绿色荧光蛋白及其在植物分子生物学研究中的应用

绿色荧光蛋白(GFP)是海洋生物水母(Aequoria victoria)体内的一种发光蛋白,近十年来成为在生物化学和细胞生物学研究和应用中用得最广泛的蛋白质之一。文章就绿色荧光蛋白的特性及其在植物分子生物学中应用的研究进展作了概述。     

红曲菌产荧光物质的研究进展

荧光物质在医疗和工业上已具有广泛的应用前景。近年来,从红曲米中发现新的荧光代谢产物已成为研究热点之一。本文在介绍荧光物质的种类、机理的同时着重介绍红曲米中荧光物质的种类、结构和功能的最新进展以及生物体发光的合成机制,并对荧光代谢产物未来研发的方向作了介绍。在肯定荧光代谢物的研究取得明显进展的基础上,分析指出研究工作存在的问题和不足。     

荧光分析及其应用

由于六十年代后期荧光仪器技术的发展,使荧光分析方法日趋完善。目前荧光分析方法已广泛地应用到工农业、环境保护、粮食保存、临床化验、药物分析、食品分析、医学和生物学基础研究等各个领域,分析对象有无机离子和化合物、有机化合物、氨基酸、蛋白质、酶、核酸、糖、维生素和甾族化合物等等。近年来,我国各个研究领域对荧光分析法比较重视,在这方面做了不少工作,取得一定的成绩。本文就荧光分析法和它的应用作一简单的介绍。     

绿色荧光蛋白及其应用

许多海洋无脊椎动物体内都含有绿色荧光蛋白,这种蛋白质结构很特殊,在受到激发时可以发射绿色或蓝色荧光。虽然对它的研究从本世纪六十年代才开始,但是它独特的性质逐渐引起了生物学界的广泛关注。本文将就绿色荧光蛋白的结构、性质及其应用前景作一综述。     

实时荧光定量PCR技术在鱼类病害研究中的应用

实时荧光定量PCR技术是一种新的核酸定量技术,通过检测PCR产物中荧光信号强度达到定量的目的,与常规PCR相比,具有无污染、特异性强、检测灵敏、定量准确等特点,该技术在分子诊断、动植物检疫等方面得到了广泛的应用.目前水产养殖业处于飞速发展时期,其中鱼类的病害问题也日益突出,为了预防和控制鱼类病害,实时荧光定量PCR技术已逐渐应用于鱼类病害的研究中.该文将从实时荧光定量PCR的技术原理、主要类型以及实时荧光定量PCR技术在鱼类病害研究的应用研究作一综述.     

荧光定量PCR技术在植物研究中的应用

荧光定量PCR技术是近年发展起来的一种用于基因定量分析的PCR方法,自同世以来在基础科学研究、临床诊断、疾病研究及药物研发等领域应用较为广泛.在植物研究方面应用起步较晚,现已开始用于植物基因表达分析、外源基因基因鉴定等方面的研究.介绍了实时荧光定量PCR技术的原理、优缺点和试验中潜在问题和条件的优化,并对其在植物研究中的应用及前景作了探讨.     

荧光单分子检测技术在生命科学中的应用

荧光单分子检测技术是用荧光标记来显示和追踪单个分子的构象变化、动力学,单分子之间的相互作用以及单分子操纵的研究。过去对于生命科学分子机制的研究,都是对分子群体进行研究,然后平均化来进行单分子估测。因此,单个分子的动态性和独立性也被平均化掉而无法表现出来。荧光单分子检测技术真正实现了对单个分子的实时观测,将过去被平均化并隐藏在群体测量中不能获得的信息显示出来。近几年来,荧光单分子检测技术的飞速发展,为生命科学的发展,开辟了全新的研究领域。现就荧光单分子检测技术在研究动力蛋白、DNA转录、酶反应、蛋白质动态性和细胞信号转导方面的应用进展作一综述。     

荧光显微分析技术在植物细胞学研究中的应用

论述了在植物细胞学研究中应用荧光显微分析技术时常用的荧光分析仪器以及近年来荧光显微分析技术在植物细胞学研究中的应用,并对其应用前景进行了展望.     

