一种利用荧光标记检测氨肽酶N抑制剂与肿瘤细胞结合的方法

目的：建立利用荧光标记法检测氨肽酶抑制剂和肿瘤细胞结合的方法。方法：以异硫氰酸荧光素（FITC）标记氨肽酶N抑制剂LYRM03和Bestatin,制备荧光探针F1TC—LYRM03、FITC—Bestatin,应用荧光显微成像观察和流式细胞仪检测标记化合物FITC—LYRM03、FITC-Bestatin对肿瘤细胞的结合与氨肽酶N抑制活性的相关性。结果：化合物LYRM03和Bestatin具有肿瘤细胞的氨肽酶N抑制活性,荧光标记化合物FITC—LYRM03、FITC-Bestatin能与肿瘤细胞有不同程度的结合。结论：标记化合物FITC—LYRM03、FITC—Bestatin和肿瘤细胞的结合与对肿瘤细胞的氨肽酶N抑制活性相一致。     

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。     

定量研究细胞表面蛋白质分子与其配位体(抗体)之间的动态结合

以定量方法确定细胞表面蛋白质分子的动态行为,可深入了介细胞的生命活动。 本文研究FITC—11—4—1Fab和11—4—1Fab同时与细胞表面糖蛋白分子进行竞争性抑制结合,测定一定时间内FITC—11—4—1Fab与Clld细胞表面糖蛋白分子的专一性结合的机率,这有助于了介细胞表面糖蛋白分子的结构和运动。 用荧光分光光度计定量的测量FITC—11—4—1Fab和11—4—1Fab对Clld细胞表面糖蛋白分子进行竞争性抑制结合后所发出的相对荧光强度,再经过计算得知每一Clld细胞表面所能确切结合的FITC—11—4—1Fab分子的真正数目。 实验结果证明FITC—11—4—1Fab的浓度增大,则每一Clld细胞表面糖蛋白分子真正能结合的FITC—11—4—1Fab数目也增多。     

近场扫描光学成像结合量子点标记的纳米技术在细胞生物学中的应用

近场扫描光学显微镜（NSOM）对传统的光学分辨极限产生了革命性的突破,可在超高光学分辨率下无侵人性和无破坏性地对生物样品进行观测。量子点（QDs）具有极好的光学性能,如荧光寿命长、激发谱宽、生物相容性强、光稳定性好等优点,适合先进的生物成像。NSOM结合QDs标记的纳米技术被应用在细胞生物学中。通过纳米量级NSOM免疫荧光成像（50nm）对特定蛋白分子在细胞表面的动态分布进行可视化研究和数量化分析,阐明了蛋白分子在不同细胞过程中的作用机制。因此,NSOM／QD基成像系统提供了单个蛋白分子最高分辨率的荧光图像,为可视化研究蛋白分子机制的提供了一种强有力的工具。     

IgG-Eu-IDPA对抗原IOV的时间分辨荧光免疫检测

镧系螯合物已经被广泛应用于高灵敏度的时间分辨荧光免疫分析.但是,使用紫外光激发镧系螯合物会对生物分子和细胞产生很大的损害.合成了能够被可见光(最大波长565nm)激发的IgG-Eu-IDPA,并测量了IgG-Eu-IDPA的光谱属性,如荧光寿命、在不同pH值和不同浓度环境下的荧光强度.在自制的时间分辨荧光仪上使用IgG-Eu-IDPA作为荧光探针,检测IOV抗原.数据显示,它的灵敏度远远高于传统的荧光仪.结果表明,IgG-Eu-IDPA能够在高灵敏度的原位和活体分析中作为一种新的、有潜力的荧光探针.     

