紫杉醇诱导不依赖于Caspase-3细胞类似Paraptosis的荧光光谱分析

研究了紫杉醇(Taxol)诱导人肺腺癌细胞(ASTC-a-1)类似Paraptosis的特征和机理。采用CCK-8技术检测了抑制细胞活性的特性,结果表明大于70 μmol·L-1浓度的紫杉醇可以显著抑制细胞活性;采用激光共聚焦扫描荧光成像从形态上检测了紫杉醇诱导细胞死亡的形态特征,表明紫杉醇诱导了类似副凋亡(Paraptosis)特征的细胞肿胀和细胞质空泡化,而且没有细胞膜皱褶、细胞核浓缩等细胞凋亡的特征;通过基因质粒转染在细胞转染稳定表达基于荧光共振能量转移(FRET)质粒SCAT3,并利用FRET受体光漂白技术和荧光光谱检测分析技术研究了紫杉醇诱导细胞类似Paraptosis过程中Caspase-3的活化特性,结果表明在紫杉醇诱导细胞质肿胀和细胞死亡的过程中Caspase-3没有被活化。以上研究结果表明紫杉醇可以通过类似Paraptosis的方式明显诱导细胞程序性死亡,该过程不依赖于Caspase-3的活性。     

基于毛细管电泳的量子点荧光共振能量转移检测

荧光共振能量转移（FRET）技术作为一种能在生物活体和体外检测纳米级距离变化的工具,为研究生物大分子内部结构、性质、反应机理及其动态监测,乃至定量分析等提供了一条快速简便的途径。由于量子点（QDs）具有独特的光学性质（宽吸收、窄发射、抗光漂白及荧光可调）,近年来基于QDs的FRET体系已在生物医学传感、免疫及活细胞内生物大分子的相互作用方面得到了广泛应用。     

用于在线E-FRET定量成像的自动背景识别与数据筛选

因灵敏性高、无损伤和测量速度快等特性,基于3-cube的荧光能量共振转移(E-FRET)显微成像术是目前最流行的活细胞定量FRET成像技术。为了实现活细胞在线实时FRET定量成像,首先提出了一种细胞图像背景自动识别与图像阈值设定的方法:逐像素统计灰度值出现的次数,第1个峰值处的灰度值确定为背景值;将背景值的β(经验常数)倍设为阈值,将扣除背景的供体激发供体探测通道图像和受体激发受体探测通道图像再次扣除阈值,负值置零后进行逻辑与运算制作用于数据筛选的布尔逻辑模板,并将其用于FRET效率和供受体浓度比的数据筛选。利用所提出的方法对转染了不同FRET质粒的细胞进行活细胞在线动态定量E-FRET成像,得到了与期望值一致的测量结果。     

基于近红外量子点的荧光共振能量转移生物探针构建及应用

本论文构建了基于近红外量子点In P/Zn S和Cy7(C45H44K3N3O16S4)的荧光共振能量转移(FRET)体系,完成了不同p H值和不同浓度下的FRET体系转换效率的检测。检测结果显示:当量子点浓度保持不变时,随着染料浓度的增加,体系转换效率也随之增加,当In P/Zn S量子点与Cy7浓度比为1∶250时,转换效率高达68%。细胞测试结果表明,FRET体系对p H值有较高敏感度,对细胞微环境p H值的检测精度可达0.1,该体系可以作为敏感型FRET探针用于生物微环境检测。     

活细胞核浆粘滞系数的荧光相关谱研究

细胞核浆粘滞系数直接反映一定生理状态下核内微环境的特性,是判断病变细胞和研究核内分子功能机制的重要依据。为了实时、灵敏和无损地测定单个活细胞的核浆粘滞系数,提出了一种基于荧光相关谱(fluorescence correlation spectroscopy, FCS)技术的新型检测方法。研究中利用FCS技术测定绿荧光蛋白分子(EGFP)在细胞核内的扩散系数,并且根据Stokes-Einstein关系式计算出核浆粘滞系数,同时实现了对特定生理条件下核浆粘滞系数的跟踪测量。结果表明：在pH 7.4以及37 ℃的条件下,人肺腺癌细胞(ASTC-a-1)和人宫颈癌细胞(HeLa)的核浆粘滞系数分别是(2.55±0.61)cP和(2.04±0.49)cP, 与传统方法的测量结果一致,并且发现核浆粘滞系数大于细胞质的粘滞系数。以上研究表明FCS技术可精确无损地检测单个活细胞内达飞升量级的微观环境,是探索细胞内生物分子动态行为的有力工具。     

