超分辨成像中基于模板函数对多个荧光分子定位

在生物结构和功能成像领域里,超分辨成像成为近年来研究的热点,而荧光分子定位,特别是多荧光分子的定位,是超分辨成像的一个重要环节.本文基于模板函数和非线性拟合给出多个荧光分子定位算法,并利用该算法分析信噪比和两个荧光分子距离对荧光分子定位精度的影响,指出了只有信噪比大于5,两个荧光分子的距离大于8个像素时,才能获得精确的荧光分子的定位.结果证明该方法的合理性和有效性.     

DNA单分子近场光学成像与荧光探测

介绍了扫描近场光学(SNOM-Scanning Near-Field Optical Microscope)／原子力显微镜（AFM－Atomic Force Microscope)系统（SNO／AM）的工作原理。在AFM模式和SNOM模式下对DNA分子进行成像和荧光探测,得到了清晰的DNA单分子的形貌像和荧光像。由形貌圆像得到的DNA分子尺寸横向为20nm,高度为2nm,其中包含了探针形貌的影响。实验中采Tapping模式的AFM成像,样品经多次搜索扫描无明显损坏。AFM模式的分辨率优于1nm。SNOM模式下DNA分子形貌像和荧光像清晰,由近场荧光分布可以确定分子取向和浓度。用YOYO－1染料对λDNA分子进行染色和荧光探测。通过对DNA分子多个截面进行测量,分析染料 与DNA结合状态。     

双光子激发荧光各向异性度的成像

荧光各向异性度 (fluorescence anisotropy) 测量可以获得荧光分子的转动速度信息,进而了解分子质量、结构、以及与周边环境的相互作用情况 . 围绕一台双光子激发扫描荧光成像系统,通过改变外光路和图像记录与处理程序,从而实现了双光子激发荧光各向异性度成像,并针对一些典型样品和体系,展示了该方法的应用 . 实验中观察了 FITC 荧光分子、 FITC 结合的 CD44 抗体分子及与肿瘤细胞表面受体结合的 FITC-CD44 抗体分子 . 测量结果表明,不同分子质量、不同微观环境状态下的荧光分子,其各向异性度大小不同,在各向异性度图中能够被明显区分 . 荧光各向异性度成像能够定量测量样品微区的各向异性度值,并以二维图像的形式直观表达,是各向异性度测量与成像技术的良好结合 .     

BK通道在细胞膜上的单分子定位及空间分布*

摘要目的：研究大电导、钙离子和电压激活的钾离子通道（BK通道）在HEK293 细胞膜上的单分子定位及其总体空间分布情况。 方法：分别用mEos2、Dronpa 等荧光蛋白标记BK通道的α亚基和辅助性β2 亚基,将这些质粒在HEK293 细胞内瞬时转染以表 达通道蛋白,然后用激光共聚焦荧光显微成像、全内反射荧光显微成像、光敏定位荧光成像等技术观察BK通道的亚细胞定位及 单分子分布,并用电生理实验技术检测荧光蛋白对BK通道有影响。结果：激光共聚焦荧光显微成像和全内反射荧光显微成像技 术只能在亚细胞水平定位通道蛋白,BK 通道在细胞膜上聚集并形成不规则的蛋白簇,它的α亚基和β2 亚基在细胞膜上完全共 定位;光敏定位荧光成像技术成功定位BK通道蛋白簇里面的单分子,虽然α和β2 亚基紧紧靠在一起,它们之间依然存在空间 距离;BK通道的质膜表达和功能特性不受荧光蛋白的影响。结论：BK通道蛋白簇里面包含大量的α和β2 亚基的蛋白单分子, 它们紧密地聚集在一起,但是并没有完全共定位,在分子水平上揭示了BK通道α和β亚基功能耦合的结构基础,为以后研究大 分子蛋白质间的相互作用机制提供了很好的分子模型,光敏定位荧光成像技术作为一种全新的单分子荧光成像手段,在基因表 达、信号通路、蛋白质相互作用等许多重要生命活动的研究中发挥重要作用。     