Stokes参量共焦显微术用于弱各向异性物质的成像研究

提出将基于Stokes参量的偏振共焦显微成像技术应用于弱各向异性物质的成像研究。通过将分振幅Stokes参量测量法与共焦扫描成像技术相结合的方法,得到了基于Stokes参量测量的偏振共焦显微成像系统。利用该系统对具有弱各向异性的生物组织样品进行逐点测量,通过四个通道同时测量获得全部的Stokes参量。再以计算得到的偏振参量作为成像物理量进行图像重建,获得对应Stokes参量、偏振度、相位差、方位角和椭率角的空间分布图像,从而对生物组织实现细胞水平的偏振显微成像研究。实验结果表明:基于Stokes参量的偏振共焦显微成像技术能够获得弱各向异性的生物组织样品的显微图像,并通过比较样品的Stokes参量及相关偏振参量的分布图像,提取样品全部的偏振信息,从而为生物组织的特性研究提供更丰富的信息。     

遗传密码扩充技术及其在蛋白质功能研究及标记成像中的应用

遗传密码扩充(genetic code expansion, GCE)技术利用终止密码子将非天然氨基酸掺入到蛋白质中,再结合点击反应对蛋白质实现定点标记。相较于荧光蛋白、标签抗体等其他标记工具,该技术在蛋白标记中使用的化合物分子较小、对蛋白空间结构影响较小,且能通过点击反应实现蛋白分子与染料分子1︰1的化学计量比,从而能够依据荧光强度对蛋白质定量。因此,在活细胞单分子追踪和超分辨率显微成像等需要细胞长时间暴露在高激光功率下的研究中,GCE技术具有极大的优势。同时,该技术也为提高活细胞成像过程中的定位精度和分子计数准确度奠定了基础。文中旨在总结近年来GCE技术在蛋白质研究中的应用进展,特别是在蛋白质标记成像方面的应用进展。     

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。     

对同一细胞内DNA、RNA和蛋白含量相关测定的显微荧光光度法

用荧光染料DAPI、PyroninY和FITC分别染同一细胞内DNA、RNA和蛋白.在紫外光、绿光和兰光顺序激发后,用MPVⅡ显微荧光光度计测量反映单个细胞内DNA、RNA和蛋白含量的荧光强度.根据荧光发射光谱分析,每种染料荧光之间的干扰是可以忽略的.测量结果与FCM获得的结果是一致的.显微荧光光度术对单个细胞多参数相关测量的优点是简单、便宜,并可用于对活细胞获得形态学和定量细胞化学的组合信息.     

A蛋白定向固定抗体用于椭偏光学生物传感器免疫检测

椭偏光学生物传感器是在椭偏光学显微成像技术的基础上发展的一项生物传感技术。它能够直接观测固体表面上的生物分子面密度,毋需任何标记辅助,适合发展成为一种无标记免疫检测技术。研究了在硅片表面上通过A蛋白定向固定抗体分子用于椭偏光学生物传感器免疫检测的可能性。实验结果表明,通过A蛋白固定抗体得到的抗体膜层的均一性和固定量的重复性能够保证椭偏光学生物传感器免疫检测结果的质量。通过A蛋白定向固定的抗体的抗原结合位点趋向一致,显著提高了抗体与抗原结合的能力。此外,通过蛋白A固定的免疫球蛋白G分子能够结合更多的多克隆抗体分子说明通过A蛋白固定的蛋白质分子能够较好地保持其空间构象。     

In vivo imaging of the actin polymerization state with two-photon fluorescence anisotropy

Using two-photon fluorescence anisotropy imaging of actin-GFP, we have developed a method for imaging the actin polymerization state that is applicable to a broad range of experimental systems extending from fixed cells to live animals. The incorporation of expressed actin-GFP monomers into endogenous actin polymers enables energy migration FRET (emFRET, or homoFRET) between neighboring actin-GFPs. This energy migration reduces the normally high polarization of the GFP fluorescence. We derive a simple relationship between the actin-GFP fluorescence polarization anisotropy and the actin polymer fraction, thereby enabling a robust means of imaging the actin polymerization state with high spatiotemporal resolution and providing what to the best of our knowledge are the first direct images of the actin polymerization state in live, adult brain tissue and live, intact Drosophila larvae.     

Fluorescence generalized polarization of cell membranes: a two-photon scanning microscopy approach.

