谱成像,通过手性荧光探针分子对氨基酸的识别区分叶酸受体两种亚型,实现可视化区分肿瘤细胞和炎症巨噬细胞的识别难题,进而提高癌细胞识别的准确性;文章介绍了手性荧光探针识别氨基酸原理及结构优化设计进展;近年来手性量子点对氨基酸不同对映体的研究备受关注。对无机手性量子点手性产生的本质特征和氨基酸对映体的识别特性进行了总结分析。最后对荧光探针及手性识别领域进行了展望。     

荧光光谱及其成像技术在光活检中的应用

分析了人体组织自体荧光的主要物质来源和荧光特性,同时总结了国内外常见的第二代新型光敏剂及其临床应用情况。在此基础上,全面比较了各种应用于早期肿瘤光活检的荧光光谱及其成像技术,并重点讨论了这些技术的基本原理和临床应用现状,以及它们今后的发展方向。     

NO激光诱导荧光对高焓气流温度的测量

由于真实气体效应,高超声速流场的研究仍然依赖于大量的实验.流场温度是实验的重要参数,目前只能通过具有非入侵性质的光学测量手段获得,然而,由于多方面的难题,鲜有对高焓流场参数测量的报道.文章介绍了利用激光诱导荧光（laser-induced fluorescence,LIF）技术对JF-10氢氧爆轰激波风洞产生的高焓实验气流温度的测量工作.搭建了用于脉冲式风洞的LIF测量系统,使用了NO分子作为荧光组分.因为高焓流场实验条件苛刻,本实验对传统的激光设置进行了调整,使用了平行于拍摄方向的竖直平面激光激发NO,使荧光信号更为集中,获得了清晰的LIF图像.利用双线测温法（two-line thermometry,TLT）测量高焓自由流中NO分子的转动温度,从而确定气流的平转温度.测量结果表明,JF-10实验气流的平转温度为600 K.      

Measurement of Light and pH Dependence of Single-Cell Photosynthesis by Fluorescence Microscopy

Measurement of algal photosynthetic performance with conventional methods requires thousands of cells obtained by isolation and subsequent cultivation. This is a time-consuming process for many species. We describe a new method to study photosynthetic performance of single algal cells under various environmental conditions by a combination of modulated chlorophyll fluorescence, light microscopy, and sample manipulation techniques. Single cell fluorescence was measured with a modulated microfluorometer integrated in an inverted microscope. The algal cell was sucked onto the tip of a glass microcapillary and positioned in the center of the field of view of the microscope by a micromanipulator. A superfusion device was used to generate a flow of experimental solution of variable composition along the alga. The light dependence of Scenedesmus obtusiusculus single-cell photosystem II (PSII) electron flow was measured at various pH. At a high light intensity PSII electron flow was inhibited at pH 6.5 and higher, while at a low light inhibition occurred at pH 9.5. This is in agreement with inhibition of photosynthesis by substrate (CO₂) limitation at alkaline pH. This approach can easily be extended to study the in vivo effects of other abiotic parameters (temperature, nutrients, toxicants, oxygen) on the photosynthetic performance of algae.     

氧化铝的X射线荧光光谱分析方法研讨

简要论述了氧化铝的X射线荧光光谱分析方法原理、样品处理、设备特点;在德国布鲁克公司的S4型X射线荧光光谱仪上用融片法分析氧化铝中的杂质含量,重点论述了用X射线荧光光谱法代替传统的化学分析方法的优势和不足.     

饱和激发下叶绿素荧光非线性变化及影响分析

叶绿素浓度是海洋初级生产力的重要指标之一,激光诱导荧光技术可以实现海水叶绿素浓度的快速测量。测量叶绿素浓度的传统激光诱导荧光原理,是利用叶绿素荧光与水体Raman散射的强度比值（I_F/R）进行反演,即叶绿素浓度n_chl＝CI_F/R,其中C为系统常量。这是依据叶绿素荧光685 nm、水体Raman散射强度都与激发光强呈线性关系。然而,该理论并没有考虑诱导荧光饱和现象的存在。当诱导激光强度达到一定程度后,685 nm荧光强度随激发光强非线性变化。另外,值得注意的是,水体Raman散射并不存在信号饱和现象。为了探讨饱和激发造成荧光非线性变化的影响,在激光诱导荧光技术测量叶绿素浓度的实验中,设计两种测量方案,即：不同激光功率诱导单一浓度样本的荧光测量,和固定激光功率时不同浓度样本的荧光测量。实验中利用Nd∶YAG三倍频激光355 nm激发获得叶绿素溶液的404 nm处 Raman散射和685 nm荧光。实验结果分为2部分进行讨论：（1）为了分析饱和激发造成荧光变化的非线性特性,通过调节激发光功率测量溶液的受激发射光谱,发现水体Raman散射强度与激发光强呈线性关系,而685 nm荧光强度出现饱和激发下的非线性变化。而且,随叶绿素浓度的增加,685 nm荧光的非线性趋势更为明显,Raman散射强度与激发光强的线性关系中斜率变小。数据分析表明,685 nm荧光数据拟合的4阶多项式和Raman散射效率值,可以定性地表征685 nm荧光的饱和程度。（2）考虑实际海洋激光雷达探测叶绿素浓度应用中存在饱和激发荧光非线性现象,为了分析荧光非线性对传统叶绿素浓度反演理论适用性的影响,在固定激发光强情况下对不同浓度叶绿素溶液的发射光谱进行测量。将激发光功率调节至52.00,80.70,132.10和197.30 mW·cm-2,获取相应激发光强下685 nm荧光与水体Raman散射的强度比值和叶绿素浓度之间的关系。实验表明,激发光强不变的情况下,685 nm荧光与水体Raman散射的强度比值,与叶绿素浓度仍满足线性关系。但是,在较高光强激发时,饱和激发造成的叶绿素荧光非线性变化,导致利用传统激光诱导荧光理论反演的叶绿素浓度值偏小。因此,需要对饱和激发下荧光非线性的影响进行修正,其关系为I_F/R=n_chl/C+C_F,修正值C_F不可忽略。另外,值得一提的是,修正关系中系统常量C随激发光强增加而增大。研究表明,饱和激发造成的荧光非线性,会对激光诱导荧光技术测量叶绿素浓度产生影响,但由于造成荧光非线性因素的复杂性,仅通过荧光数据拟合获得的多项式,无法定量说明其影响权重。然而,当激发光强不变时,可以实验测量获得基于激光诱导荧光原理的修正关系,从而准确反演叶绿素浓度。     

