活细胞内的单分子荧光成像方法

文章介绍近年来新发展的几种重要的活细胞内单分子荧光成像方法,如转盘式共聚焦显微术、全内反射荧光显微术、荧光共振能量转移技术等。通过介绍它们的原理、特点和在活细胞内单分子行为研究中的应用实例,展示了这些新方法在生命科学领域广阔的应用前景。     

基于ICCD的快速荧光显微成像技术及在活细胞研究中的初步应用

利用像增强型CCD、氩离子激光器和氙灯等建立了一套快速荧光显微成像系统,并初步应用于活细胞研究。实时观测和拍摄了大鼠脑微血管内皮细胞增殖分裂过程中细胞内钙敏感荧光探针Fluo-3标记的钙离子浓度分布快速变化的图像,并选取动态图像中四个典型的点给出荧光强度灰度值的变化曲线。该系统可用于高灵敏实时记录活细胞内基于荧光显微成像的快速变化过程,为活细胞的研究提供了一种很好的观察和分析手段。     

拉直的单个DNA分子的全内反射荧光实时成像研究

全内反射荧光(TIRF)成像技术利用穿透深度仅200 nm左右的隐失波来激发诱导荧光,探测灵敏度和图像信噪比大大提高,成为单分子研究的有力工具。分子梳技术利用DNA末端与固体表面的结合力和周围流体流动产生的侧向力将DNA分子拉伸并平铺在表面上。结合这两种技术,对分子梳拉直的单个DNA分子进行了清晰的实时荧光成像,发现TIRF成像条件下DNA分子与荧光探针YOYO-1组成的复合体可自然避免发生光敏断裂现象;实时监测了单个DNA-YOYO-1复合体的光漂白过程,通过对激发光照射时间与探测器曝光时间进行同步控制,可大幅降低光漂白程度,为拉直的单个DNA分子的长时间实时观察和成像研究优化了实验条件,为实时、可视化地研究其与蛋白质相互作用的动力学过程奠定了基础。     

活细胞应激反应过程中线粒体和核仁微环境动力学的荧光寿命成像研究

核仁和线粒体在维持细胞平衡发挥重要作用,研究其生理过程有助于深入了解生物学功能.本文采用一种红色荧光的芘罗丹明荧光探针在不同条件下靶向标记细胞线粒体和核仁.通过激光共聚焦成像和荧光寿命成像技术分析HeLa细胞在光照和药物刺激下细胞凋亡的形态变化,并利用相图定量分析了线粒体与核仁的微环境变化,确定在稳态HeLa细胞中探针标记到的线粒体的平均荧光寿命约为3.65 ns,线粒体黏度约为66×10–3 Pa·s.在激光光照后,探针标记到HeLa细胞线粒体的荧光寿命降至3.61 ns,对应线粒体黏度增至约131×10–3 Pa·s;使用紫杉醇和秋水仙碱诱导细胞凋亡,观察到探针标记于HeLa细胞核仁的荧光寿命先增加后降低,反映了在HeLa细胞凋亡过程中核仁微环境的变化,证明HeLa细胞在非稳态情况下核仁和线粒体的功能变化,为线粒体和核仁功能障碍相关疾病研究提供了新的研究方法.     

联合动静态图像分析技术研究鳄鱼血红细胞形态功能随pH值的变化

采用多维显微成像技术,结合有关动、静态图像分析方法,通过测定鳄鱼血红细胞在不同pH值下其形态参数(包括红细胞的接触面积、周长、长轴、短轴、伸长率和圆度)和反映细胞变形能力的频颤变化,揭示了鳄鱼血红细胞形态结构与有关生理功能随环境pH值的变化情况.结果表明,鳄鱼红细胞无论形态结构还是变形能力都比人体的红细胞更耐受酸性环境...     