基于近红外上转换荧光共振能量传递体系的均相免疫分析

在上转换纳米晶(UCNPs)为供体的荧光共振能量传递(FRET)生物均相检测体系中,弱的供体光强度使FRET信号难于检测,同时还有来自生物基质的自发荧光干扰。这使得UCNPs不产生背景荧光和散射光的优点不能够充分地体现。针对这个问题,作者利用在800nm处有强近红外光的NaYF4:Yb3+,Tm3+UCNPs作为供体,在784nm处有表面等离子共振吸收带的金纳米粒子(GNPs)作为受体构建了新型的FRET体系。UCNPs偶联抗体(goat antihumanIgG)及GNPs偶联抗原(humanIgG),在抗原抗体免疫亲和作用下两者距离靠近;UCNPs荧光光谱和GNPs吸收光谱有效交叠,使FRET发生。当体系中加入单纯humanIgG,竞争性地争夺与goatantihumanIgG结合位点,破坏FRET构建,供体近红外光增强。根据此对应关系,确定humanIgG检测限为5μg·mL-1。这种方法可适用于更广泛的荧光分析。     

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。     

多糖对白细胞荧光发射特性及变化机制研究

细胞微环境的稳定是保持细胞正常增殖、代谢和功能活动的重要条件,微环境成分的异常变化可使细胞发生病变。采用荧光光谱技术研究离体白细胞在多糖微环境中荧光发射特性的变化规律和发光机制,并进一步的分析了多糖对白细胞的生物活性的影响。实验结果表明：当白细胞受波长为407 nm的激光照射时,发射位于450 nm的荧光。在加入脂多糖或葡聚糖时,白细胞的荧光发射峰的位置不会变化,峰值受到影响。脂多糖的加入会减弱白细胞荧光峰强度,且荧光强度随脂多糖浓度（0～500 μg·mL-1范围内）的增加而持续减弱。而葡聚糖可以一定程度增加白细胞的荧光强度,浓度越高,荧光强度越大。分析认为白细胞发射的450 nm荧光来自发射物质烟酰胺腺嘌呤二核苷酸(NADH)。白细胞内NADH随着离体时间的增长,被氧化成不发荧光的烟酰胺腺嘌呤磷酸二核苷酸（NAD+）,导致细胞荧光峰值下降,从而引起细胞凋亡。加入脂多糖产生的羟自由基（·OH）会与NADH发生氧化反应,因此脂多糖加速了NADH的消耗,导致白细胞荧光减弱,加快细胞凋亡。而葡聚糖主要是由葡萄糖单体组成,葡聚糖的加入会将NAD+还原成NADH,因此延缓了白细胞的凋亡。分析认为脂多糖可以加速白细胞的凋亡,提高细胞发生炎症甚至是肿瘤的机率,而葡聚糖对白细胞有保护作用。该研究为研究肿瘤的发生和发展过程以及治疗提供有价值的参考。     

量子点尺寸对量子点和金纳米粒子之间的荧光共振能量转移的影响

量子点（QDs）所具有的一元激发多元发射的特点预示着QDs在高通量的多元量化生物信息处理领域具有应用前景。采用柠檬酸钠直接还原法制备了粒径为15 nm的Au纳米粒子,利用静电自组装的方法构建了Au与QDs的荧光共振能量转移（FRET）系统。采用该系统研究了QDs的尺寸变化对FRET效率的影响,获得了FRET效率和给体与受体之间交叠积分的关系。结果证明,QDs是构建多元均相免疫检测技术的一个有效给体。     

利用拉曼光谱分析顺铂诱导的胃癌细胞凋亡

利用激光拉曼光谱对顺铂诱导的胃癌细胞凋亡进行分析,胃癌细胞SGC7901经10 μg·mL-1顺铂处理24, 48, 72 h后,一部分用于荧光染色,另一部分用扫描方式收集细胞的拉曼光谱。光谱结果依次经背景扣除、平滑、归一化、基线校正、峰拟合等方法预处理。荧光染色结果显示对照组细胞核染色均匀,而药物处理72 h后,核碎裂,呈致密浓染。拉曼光谱结果显示顺铂处理24, 48, 72 h后,与核酸及蛋白质相关的峰均有降低,其中783, 1 002, 1 343 cm-1峰72 h后依次降低为初始值的52%,64%,76%,表明顺铂能够诱导胃癌细胞凋亡,凋亡细胞内核酸和蛋白质含量降低。以上结果说明,拉曼光谱能提供生物细胞内物质变化的丰富信息,是实时检测细胞凋亡过程的有效手段。     

