有机荧光团及荧光标记细胞的阴极荧光成像研究

目的 阴极荧光（CL）成像是一种以电子束为激发源的高分辨荧光成像技术,但生物材料对电子束的敏感性限制了CL技术在生命科学中的广泛应用。为了研究和发展CL技术在生物样品中的应用,本文旨在通过探究电子辐照引起碳基材料的结构损伤、有机基团的降解及荧光猝灭等问题,深入理解电子源对有机荧光团的激发特性。方法 本研究应用扫描电镜（SEM）和阴极荧光谱仪系统（SEM-CL）,研究电子源对有机荧光团及荧光探针标记细胞的激发特性,观测了有机物的CL信号的发射特性、强度衰减、成像方式及特点。结果 实验结果显示,在低能量（2.5～5 keV）和低束流（～10 pA）电子辐照下,有机荧光微珠发射出较强的荧光,CL像分辨率达到～30 nm。荧光微珠经过12 min辐照,信号强度衰减了25%,CL像仍保持了可接受的发光强度和足够的信噪比。此外,还获得了从细胞表面到内部一定深度内,荧光标记的亚细胞结构信息。结论 在SEM-CL系统中,可以同时获得由电子束激发产生的电子像和CL像,实现阴极荧光与电子显微镜关联（CCLEM）成像。本实验的研究结果为CCLEM技术应用于生物结构研究提供了数据及技术支持。     

利用调制荧光仪在线监测叶绿素荧光

介绍了利用便携式调制荧光仪PAM-2100在线监测叶绿素荧光的技术。该技术不影响植物的自然光合状态,司以在线监测Ft、F′m、Y、rETR、qP、qN或NPQ、PAR和叶温等指标。以凤眼莲为例进行了在线监测,每隔5min监测一次,共进行了225min的监测。结果表明叶绿索荧光参数的变化依赖于PAR的变化。Ft、rETR、qN和NPQ的变化与PAR的变化趋势一致,F′m、Y和qP的变化与PAR的变化相反。通过对风眼莲的在线监测,说明该技术是可靠的,具有简单、快速、灵敏等特点。随着新型调制荧光仪的出现,该技术可能在植物生态学领域得到广泛应用。     

用荧光雄鼠成功繁殖荧光鼠

GFP转基因鼠在医学实验中应用较广,由于这种鼠全身所有的细胞都带荧光,可取这种鼠的细胞在体外做各种处理,然后回输照射鼠,可以在照射鼠体内跟踪这些细胞的转化与分布,因此荧光鼠有着广泛的用途.我们在只有荧光雄鼠的情况下,繁殖了荧光鼠后代.由于荧光鼠全身是黑毛,为了更好地区别杂交后的子代是否是荧光鼠,我们用白色的杂交鼠与荧光雄鼠杂交,结果所生的子代全部是黑毛,经过回交,得到的荧光鼠后代,外周血中GFP阳性细胞百分比达到了95.8％,达到了做照射鼠治疗的研究要求,现报道如下.     

绿色荧光蛋白

来源于水母Aequorea victoria的绿色荧光蛋白(green fluorescent protein, GFP)现已成为在生物化学和细胞生物学中研究和开发应用得最广泛的蛋白质之一. 其内源荧光基团在受到紫外光或蓝光激发时（λ_max=395 nm, 小峰在479 nm）可高效发射清晰可见的绿光. GFP的高分辨率晶体结构为了解和研究蛋白质结构和光谱学功能关系提供了一个极好的机会. GFP已成为一个监测在完整细胞和组织内基因表达和蛋白质定位的理想标记. 通过突变和蛋白质工程构建的GFP嵌合蛋白在生理指示剂、生物传感器、光化学领域以及生产发光纤维等方面展示了广阔前景.     