基于光谱成像技术的宫颈癌TBS与细胞DNA定量分析联合筛查方法研究

传统的宫颈癌筛查方法主要有TBS分类筛查法和基于细胞DNA的定量分析筛查法,TBS分类筛查法诊断率高但需要经验丰富的医生参与且敏感度低,难以实现宫颈癌早期筛查;基于细胞DNA的定量分析筛查法仅染色细胞核,实现了定量化与自动化分析,敏感度高但特异性差。因此实现宫颈癌TBS与DNA定量分析联合筛查十分必要,但目前TBS与DNA定量分析联合筛查方法均是使用两张不同的细胞涂片分别进行,费时费力费材极为不便,国内外还没有在一张宫颈细胞涂片上联合使用这两种方法的宫颈癌筛查方法。为此提出了一种在一张细胞涂片上同时使用TBS分类法和细胞DNA定量分析法的宫颈癌筛查方法:在同一张细胞涂片上对细胞进行巴氏染色和Feulgen染色,为解决多重染色带来的DNA物质的吸光度干扰问题,建立一套多光谱成像系统,并提出基于线性多元回归的DNA吸光度剥离模型,通过该模型解算出DNA物质的真实吸光度从而实现DNA物质的定量分析;选取接近RGB波长的3个波段细胞图像合成伪彩色图像进行TBS分类筛查。实现了一张细胞涂片上的宫颈癌TBS与DNA定量分析联合筛查,DNA定量分析模型稳定度高、误差小,诊断率高,用于TBS筛查的伪彩色图像颜色明亮、细胞核清晰可见、细胞质边缘明确,该方法在宫颈癌的诊断和筛查中具有很强的实用性。     

Electroporative Adjustment of pH in Living Yeast Cells: Ratiometric Fluorescence pH Imaging

A number of vital cell functions including modulation of signaling pathways and regulation of the cellular transport critically depends on the cytoplasmic pH. Many pathological cellular changes are related to the abnormal cytosolic pH as well. Reliable and well-calibrated methods for quantification of the cytosolic pH are therefore of high importance. The pH calibration is particularly difficult in walled cells since standard methods fail. In this report we evaluated the new electroporative calibration method of the cytosolic pH in yeasts by the fluorescence microscopy. The calibration was done on living cells using pyranine as a ratiometric pH-sensitive probe. The probe was electroporatively delivered to the cytosol. We have shown that unlike the measurements in suspension the fluorescence microscopy reveals cell subpopulations with different sensitivity to the pH calibration. While the majority of the cells were well calibrated, there was found subpopulation of uncalibrated cell as well as singular cells exhibiting anomalous pH calibration due to the staining of acidic organelles. Resolution of cell subpopulations helps to achieve better pH calibration compared to the calibration in cuvette on a cell suspension.     

单个牛视网膜神经细胞内游离Ca2+动态变化的实时观测方法

为观测神经营养因子对胞内Ca2+浓度的影响,将牛视网膜神经细胞内的Ca2+用Fluo-3标记,用搭建的活细胞实时成像荧光显微系统对其成像,并观测在脑源性神经营养因子BDNF等四种神经营养因子的作用下细胞内游离Ca2+浓度([Ca2+]_i)随时间的变化的规律。由于荧光分子有自发衰减效应,故对得到的细胞内荧光强度采用“去衰减效应修正”的处理方法,再现了较真实地代表[Ca2+]_i的荧光强度。修正后的数据显示,加入四种神经因子后,[Ca2+]_i皆有不同程度的升高,与相关文献所描述的类似实验具有相似结果,说明此种对活细胞内荧光标记物的实时动态成像的实验方法可行,去衰减效应修正的数据处理方法可靠。     