绿色荧光蛋白及其应用

许多海洋无脊椎动物体内都含有绿色荧光蛋白,这种蛋白质结构很特殊,在受到激发时可以发射绿色或蓝色荧光。本文将就绿色荧光蛋白的结构,性质及其应用前景作一综述。     

荧光蛋白研究进展

荧光蛋白在生物学众多研究领域中有着广泛的应用,基于荧光蛋白的分子探针和标记方法已成为活细胞或活体内动态成像研究生物大分子或细胞功能的重要工具。本文对现有荧光蛋白的种类和理化特性,及其在生物学研究中的应用进行了综述介绍。重点介绍了近年来荧光蛋白在亮度、Stokes位移、光谱改变等方面的研究进展,介绍了光转换与光活化荧光蛋白及其在超分辨荧光成像技术中的应用。最后对荧光蛋白未来的发展方向进行了展望。     

时间分辨荧光免疫分析及其在临床检测中的应用

本文介绍了时间分辨荧光免疫分析法的检测原理、检测方法,分析了时间分辨荧光免疫分析仪的结构并介绍了其在临床检测方面的应用。     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

量子点荧光标记技术的研究热点及面临的挑战

半导体量子点作为新型荧光标记物,在生物医学领域具有重要应用．与传统的有机染料及荧光蛋白等荧光标记物相比,半导体量子点具有发光颜色可调、激发范围宽、发射光谱窄、化学及光稳定性好、表面化学丰富以及生物偶联技术成熟等诸多优势,为生命体系的靶向示踪,高灵敏、原位、实时、动态荧光成像,DNA及蛋白质检测,靶向药物,临床医学,生物芯片和传感器等研究提供了新的发展契机．基于作者在半导体量子点生物荧光成像和安全性评价研究的基础,综述了半导体量子点荧光标记物在生命科学与医学领域应用的研究热点,并对半导体量子点荧光标记技术走向实用面临的挑战进行了评述．     

光片荧光显微镜特点及其应用

传统荧光显微镜由于对某些荧光分子存在光毒性、光损伤等方面的缺陷,无法满足对部分活体样本进行长时间观测的需求。光片荧光显微镜（light sheet fluorescence microscope,LSFM）是一种新型荧光显微镜,有别于激光共聚焦显微镜,其特殊的正交光路设计和高效的信号采集装置,使其具备低光毒性、低光漂白、低光损伤和高时空分辨率等优良特性,从而能对细胞及大尺度生物组织样本进行时空连续性较好的记录,尤其适宜于活体生物样品。基于此,概述了光片荧光显微镜的成像原理、成像优势、成像效果的改进与优化历程及其在生命科学领域应用所取得的研究成果,重点对近三年相关应用进行了汇总,并简要介绍了其在神经生物学、发育生物学、动物细胞生物学和植物科学领域中一部分代表性研究内容,最后,总结了光片荧光显微镜的优点与发展至今仍存在的不足,并对其在光遗传学和多组学研究中的潜在应用进行了展望,以期为研究人员提供较为系统的光片荧光显微镜相关基础知识、最新的研究应用进展以及未来的潜在应用方向,为研究人员提供参考。     

Fluorescent biosensors — Probing protein kinase function in cancer and drug discovery

One of the challenges of modern biology and medicine is to visualize biomolecules in their natural environment, in real-time and in a non-invasive fashion, so as to gain insight into their physiological behavior and highlight alterations in pathological settings, which will enable to devise appropriate therapeutic strategies. Fluorescent biosensors constitute a class of imaging agents which have provided major insights into the function and regulation of enzymes in their cellular context. GFP-based reporters and genetically-encoded FRET biosensors, have been successfully applied to study protein kinases in living cells with high spatial and temporal resolution. In parallel, combined efforts in fluorescence chemistry and in chemical biology have enabled the design of non-genetic, polypeptide biosensors coupled to small synthetic fluorescent probes, which have been applied to monitor protein kinase activities in vitro and in more complex biological samples, with an equally successful outcome. From a biomedical perspective, fluorescent biosensor technology is well suited to development of diagnostic approaches, for monitoring disease progression and for evaluating response to therapeutics. Moreover it constitutes an attractive technology for drug discovery programs, for high content, high throughput screening assays, to assess the potency of new hits and optimize lead compounds, whilst also serving to characterize drugs developed through rational design. This review describes the utility and versatility of fluorescence biosensor technology to probe protein kinases with a specific focus on CDK/cyclin biosensors we have developed to probe abundance, activity and conformation. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

What precision‐protein‐tuning and nano‐resolved single molecule sciences can do for each other

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard‐ and software developments, but also by the availability and suitability of protein‐engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints.     