BK通道在细胞膜上的单分子定位及空间分布

目的：研究大电导、钙离子和电压激活的钾离子通道（BK通道）在HEK293细胞膜上的单分子定位及其总体空间分布情况。方法：分别用mEos2、Dronpa等荧光蛋白标记BK通道的α亚基和辅助性β2亚基,将这些质粒在HEK293细胞内瞬时转染以表达通道蛋白,然后用激光共聚焦荧光显微成像、全内反射荧光显微成像、光敏定位荧光成像等技术观察BK通道的亚细胞定位及单分子分布,并用电生理实验技术检测荧光蛋白对BK通道有影响。结果：激光共聚焦荧光显微成像和全内反射荧光显微成像技术只能在亚细胞水平定位通道蛋白,BK通道在细胞膜上聚集并形成不规则的蛋白簇,它的仅亚基和β2亚基在细胞膜上完全共定位;光敏定位荧光成像技术成功定位BK通道蛋白簇里面的单分子,虽然α和β2亚基紧紧靠在一起,它们之间依然存在空间距离;BK通道的质膜表达和功能特性不受荧光蛋白的影响。结论：BK通道蛋白簇里面包含大量的α和β2亚基的蛋白单分子,它们紧密地聚集在一起,但是并没有完全共定位,在分子水平上揭示了BK通道α和p亚基功能耦合的结构基础,为以后研究大分子蛋白质间的相互作用机制提供了很好的分子模型,光敏定位荧光成像技术作为一种全新的单分子荧光成像手段,在基因表达、信号通路、蛋白质相互作用等许多重要生命活动的研究中发挥重要作用。     

量子点荧光探针在生物成像中的应用进展

量子点荧光探针是近几年发展起来的一种新型荧光标记物,拥有荧光染料及荧光蛋白所不能比拟的独特优势,已经在细胞功能研究及细胞表面和内部功能分子的探测、组织的成像和病灶的定位等方面得到了较为广泛的应用。本文对量子点的光学特性、生物化修饰及其在生物成像等方面的应用进展进行了较为详细的介绍,并展望了其应用发展。     

基于单分子定位的超分辨显微中荧光激发密度的优化

基于单分子定位的超分辨显微技术中,荧光点的中心位置可通过对每个荧光点进行单点扩散函数(point spread function,PSF)逐个拟合定位或对多个荧光点进行多PSF同时拟合定位获得。定位误差以及图像采样时间与荧光激发密度直接相关。为定量得到基于这两种算法的最优荧光激发密度,模拟了成像与定位过程,比较了常见的几种荧光分子在采用基于单PSF逐个拟合和多PSF同时拟合两种算法定位时的中心定位误差、荧光获取比率、获取的有效荧光点数与荧光激发密度间的关系,进而得到了采用这两种算法对不同荧光染剂进行超分辨成像时的最优荧光激发密度。该结果对单分子成像过程中荧光激发密度的选择和激发激光的强度控制具有指导意义。     

PNAS：肿瘤活体实时成像技术新思路

在国家自然科学基金等的资助下,湖南大学化学生物传感与计量学国家重点实验室王柯敏课题组在核酸适配体的肿瘤活体荧光分子成像研究中。首次提出了基于细胞膜蛋白触发构型变化的”激活式核酸适配体探针”概念,设计合成了一种针对肿瘤细胞特异性表达蛋白的探针。显著提高了肿瘤细胞成像反差,缩短了检测时间,并成功用于裸鼠肿瘤活体实时荧光成像。     

生物分子水溶液的蒙特卡罗模拟——Ⅱ.缬氨酸溶液

本文对由115个水分子包围一个中性缬氨酸分子所组成的分子集团做了蒙特卡罗模拟.模拟的温度是298K.对缬氨酸羧基区、氨基区和异丙基区分别提取了平均水—水和水—缬氨酸分子的相互作用能的径向分布函数,水分子的氧原子和氢原子的径向分布函数以及水分子偶极矩的取向关联函数.此外,我们还求出了所模拟溶液的构型比热和缬氨酸分子分区及全体的第一个水化层的平均水分子数.     

基于单分子量子相干调制的细胞温度成像技术

目的 细胞温度成像可以帮助科学家研究和理解细胞内部的温度分布,揭示细胞代谢和生物化学过程的关键信息。目前,基于荧光温度探针的细胞温度成像技术存在低温度分辨率和有限测量范围等限制。本文旨在利用单分子量子相干过程依赖温度的特性,开发一种单细胞温度成像和实时检测技术。方法 基于飞秒脉冲激光制备延时和相位可调的飞秒脉冲对,调制的脉冲对通过显微系统激发细胞内标记的荧光单分子,之后收集并记录每个荧光光子的到达时间。利用单分子相干过程与周围环境温度的关系,定义单分子量子相干可视度（V）,建立V与环境温度的对应关系。通过调制解调荧光光子的到达时间,获取单分子周围环境温度,结合扫描成像,实现细胞的温度成像和实时检测。结果 该方法可以实现高精度（温度分辨率<0.1℃）和大范围温度（10～50℃）的温度成像和测量,并观测到了单个细胞代谢相关的温度变化。结论 该研究有助于深入了解细胞代谢、蛋白质功能和疾病机制,为生物医学研究提供重要工具。     

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

The use of fluorescence anisotropy decay of poly d(A-T) ethidium bromide complex to estimate the unwinding angle of the double helix.