基于激光光解诱导荧光技术实现燃烧反应区及中间产物CH_3瞬态可视化

实现火焰反应区和不同中间组分的在线二维瞬态成像,在湍流燃烧的基础研究中具有十分重要的意义。用Nd∶YAG激光器的5倍频输出(212.8nm)作为光源,通过激光光解诱导荧光技术在甲烷/空气预混火焰中,成功实现了火焰反应区的瞬态成像,并首次采用该技术实现了CH_3的在线瞬态成像测量。分析了该方法同其他荧光标示物在反应区二维瞬态成像方法的优势,并研究了火焰燃烧过程中其他燃烧中间产物和不同燃空比对CH_3单脉冲成像的影响,讨论了现有条件下该技术的应用范围。根据实验结果,在燃空比Φ=1.2的条件下,在反应区我们获得了信噪比约为8的单脉冲成像,分析火焰中CH_3的单脉冲成像结果可知火焰燃空比在1.0~1.4之间时,或者火焰中CH_3的浓度大于9.3×1015 molecules·cm-3信噪比较好。该项技术在动力机械及其他研究领域的应用有十分重要的参考价值。     

Fluorescence Polarization as a Functional Parameter in Monitoring Living Cells: Theory and Practice

The use of fluorescence polarization as a functional parameter in monitoring cellular activation calls for the reliable and accurate measurement of the fluorescence intensity and polarization (FI and FP) of microscopic objects. The relevant experimental parameters that enter such measurements are thoroughly discussed. The possibility of executing FP measurements properly by flow-through systems is compared with that of static cytometry. Remarks on the effects of high-power excitation on markers and cells conclude the paper.     

应用于原子荧光光谱分析的空心阴极灯-As灯电流与激发光强度及延时关系的研究

用XGY-6080型全自动双通道原子荧光光谱仪和高速数据采集系统,采集了As空心阴极灯激发光强度和灯电流波形,分析和讨论了As空心阴极灯激发光强度与灯电流幅度和时序之间的关系,为原子荧光光谱分析中选择空心阴极灯电流及脉宽提供一些参考依据.     

Probing DNA Hybridization in Homogeneous Solution and at Interfaces via Measurement of the Intrinsic Fluorescence Decay Time of a Single Label

The hybridization of DNA oligomers including molecular beacons can be detected by measurement of either the decay time or the intensity of a single fluorescent label attached to the end of the respective oligonucleotide. The method works both in solution and solid phase and can distinguish between fully complementary and mismatch sequences as demonstrated for a 15-mer oligonucleotide and a 25-mer molecular beacon. The fluorescence lifetime method is advantageous in (a) requiring a single label (and therefore a single labeling step) only; and (b), being based on measurement of a self-referenced magnitude that is hardly affected by parameters such as fluctuations in light intensity that make measurement of intensity more prone to interferences.     

Analytical Study on the Rate of Sound Transmission Loss in Single Row Honeycomb Sandwich Panel Using a Numerical Method

Honeycomb structures have recently, replaced with conventional homogeneous materials. Given the fact that sandwich panels containing a honeycomb core are able to adjust geometric parameters, including internal angles, they are suitable for acoustic control applications. The main objective of this study was to obtain a transmission loss curve in a specific honeycomb frequency range along with same overall dimensions and weight. In this study, a finite element model (FEM) in ABAQUS software was used to simulate honeycomb panels, evaluate resonant frequencies, and for acoustic analysis. This model was used to obtain acoustic pressure and then to calculate the sound transmission loss (STL) in MATLAB software. Vibration and acoustic analysis of panels were performed in the frequency range of 1 to 1000 Hz. The models analyzed in this design includes 14-single row-honeycomb designs with angles of −45°, −30°, −15°, 0°, +15°, +30°, +45°. The results showed that a-single row and −45°cell angle honeycomb panel in the frequency range of 1 to 1000 Hz had the highest STL as well as the highest number of frequency modes (90 mods). Furthermore, the panel had the highest STL regarding the area under the STL curve (dB∙Hz). The panels containing more frequency mods, have a higher transmission loss. Moreover, the sound transmission loss is more sensitive to the cell angle variable (θ). In other studies, the STL was more sensitive to the number of honeycomb cells in the horizontal and vertical directions, as well as the angle of cells.     