Fluorescent nanoparticles as tools in ecology and physiology

Fluorescent nanoparticles (FNPs) have been widely used in chemistry and medicine for decades, but their employment in biology is relatively recent. Past reviews on FNPs have focused on chemical, physical or medical uses, making the extrapolation to biological applications difficult. In biology, FNPs have largely been used for biosensing and molecular tracking. However, concerns over toxicity in early types of FNPs, such as cadmium-containing quantum dots (QDs), may have prevented wide adoption. Recent developments, especially in non-Cd-containing FNPs, have alleviated toxicity problems, facilitating the use of FNPs for addressing ecological, physiological and molecule-level processes in biological research. Standardised protocols from synthesis to application and interdisciplinary approaches are critical for establishing FNPs in the biologists’ tool kit. Here, we present an introduction to FNPs, summarise their use in biological applications, and discuss technical issues such as data reliability and biocompatibility. We assess whether biological research can benefit from FNPs and suggest ways in which FNPs can be applied to answer questions in biology. We conclude that FNPs have a great potential for studying various biological processes, especially tracking, sensing and imaging in physiology and ecology.     

Plasmonics in Biology and Plasmon-Controlled Fluorescence

Fluorescence technology is fully entrenched in all aspects of biological research. To a significant extent, future advances in biology and medicine depend on the advances in the capabilities of fluorescence measurements. As examples, the sensitivity of many clinical assays is limited by sample autofluorescence, single-molecule detection is limited by the brightness and photostability of the fluorophores, and the spatial resolution of cellular imaging is limited to about one-half of the wavelength of the incident light. We believe a combination of fluorescence, plasmonics, and nanofabrication can fundamentally change and increase the capabilities of fluorescence technology. Surface plasmons are collective oscillations of free electrons in metallic surfaces and particles. Surface plasmons, without fluorescence, are already in use to a limited extent in biological research. These applications include the use of surface plasmon resonance to measure bioaffinity reactions and the use of metal colloids as light-scattering probes. However, the uses of surface plasmons in biology are not limited to their optical absorption or extinction. We now know that fluorophores in the excited state can create plasmons that radiate into the far field and that fluorophores in the ground state can interact with and be excited by surface plasmons. These reciprocal interactions suggest that the novel optical absorption and scattering properties of metallic nanostructures can be used to control the decay rates, location, and direction of fluorophore emission. We refer to these phenomena as plasmon-controlled fluorescence (PCF). We predict that PCF will result in a new generation of probes and devices. These likely possibilities include ultrabright single-particle probes that do not photobleach, probes for selective multiphoton excitation with decreased light intensities, and distance measurements in biomolecular assemblies in the range from 10 to 200 nm. Additionally, PCF is likely to allow design of structures that enhance emission at specific wavelengths and the creation of new devices that control and transport the energy from excited fluorophores in the form of plasmons, and then convert the plasmons back to light. Finally, it appears possible that the use of PCF will allow construction of wide-field optical microscopy with subwavelength spatial resolution down to 25 nm.     

二次谐波显微成像技术

二次谐波非线性显微成像技术是近年发展起来的一种新型光学成像方法,已广泛应用于生物医学的各个领域。介绍了光学二次谐波产生的原理、成像装置及其技术发展,描述了二次谐波的成像特点和它与双光子荧光成像的异同,并对其在生物医学上的应用及发展前景做出展望。     

A new xanthene‐based two‐photon fluorescent probe for the imaging of 1,4‐dithiothreitol (DTT) in living cells

1,4‐Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two‐photon fluorescence probe (Rh‐DTT) to detect DTT in living systems for the first time. Rh‐DTT showed high selectivity and sensitivity to DTT. Rh‐DTT can be successfully used for the two‐photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.