We use the lipophilic fluorescence probe Laurdan to study cell membranes. The generalized polarization (GP) of Laurdan-labeled cells contains useful information about membrane fluidity and polarity. A high GP is usually associated with low fluidity, low polarity, or high cholesterol content of the membranes, and a low GP is the opposite. We have combined the GP method and two-photon fluorescence microscopy to provide an alternative approach to study cell membranes. Using two-photon excitation in a conventional microscope offers great advantages for studying biological samples. These advantages include efficient background rejection, low photodamage, and improved depth discrimination. We performed GP measurements on mouse fibroblast cells and observed that both intensity and GP images are not spatially uniform. We tested for possible GP artifacts arising from cellular autofluorescence and lifetime quenching, using a procedure for background fluorescence subtraction and by direct lifetime measurements in the microscope. GP measured in a single cell displays a broad distribution, and the GP of 40 different cells grown on the same cover glass is also statistically distributed. The correlations between intensity and GP images were analyzed, and no monotonic dependence between the two was found. By digitally separating high and low GP values, we found that high GP values often associate with the regions of the plasma membrane and low GP values link with the nuclear membranes. Our results also show local GP variations within the plasma and nuclear membranes.     

Improved quantification of collagen anisotropy with polarization‐resolved second harmonic generation microscopy

Imaging tissue samples by polarization‐resolved second harmonic generation microscopy provides both qualitative and quantitative insights into collagen organization in a label‐free manner. Polarization‐resolved second harmonic generation microscopy goes beyond simple intensity‐based imaging by adding the laser beam polarization component and applying different quantitative metrics such as the anisotropy factor. It thus provides valuable information on collagen arrangement not available with intensity measurements alone. Current established approaches are limited to calculating the anisotropy factor for only a particular laser beam polarization and no general guidelines on how to select the best laser beam polarization have yet been defined. Here, we introduce a novel methodology for selecting the optimal laser beam polarization for characterizing tissues using the anisotropy in the purpose of identifying cancer signatures. We show that the anisotropy factor exhibits a similar laser beam polarization dependence to the second harmonic intensity and we combine it with the collagen orientation index computed by Fast Fourier Transform analysis of the recorded images to establish a framework for choosing the laser beam polarization that is optimal for an accurate interpretation of polarization‐resolved second harmonic generation microscopy images and anisotropy maps, and hence a better differentiation between healthy and dysplastic areas.

SHG image of skin tissue (a) and a selected area of interest for which we compute the SHG intensity (b) and anisotropy factor (c) dependence on the laser beam polarization and also the FFT spectrum (d) to evaluate the collagen orientation index.     

One- and two-photon-induced fluorescence from recombinant green fluorescent protein

The fluorescence spectral properties of recombinant green fluorescent protein (rGFP) were examined with one- and two-photon excitations using femtosecond pulses from a Ti:sapphire laser. Intensity-dependent properties of the two-photon-induced fluorescence from rGFP excited by an 800-nm, 100-fs laser beam were reported, and the two-photon excitation cross section of rGFP was measured at 800 nm as about 160 x 10(-50) cm(4)s/photon. The possible excited-state proton transfer between two electronic states at about 400 nm in protonated (RH) species and 478 nm in deprotonated (R(-)) species in rGFP was confirmed by fluorescence and fluorescence excitation anisotropy spectra. A subelectronic state (or vibronic progression) at about 420 nm in RH species was identified, which was relatively stable and not involved in the excited state proton transfer in rGFP upon irradiation.     

Membrane order and molecular dynamics associated with IgE receptor cross-linking in mast cells

Cholesterol-rich microdomains (or "lipid rafts") within the plasma membrane have been hypothesized to exist in a liquid-ordered phase and play functionally important roles in cell signaling; however, these microdomains defy detection using conventional imaging. To visualize domains and relate their nanostructure and dynamics to mast cell signaling, we use two-photon (760 nm and 960 nm) fluorescence lifetime imaging microscopy and fluorescence polarization anisotropy imaging, with comparative one-photon anisotropy imaging and single-point lifetime and anisotropy decay measurements. The inherent sensitivity of ultrafast excited-state dynamics and rotational diffusion to the immediate surroundings of a fluorophore allows for real-time monitoring of membrane structure and organization. When the high affinity receptor for IgE (FcepsilonRI) is extensively cross-linked with anti-IgE, molecules associated with cholesterol-rich microdomains (e.g., saturated lipids (the lipid analog diI-C(18) or glycosphingolipids)) and lipid-anchored proteins coredistribute with cross-linked IgE-FcepsilonRI. We find an enhancement in fluorescence lifetime and anisotropy of diI-C(18) and Alexa 488-labeled IgE-FcepsilonRI in the domains where these molecules colocalize. Our results suggest that fluorescence lifetime and, particularly, anisotropy permit us to correlate the recruitment of lipid molecules into more ordered domains that serve as platforms for IgE-mediated signaling.     