A Method for Measuring the Three-Dimensional Refractive-Index Distribution of Single Cells Using Proximal Two-Beam Optical Tweezers and a Phase-Shifting Mach—Zehnder Interferometer

This paper describes a method for measuring the three-dimensional (3D) refractive-index distribution in a single cell. The method can be used to observe the distribution of cell components without fluorescence staining. The two-dimensional optical path length distributions from multiple directions are obtained by non-contact rotation of the cell. These optical path lengths are converted into the line integrals of the refractive index, and the 3D refractive-index distribution is reconstructed by means of computed tomography. The refractive-index distribution in a breast cancer cell can be measured using a phase-shifting Mach—Zehnder interferometer in conjunction with proximal two-beam optical tweezers.     

新型光敏剂二氢卟吩e_6-C_(15)单甲酯荧光光谱特性及其体内分析应用

研究了二氢卟吩e_6-C_(15)单甲酯在不同溶剂体系中的荧光光谱特性,据此建立了检测血浆中二氢卟吩e_6-C_(15)单甲酯的荧光分析法。首先,通过比较分析二氢卟吩e_6-C_(15)单甲酯在甲醇、乙醇、丙酮、乙腈、磷酸盐缓冲液和水六种不同溶剂体系中的荧光光谱特征,考察了溶剂体系对二氢卟吩e_6-C_(15)单甲酯荧光光谱特性的影响,结果发现甲醇和乙腈是比较理想的溶剂体系,而磷酸盐缓冲液优于水溶液。通过进一步考察有机溶剂含量和溶液pH对二氢卟吩e_6-C_(15)单甲酯发射波长和荧光强度的影响,结果表明pH 7.2磷酸盐缓冲液-乙腈(3:7)混合溶液中二氢卟吩e_6-C_(15)单甲酯荧光强度大、性质稳定。采用pH 7.2磷酸盐缓冲液-乙腈(3:7)混合溶液沉淀SD大鼠血浆蛋白,选择498.00和664.05 nm作为激发波长和发射波长,建立的SD大鼠血浆样品中二氢卟吩e_6-C_(15)单甲酯荧光分析法专属性好,灵敏度高,线性范围为0.50~50.00μg·mL~(-1)(R~2≥0.999),批内和批间精密度RSD均小于10%,提取回收率大于90%。该方法简便、快速、可靠,成功地应用于二氢卟吩e_6-C_(15)单甲酯在SD大鼠体内的药代动力学研究。     

艾比湖流域地表水二维荧光峰值与水质指标关系初探

随着技术的进步与发展,国内外学者早已利用不同的技术手段对水质指标的评价与估算开展了大量研究,但是利用二维荧光峰值估算水质指标的方法尚不多见。以艾比湖流域地表水为研究对象,利用多元统计分析和逐步回归分析方法,对二维荧光峰值和水质指标建立估算模型以初步探究二者的相互关系。结果表明：(1)艾比湖流域不同河流的水质状况不同,博尔塔拉河(简称博河)的TN含量最高,水库及水渠的TP含量最高。(2)同一水样点有三个荧光峰(Peak1,Peak2和Peak3)且强度逐渐减弱,不同河流的荧光峰强度不同,其中博河、水库及水渠的荧光峰强度变幅最大。(3)经过荧光峰值与水质指标的相关分析发现Peak1与BOD₅,Peak2与BOD₅,Peak2与COD,Peak2与DO具有较好的相关性,相关系数分别为：0.479,0.371,0.655和0.618。将单荧光峰值和水质指标建立估算模型,经验证四组估算模型的精度较差,单峰值建模无法达到监测要求。因此,在单荧光峰值建模的基础上,将每个水样的三组荧光峰值和水质指标进行综合建模,建立多元回归方程,经模型验证得出三组荧光峰值与DO的综合模型效果最好,R=0.756, RMSE=1.001。因此二维荧光峰值与水质指标DO的综合估算模型是可行的,对干旱区水质指标监测具有一定的参考意义,为艾比湖流域水资源的开发、利用及保护提供科学依据。