Quantitative analysis of caspase-3 activation by fitting fluorescence emission spectra in living cells

Confocal fluorescence imaging and fluorescence resonance energy transfer (FRET) technology have been widely used to study protein–protein interactions in living cells. However, it is very difficult to quantitatively analyze FRET efficiency due to the excitation spectral crosstalk and emission spectral crosstalk between donor and acceptor. In this study, we developed a novel method to quantitatively obtain the FRET efficiency by fitting the emission spectra (FES) of donor–acceptor pair, and this method is free from both excitation and emission spectral crosstalk. We used the FES method to quantitatively monitor the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by FES are consistent with that by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM) in living cells stably expressing SCAT3. In this study, the FES was also used to analyze the caspase-3 activation in living cells during anti-cancer drug such as taxol, Artesunate (ART) or Dihydroartemisinin (DHA) treatment. Our results showed that ART or DHA induced apoptosis by a caspase-3-dependent manner, while caspase-3 was not involved in taxol-induced cell death.     

Photobleaching-Based Quantitative Analysis of Fluorescence Resonance Energy Transfer inside Single Living Cell

The current advances of fluorescence microscopy and new fluorescent probes make fluorescence resonance energy transfer (FRET) a powerful technique for studying protein-protein interactions inside living cells. It is very hard to quantitatively analyze FRET efficiency using intensity-based FRET imaging microscopy due to the presence of autofluorescence and spectral crosstalks. In this study, we for the first time developed a novel photobleaching-based method to quantitatively detect FRET efficiency (Pb-FRET) by selectively photobleaching acceptor. The Pb-FRET method requires two fluorescence detection channels: a donor channel (CH ₁ ) to selectively detect the fluorescence from donor, and a FRET channel (CH ₂ ) which normally includes the fluorescence from both acceptor and donor due to emission spectral crosstalk. We used the Pb-FRET method to quantitatively measure the FRET efficiency of SCAT3, a caspase-3 indicator based on FRET, inside single living cells stably expressing SCAT3 during STS-induced apoptosis. At 0, 6 and 12 h after STS treatment, the FRET efficiency of SCAT3 obtained by Pb-FRET inside living cells was verified by two-photon excitation (TPE) fluorescence lifetime imaging microscopy (FLIM). The temporal resolution of Pb-FRET method is in second time-scale for ROI photobleaching, even in microsecond time-scale for spot photobleaching. Our results demonstrate that the Pb-FRET method is independent of photobleaching degree, and is very useful for quantitatively monitoring protein-protein interactions inside single living cell.     

Live morphological analysis of taxol-induced cytoplasmic vacuolization [corrected] in human lung adenocarcinoma cells

Taxol (paclitaxel), one of the most active cancer chemotherapeutic agents, can cause programmed cell death (PCD) and cytoplasmic vacuolization. The objective of this study was to analyze the morphological characteristics induced by taxol. Human lung adenocarcinoma (ASTC-a-1) cells were exposed to various concentration of taxol. CCK-8 was used to assay the cell viability. Atomic force microscopy (AFM), plasmid transfection and confocal fluorescence microscopy were performed to image the cells morphological change induced by taxol. Fluorescence resonance energy transfer (FRET) was used to monitor the caspase-3 activation in living cells during taxol-induced cell death. Cells treated with taxol exhibited significant swelling and cytoplasmic vacuolization which may be due to endoplasmic reticulum (ER) vacuolization. Caspase-3 was not activated during taxol-induced cytoplasmic vacuolization and cell death. These findings suggest that taxol induces caspase-3-independent cytoplasmic vacuolization, cell swelling and cell death through ER vacuolization.     