We measured the fluorescence decay under polarized light, of ethidium bromide bound to the poly d(A-T) isolated from Cancer Pagurus. The decay of the whole fluorescence is a single exponential function revealing a good homogeneity of the binding sites. The anisotropy decay due to energy transfers between the ethidium bromide molecules bound to a same poly d(A-T) molecule has been analysed, with a Monte Carlo calculation. We found the dye unwinds the poly d(A-T) duplex by an angle of 17 degrees plus or minus 2 degrees. This result is in agreement with the value previously found in the case of calf thymus DNA-ethidium bromide complex, although the base compositions of the two nucleic acids are different.     

Single-molecule chiroptical spectroscopy: Fluorescence excitation of individual helicene molecules in polymer-supported thin-films

We present results of fluorescence excitation circular dichroism studies of the chiroptical response of single (bridged triarylamine) helicene molecules immobilized at a polymer interface. We extract directly dissymmetry parameters, and corresponding probability distributions, associated with the single-molecule fluorescence excitation associated with modulation of a circular polarized excitation field for three different excitation wavelengths (405, 440, 457 nm) showing circular dichroism in bulk films. The observed single molecule chiroptical response is anomalously large in comparison with the results of time-dependent density functional calculations, and the observed defocused emission patterns seem to indicate a higher multipole nature to the transition probed. Our results provide new insights into chiroptical properties of chiral fluorophores that are hidden under the extensive averaging associated with conventional chiroptical probes.     

A fluorescence photobleaching study of the microsecond reorientational motions of DNA.

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.     

Orientation and spectral properties of two stilbazolium merocyanine dyes in stretched and unstretched polyvinyl alcohol films

Spectral properties (anisotropy coefficients calculated for absorption, emission and fluorescence decay time) of two stilbazolium merocyanine dyes have been determined to evaluate the applicability of these dyes as sensitizers in photodynamic therapy. The dyes were embedded in an anisotropic polymer matrix. Analysis of the emission decay components measured in polarized light provides information on the interactions of the dye molecules with the polymer matrix being a model of an anisotropic biological system. Different values of the emission anisotropies obtained from various polarized components of fluorescence decays have shown that the orientations of the dye molecules influence their interactions with the polymer. This means that differently oriented dye molecules located in biological systems should exhibit different interactions with membranes. The chain length and type of side groups attached as well as the salt form of the dye molecule were shown to influence the dye-polymer interactions and should be taken into account before the application of merocyanine dyes in medicine. These dyes seem to be promising optical sensors with spectral properties, including the calculated anisotropy coefficients, sensitive to the molecular environment, useful to study orientation and interaction with neighbouring molecules in biological membranes.     

Measurement of rotational motion in membranes using fluorescence recovery after photobleaching.

A method has been developed for the measurement of the rotational motion of membrane components. In this method fluorescent molecules whose transition dipole moments lie in a given direction are preferentially destroyed with a short intense burst of polarized laser radiation. The fluorescence intensity, excited with a low intensity observation beam of polarized laser radiation, changes with time as the remaining fluorescent molecules rotate. The feasibility of the method has been demonstrated in a study of the rotation of the fluorescent lipid probe, dil ([bis,-2-(N-octadecyl-3,3-dimethyl-1-benzo[b]pyrrole]-trimethincyanine iodide) incorporated into membranes composed of distearoylphosphatidylcholine (DSPC) or dipalmitoylphosphatidylcholine (DPPC) and 0.20 mol% cholesterol, below the main chain-melting transition temperatures of the phosphatidylcholines. Rotation times in the 0.6-800 s range were observed. The fluorescence recovery (or decay) curves are in satisfactory agreement with theoretical calculations.     

Packing of binding stained DNA strands in bacteriophage lambda

Distribution of stainable DNA strands in phage lambda has been studied by polarized fluorescence. The effect of tight DNA-packing on fluorescence depolarization of complex dye-DNA was calculated. It is shown that stainable DNA in the phage is not concentrated in the central region. The arrangement of acridine orange molecules on the surface layers of the packed DNA is the most probable one.     

Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast

We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.