Differential polarization imaging. II. Symmetry properties and calculations

Various differential polarization images or Mueller images of model objects are generated using the equations derived in the previous paper (paper I of this series). These calculated images include models of the higher-order organization of metaphase chromosomes, and show the applicability of the differential polarization imaging method to the elucidation of complex molecular organizations. Then, the symmetry behavior of the Mueller matrix elements upon infinitesimal rotations of the optical components about the optical axis of the imaging system is presented. It is shown that the rotational properties of the Mueller images can be used to eliminate the linear polarization contributions to the M14 and M44 images, which appear when these images are generated with imperfect circular polarizations. The relationships between the 16 bright-field Mueller images for four different media, i.e., linearly and circularly isotropic, circularly anisotropic, linearly anisotropic, and linearly and circularly anisotropic, are also derived. For the first three cases simple relationships between the Mueller images are found and phenomenological equations in terms of the optical coefficients are derived. In the last case there are no specific relationships between the Mueller images and instead we briefly present Schellman and Jensen's method for treating this type of medium. The criterion of spatial resolution between adjacent domains of different optical anisotropy is then derived. It is found that in transitions between domains of opposite anisotropy the classical Rayleigh limit must be replaced by a magnitude criterion which depends on the limits of the sensitivity of the detection. Finally, the feasibility of optical sectioning in differential polarization imaging is demonstrated.     

Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy

The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as determined by two-photon FCS measurements, increases by a factor of two, indicating dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these monomers unfold, as evidenced by the continual decrease in the tryptophan steady-state anisotropy, average lifetime, and rotational correlation time, concomitant with secondary structural changes. These results help to elucidate the unfolding pathway of the tubulin heterodimer and demonstrate the value of FCS measurements in studies on oligomeric protein systems.     

Imaging lipid lateral organization in membranes with C-laurdan in a confocal microscope

Lateral organization of biological membranes is frequently studied using fluorescence microscopy. One of the most widely used probes for these studies is 2-dimethylamino-6-lauroylnaphthalene (laurdan). The fluorescence of this probe is sensitive to the environment polarity, and thus laurdan reports the local penetration of water when inserted in membranes. Unfortunately, this probe can only be used under two-photon excitation due to its low photostability. This is a very important limitation, because there are not too many laboratories with capability for two-photon microscopy. In this work, we explored the performance of 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), a carboxyl-modified version of laurdan, for imaging biological membranes using a conventional confocal microscopy setup. We acquired generalized polarization (GP) images of C-laurdan inserted in giant unillamelar vesicles composed of binary mixtures of lipids and verified that the probe allows observing the coexistence of different phases. We also tested the performance of the probe for measurement with living cells and registered GP images of melanophore cells labeled with C-laurdan in which we could observe highly ordered regions such as filopodia. These findings show that C-laurdan can be successfully employed for studies of membrane lateral organization using a conventional confocal microscope and can open the possibility of studying a wide variety of membrane-related processes.     

Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.     

Fluorescence recovery spectroscopy as a probe of slow rotational motions.

Pump-and-probe techniques can be used to follow the slow rotational motions of fluorescent labels bound to macromolecules in solution. A strong pulse of polarized light initially anisotropically depletes the ground-state population. A continuous low-intensity beam of variable polarization then probes the anisotropic ground-state distribution. Using an additional emission polarizer, the generated fluorescence can be recorded as it rises towards its prepump value. A general theory of fluorescence recovery spectroscopy (FRS) is presented that allows for irreversible depletion processes like photobleaching as well as slowly reversible processes like triplet formation. In either case, rotational motions modulate recovery through cosine-squared laws for dipolar absorption and emission processes. Certain pump, probe, and emission polarization directions eliminate the directional dependence of either dipole and simplify the resulting expressions. Two anisotropy functions can then be constructed to independently monitor the rotations of either dipole. These functions are identical in form to the anisotropy used in fluorescence depolarization measurements and all rotational models developed there apply here with minor modifications. Several setups are discussed that achieve the necessary polarization alignments. These include right-angle detection equipment that is commonly available in laboratories using fluorescence methods.