荧光定量PCR

荧光定量PCR又可以分为TaqMan探针和SYBR Green I荧光染料两种方法。比较而言,探针杂交技术在原理上更为严格,所得数据更为精确,荧光染料技术则成本更为低廉,实验设计更为简便。也许作为确认微阵列数据的金标准,TaqMan有些名不副实。TaqMan与SYBR Green孰优孰劣？在选择实验方案时要根据实验目的,以及对数据精度的要求来决定。     

荧光细胞剂量计

小鼠经钴-60Υ射线全身照射后,外周血白细胞中出现红、黄色荧光细胞比值上升,它与照射后时间和辐照量有相依关系。本文探讨钴-60Υ,射线100伦—705伦照射后红、黄色荧光细胞比值与辐照量的关系。发现在此辐照量范围内与红、黄色荧光细胞比值有线性关系。因此红、黄色荧光细胞比值上升可作为早期急性放射损伤诊断依据之一,亦可作为生物剂量指标。材料和方法本实验应用华北制药厂动物场饲养的纯种小白鼠,体重20—23克,雄性,每组10只,共分五组。照射前后均自尾部取血,经过吖啶橙染色,用紫外光显微镜观察,检查每百个白细胞中红色荧光细胞和黄色荧光细胞(简称红、黄色荧光细胞)与绿色荧光细胞的比值。     

荧光分光光度计

一、前言早在三百多年前,人们就知道了某些液体和固体的荧光现象。利用荧光分析法进行物质的定性和定量分析已有几十年的历史,由于近年来仪器研制的迅速发展,使荧光分析法获得愈来愈广泛的应用。荧光分析法是一种灵敏度很高的定量测定法,它比一般分光光度法灵敏度可高2—3个数     

环化荧光蛋白生物探针荧光寿命成像研究进展

环化荧光蛋白探针作为一种新型的基因编码生物探针,是由荧光蛋白和对探测物有特异性响应的结合域蛋白构成的。由于其独特的构造方式使其在对探测物检测时具有较高的灵敏度和较大的动态范围,引起了越来越多科学家的兴趣,已发展成为监测生命活动的一种强有力工具。目前越来越多基于环化荧光蛋白技术开发的生物探针被广泛的应用于生物成像、药物筛选、细胞代谢物检测等领域,并发挥出了越来越大的作用。但是,这类探针目前仍然存在着开发工作量大,部分发光强度较弱及测量方法受限等问题,解决这些问题还需要科学家们不断的努力。对环化荧光蛋白生物探针进行综述,主要介绍了环化荧光蛋白生物探针的设计方法,已有探针的种类和基本性质,现阶段的发展状况以及存在的问题。另外,介绍了多种检测方法在环化荧光蛋白生物探针上面的使用,特别是近期出现的荧光寿命成像技术在其应用上的进展,比较了这些检测方法的优劣,旨在从多方面阐述环化荧光蛋白生物探针的发展现状,以便能给相关研究者提供一些参考和启发。     

甲醇固定导致绿色荧光蛋白的荧光消失

发现用甲醇固定转染了绿色荧光蛋白(GFP)基因的细胞会导致GFP的荧光消失,而当用聚甲醛固定时,GFP的荧光就没有失去。因此建议避免用甲醇对有GFP表达的细胞进行免疫组化前的固定。     

苔藓植物光合作用荧光光谱和动力学荧光的比较

本文首次比较了光合作用的荧光光谱和荧光动力学在苔藓植物的原始种类和进化种类之间的异同。原始的和进化的苔藓植物具有发射波长相同的室温荧光光谱,其发射高峰位于686-690nm(来自光系统Ⅱ)和736-740nm(来自光系统Ⅰ)。 而它们的低温(77K)荧光光谱有三个发射峰：F_687-689和F_697-699来自光系统Ⅱ,F_723-734来自光系统I。 前两个峰在原始的和进化的种类中基本相同。 按第三个发射峰可把被测的苔藓植物分为两组：发射峰在725nm左右的有细牛毛藓、长肋对齿藓、对齿藓、斜叶芦荟藓、密集匍灯藓和地钱,它们是较原始的藓类和较进化的苔类;发射峰在732nm左右的有细枝羽藓、东亚金灰藓、鼠尾藓、鳞叶藓、粗枝藓和美灰藓等较进化的藓类,也有较原始的钝叶匍灯藓。已知光系统I核心复合物CPI的77K荧光发射峰在722nm,而CPIa(核心复合物与外周天线复合物)和LHC-I(外周天线复合物)的发射峰在730nm。这说明在苔藓植物进化过程中,光系统Ⅱ比较保守;而光系统Ⅰ有所变化,原始的藓类主要含有光系统Ⅰ核心复合物,而较进化的藓类才含有较完善的外周天线复合物。光合作用荧光动力学分析表明,在原始藓类和地钱中具有较低的光系统Ⅱ活性、光系统Ⅱ的原初光能转换效率、光合碳同化和潜在的光合量子转换效率;而较进化的具有较高的活性和效率。 但是,原始的密集匍灯藓也具有较高的活性和效率,而进化的美灰藓却具有较低的活性和效率。这可能表明这两种植物是由原始向进化发展过程中的中间类型。     