Spectral measurement of acceptor-to-donor extinction coefficient ratio in living cells

This report presents a simple method named as sp-ECR to determine the molar extinction coefficient ratio (γ(λ_ex)) of acceptor-to-donor in living cells at excitation wavelength λ_ex, which is closely associated with the acceptor cross-excitation, the hardest issue of FRET quantification. sp-ECR determines γ(λ_ex) by spectrally unmixing the emission spectrum of a donor–acceptor tandem construct under λ_ex excitation without any additional references, such that this method can be performed under optimal imaging condition. We used sp-ECR to measure the γ(458) of Venus/Cerulean in living HepG2 cells on a confocal microscope, and the measured values were consistent with those obtained by lux-FRET method. We also used sp-ECR to measure the γ(458) values of Venus/Cerulean and YFP/CFP as well as YFP/GFP, the commonly used FRET FPs pairs in other two kinds of cancer cell lines on the confocal microscope, and found that the extinction coefficients of FPs depended on cell lines. After predetermining the γ(458) of Venus to ECFP, we used sp-ECR method to monitor the staurosporine (STS)-induced dynamical caspase-3 activation in single live A549 cells expressing SCAT3 by spectrally resolving the absolute FRET efficiency of SCAT3, and found that STS-induced caspase-3 activation in single cells is a very rapid process within 20 min.     

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca²⁺; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca²⁺ in biological samples, we demonstrate thatintensity-based measurements of Ca²⁺ with single-wavelength Ca²⁺ probes such as Calcium Crimson may falsely report the actual Ca²⁺ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.     

肿瘤细胞中表达的GFP蛋白的荧光漂白特性的研究

GFP作为生物源性荧光探针具有其他荧光标记物所无法比拟的优势，目前已广泛应用于生物学研究的各个领域。利用常规转染方法将带有EGFP基因的质粒载体导入人肺腺癌肿瘤细胞(ASTC-a-1)，并得到GFP稳定表达的细胞株。研究中发现，肿瘤细胞中表达的GFP长时间暴露于强激发光中会发生非常强列的荧光漂白作用，并且这种漂白作用是不可恢复的。对不同强度的激发光(饱和光源、阻断片ND4／ND8／ND16)对GFP的漂白作用进行了研究，并对冷冻保存样品的光漂白作用进行了初步的探讨。     

新型Eu~(3+)、Tb~(3+)掺杂配合物的合成与荧光性能研究

合成了稀土(Eu3+和Tb3+)与二苯甲酰甲烷(DBM)、2,2′-联吡啶(Dipy)的一系列稀土配合物EuxTb1-x(DBM)3Dipy。元素分析和红外分析确定了配合物的组成,荧光光谱研究了荧光性质。铽掺入配合物后,铽能极大地增强铕的特征荧光,铽对铕配合物的发光有协同作用。在该系列配合物中,不仅有机配体可以将吸收的能量传递给发光的铕离子使其发光,而且铽离子也可将其吸收的能量通过分子内能量传递给铕离子。     

不同有机酸配体对Tb(Ⅲ)离子荧光效率的影响

对合成的七种Tb-配合物的荧光发射光谱和激发光谱进行了比较研究,重点比较了七种Tb-配合物的荧光强度,并从配体结构、能量传递和能级匹配等方面分析了不同配体对Tb(Ⅲ)离子荧光效率的影响,同时讨论了七种Tb-配合物的红外光谱和拉曼光谱。研究表明,配体不影响Tb(Ⅲ)离子的特征发射峰位置,只影响其荧光效率。配体的共轭性、刚性越好,配合物荧光效率越高,二元酸比一元酸更有利于增强Tb(Ⅲ)离子的荧光效率。     

在铕-2-噻吩甲酸-邻菲咯啉三元配合物中La3+对Eu3+的发光影响

合成了七种不同掺杂比例的稀土高氯酸盐(铕掺镧)与2-噻吩甲酸-邻菲咯啉的固态配合物,对配合物进行了元素分析、稀土络合滴定、摩尔电导测定,确定了配合物组成为(Eu1-xLax)·L3·phen·1/2H2O(x=0·000～0·200,L为2-噻吩甲酸,phen为邻菲咯啉),并测定了配体及配合物的IR谱及荧光激发和发射光谱。摩尔电导数据表明,此类配合物为非电解质。红外光谱测定表明,配体2-噻吩甲酸羧基氧与稀土离子配位,配体1,10-邻菲咯啉两个氮原子与稀土离子配位。荧光光谱测定表明,Eu3 处于无反演对称中心格位上,Eu3 配合物发射强度增大。配合物中La3 对Eu3 的发光产生敏化增强效应,当La3 掺入量为0·005mol时敏化强度最大,随着La3 浓度的增大,对Eu3 的发光敏化强度降低。