含油岩心显微荧光成像光谱研究

发展了一种显微荧光光谱成像技术,并将其应用于天然岩心进行显微荧光成像光谱研究。利用这种技术同时采集含油岩心表面的荧光光谱信息和空间信息．并对获得的光谱图像给予光谱学和地质学解释。结果表明,不但能显示岩心形貌和组分的大致趋势,而且能揭示其精细细节．为石油地质研究提供了一种新方法,为今后的石油勘探开发工作提供了一种先进的指导手段。     

荧光共焦扫描系统成像特性的优化

对荧光共焦扫描系统用光强点扩散函数进行傅里变换得到系统三维传递函数的数学模型，并由此求得环形透镜和各种有限大小探测器系统的光学传递函数。用计算机模拟和光学传递九数值计算，分析了采用不同环形透镜及探测器对系统成像特性的影响     

Super-Resolution Two-Photon Fluorescence Tomography Through the Phase-Shifted Optical Beatings of Bessel Beams for High-Resolution Deeper Tissue 3D Imaging

A novel z-scanning-free epi-detected super-resolution two-photon fluorescence tomography (TPFT) technique enabling super-resolution deeper tissue 3D imaging is reported. To accomplish this, a unique method is conceived by generating the phase-shifted optical beatings of Bessel beams (PS-OB³) with a spatial light modulator (SLM) to break the diffraction limit for enhancing both the lateral and axial resolutions as well as improving the penetration depth in TPFT for super-resolution deeper tissue imaging. By electronically varying the optical beating frequency and the phase shifts of the beating patterns through SLM, the depth-resolved TPF signals about the volumetric tissue are encoded in the spatial frequency domain and hence, a series of depth-resolved TPF images can be retrieved by implementing inverse fast Fourier transform without a need of mechanical depth-scanning. PS-OB³ TPFT provides ≈1.3- and 2-fold improvements in lateral and axial resolutions in comparison with conventional point-scan TPF imaging. It is also illustrated that the epi-detected PS-OB³ TPFT imaging with inherent scattering-resilient properties of the Bessel beams employed gives over 2-fold improvement in imaging depth in porcine brain tissue compared to conventional point-scan Gaussian beam TPF imaging. The z-scanning-free optical sectioning ability of PS-OB³ method developed in TPFT is universal, which can be readily extended to practically any other nonlinear optical imaging modalities for super-resolution deeper 3D imaging in biological and biomedical tissues.     

基于激光共焦扫描术的亚心形扁藻显微图像与光谱

采用激光共聚焦扫描显微技术,针对亚心形扁藻开展了研究.从获得的488 nm Ar+激光单光子激发的亚心形扁藻自体荧光光谱与图像,可知细胞内有一杯状叶绿体物质,其荧光峰值为682 nm,对应叶绿体发出的红色荧光.在单通道模式下,获得800 nm fs激光双光子激发的扁藻自体荧光光谱与图像,可知每个杯状叶绿体的内部有一个自体荧光更强的圆形物质.在双通道模式下,可分别获得小圆形物质的自体荧光图像,杯状叶绿体自体荧光图像,以及两个通道图像的叠加.进一步获得了双光子藻细胞荧光图的6个主要的荧光峰.采用单光子激光激发可获得亚心形扁藻叶绿体自体荧光图像及其荧光光谱,而双光子激光激发荧光光谱的多通道以及Lambda模式下采集光谱信号与图像,不仅可观察到亚心形扁藻的内部形态结构,还可能从双光子激发荧光图中研究分析亚心形扁藻生化物质的存在,灵敏度较单光子激发高.激光扫描共聚焦显微技术,特别是双光子荧光与图像技术可为海藻的检测与研究提供一种快速、实时、有效、简便的方法.