Fluorescence microscopy

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.     

Fluorescence spectroscopy

Some practical aspects of the use of spectrophotofluorometers as applied to the organic analyses of returned lunar samples and other geological materials have been discussed. Because of the single beam nature of spectrophotofluorometers many instrumental artifacts such as grating anomalies, irregular spectral output of light sources and response characteristics of photodetectors are apparent in sample spectra. In order to avoid some of these instrumental artifacts from influencing sample spectra, the performance characteristics of each optical component have been described and the necessity of instrumental calibration has been emphasized.     

Comparative Studies on Photosynthetic Fluorescence Spectra and Fluorescence Kinetics of Bryophytes

Bryophytes are the transitional forms from water habitants to terrestrials, however, there have been only a few works on their photosynthesis. It was the first time to study on photosynthetic fluorescence spectra and fluorescence kinetics of primitive and advanced species comparatively. Both the primitive and advanced ones had the same fluorescence spectra at room temperature, which contained two maximum emissions: F_686-690 from the Photosystem II and F_736-740 from the Photosystem I. And then, there were three maximum emissions in the fluorescence spectra at 77K :F_687-689 and F_697-699 from Photosystem II, and F_723-734 from Photosystem I. The first two maximum emissions were the same for both the primitive and advanced species. According to the third maximum emission the bryophytes under study fell into two categories: The first one possessing the maximum emission around 725 nm, including Ditrichum flexicaule , Didymodon icmadophyllum , Didymodon rigidicaulis, Aloina obliquifolia, Plagiomnium confertidens and Marchantia polymorpha, which were primitive mosses and advanced liverwort. The second one possessing the maximum emission around 732nm, including Thuidium delicatulum , Pylaisia brotheri , Myuroclada maximowiczii , Taxiphyllum taxirameum, Gollania neckerella, Eurohypnum leptothallum, which were advanced mosses, and the primitive one Plagiomnium rostratum. The characteristics of fluorescence spectra implied that the Photosystem II was conservative and Photosystem I was changeable during bryophyte evolution. The primitive mosses possess mainly the PSI core complex (CPI) and then the advanced species contain both CPI and LHC-I. In analysis of photosynthetic fluorescence kinetics, Fv/(Fc+Fv) is a measure of the activity of the Photosystem II; Fv/Fm is dependent on efficiency of primary photoconversion in the Photosystem II; Fm/(Fo+Fv) is related to photosynthetic carbon assimilation; and Fd/Fs is a measure of the potential photosynthetic quantum conversion. The fluorescence kinetics of the bryophyte photosynthesis showed that the Photosystem II activity, the efficieiency of primary photoconversion in Photosystem II, the photosynthetic carbon assimilation and the efficiency of the potential photosynthetic quantum conversion in primitive species, such as Ditrichum flexicaule, Didymodon icmadophyllus, D. rigidicaulis, Plagiomnium rostratum and the liverwort Marchantia polymorpha, were lower than those in the advanced species, Myuroclada maximowiczii, Pylaisia brotheri , Gollania neckerella Taxiphyllum taxirameum , Thuidium delicatulum. However, the primitive Plagiomnium confertidens was of the high activities and efficiencies and the advanced Eurohypnum leptothallum was of low ones. It seemed that P. confertidens and E. leptothallum were an intermediatefrom the primitive to the advanced.