荧光寿命成象显微技术及其应用

本文综述了荧光寿命成象显微技术的概念、原理及实现方法,介绍了荧光寿命成象显微技术在生物物理、生物化学及临床医学诊断等领域的最新研究成果和发展现状,并就其未来的发展及应用研究进行了讨论.     

超分辨荧光显微成像染料结构与生物应用（特邀）

荧光显微成像技术的生物医学应用离不开荧光染料的设计与开发。有机小分子荧光染料因其易于修饰、生物相容性好、光物理性质优异等特点,在细胞生物成像领域受到了广泛关注。随着超分辨荧光显微镜的发展和技术的进步,使得荧光显微成像突破了光学衍射极限,可以获得更为精准的生物分子学信息,观察纳米尺度下亚细胞器之间的相互作用。根据不同的成像原理,科学家开发出了单分子定位成像技术、受激辐射损耗成像技术、结构光照明技术等超分辨荧光显微技术。这些技术在细胞荧光显微成像领域的应用与发展,同时对有机小分子荧光染料的设计与开发提出了新要求。本文介绍了主流超分辨荧光显微技术的原理,总结已发表的超分辨荧光显微成像荧光染料的结构和光物理性质特点,归纳了其设计要求,旨在为新型荧光染料的设计提供参考。     

荧光寿命数据的相量分析及其应用

荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)具有特异性强、灵敏度高、可定量测量等优点,被广泛应用于生物医学、材料学等领域的研究.为使FLIM技术更好地适用于高通量数据的快速分析,近年来涌现出多种荧光寿命分析的新算法.其中,相量分析法(phasor analysis, PA)通过将时间域的拟合转化为频率域的直接计算来获得荧光寿命值,与传统的最小二乘拟合法相比,不仅更加简便快速,适用于低光子数情形,而且便于使数据内容可视化和对数据进行聚类分析,因此越来越受到科研人员的青睐.本文详细阐述了相量分析法的基本原理及运用方法,并在此基础上介绍了该方法在细胞代谢状态测量、蛋白质相互作用研究、细胞微环境测量,以及辅助病理诊断和提高超分辨成像分辨率等方面的应用,着重讨论了PA法在这些FLIM应用实例中的优势所在,为相关领域的研究提供有益的参考.最后,对荧光寿命数据的相量分析及其应用的发展方向进行展望.     

超分辨成像及超分辨关联显微技术研究进展

光学成像系统中有限孔径对光波的衍射,使得光学显微成像技术的分辨率受到"衍射极限"限制而无法进一步提高.自1873年E.K.Abbe提出该问题以来,衍射极限就一直是学术界研究的热点.近年来,随着高强度激光、高灵敏探测器等光电器件研制技术以及新型荧光探针开发等相关领域的快速发展,光学显微技术衍射极限问题的研究迎来了新的契机,超分辨显微成像技术(super-resolution microscopy.SRM)在近十年内取得了令人瞩目的巨大成就.本文从空域和频域角度回顾了衍射极限分辨率的基本原理,并据此对目前常见的各种SRM技术"绕过"衍射极限提高分辨率的机理给予了详解,同时介绍了各类技术的发展动态和研究方向;作为SRM的一个新的重要的发展趋势,本文详细介绍了超分辨关联显微技术的最新研究进展,包括SRM与活细胞实时荧光显微、荧光寿命显微、光谱测量和成像、电子显微、原子力显微、质谱技术等的关联,着重讨论了各类超分辨关联显微技术的作用和意义;最后,对SRM技术和超分辨关联显微技术的未来发展方向进行了展望.     

多色虚拟荧光辐射差分显微成像

荧光辐射差分显微成像是一种荧光染料普适性强、光毒性较低的超分辨成像技术。然而传统荧光辐射差分成像由于受其成像原理限制,系统复杂度较高、稳定性低且成像速度受限。针对上述问题,本文设计搭建了一套多色虚拟荧光差分显微系统,并对该系统的成像方法和参数间的制约关系进行了分析,基于已有的多色虚拟荧光辐射差分显微术原理,进一步考虑了信噪比和背景噪声等的影响,建立了可通过实验验证的虚拟荧光辐射差分显微成像模型。实验表明,本系统与方法具有结构简单、背景去噪能力强、荧光染料普适性强以及光毒性低等特性,成像分辨率较共聚焦系统提升了1.9倍,成像速度较传统的荧光辐射差分显微系统提升一倍,在3个波长上均获得了良好的成像效果,并在生物细胞成像中得到实验验证。     

荧光共振能量转移-荧光寿命显微成像(FRET-FLIM)技术在生命科学研究中的应用进展

细胞是动植物结构和生命活动的基本单位.细胞过程的一个重要特点就是其生化组分在时空调控上的相互作用关系.然而,利用传统的生化方法(如酵母双杂交系统、pull-down系统等)很难在空间上评估活细胞内分子间的相互作用.光学技术的快速发展,为研究活细胞中生物分子的时空动态提供了新的遗传研究工具,其中荧光共振能量转移-荧光寿命...     

任意数量离散不规则感兴趣区域的快速荧光寿命显微成像

荧光寿命显微成像(fluorescence lifetime imaging microscopy,FLIM)技术在细胞微环境传感中具有特异性强、灵敏度高、可定量的优点,被广泛应用于生物医学研究.其中,基于时间相关单光子计数(time-corre-lated single photon counting,TCSPC)进...     

单分子定位超分辨显微成像技术研究进展及展望(特邀综述)

随着新型荧光探针、先进激光、高灵敏光电探测器等相关领域的不断发展,突破衍射极限的超分辨光学显微技术为现代生物医学研究提供了新的有力工具,其中的单分子定位技术利用荧光分子的光开关效应,实现了亚细胞结构的纳米精度超分辨成像.本文介绍了单分子定位超分辨显微技术的基本原理与实现,例举了其在细胞生物学、组织生物学以及神经科学等方面的应用,讨论了该技术目前的发展趋势及可能的改进方向,为相关领域科学研究提供参考.超分辨光学显微技术的不断创新将推动生命科学的新发展.     

时域两维荧光寿命显微测量研究

提出了一种新的时域两维荧光寿命显微测量技术,建立了一套荧光寿命成像显微系统,介绍了这种测量技术的数据处理方法。用标准样品对该系统进行了测试,实验表明,该系统的时间分辨率为2ps,在放大倍率为100倍的情况下,该系统的空间分辨率为8um。如果在现有的设备下采用更细的网格板和微位移系统,那么该系统的空间分辨率可小于1um.     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

基于激光诱导叶绿素荧光寿命成像技术的植物荧光特性研究

针对植物荧光遥感探测中信号易受干扰的问题, 提出了一种用于评估植物生长状况及环境监测的荧光寿命成像技术. 采用凹透镜对355 nm波长的激光扩束, 再照射植物激发叶绿素荧光, 由增强型电荷耦合器件接收荧光信号. 采用时间分辨测量法, 连续用相同激光脉冲照射植物以激发相同的荧光信号, 同时不断改变激光脉冲触发探测器启动的延时时间, 从而能够得到完整的离散荧光信号分布图像. 对植物特定位置点产生的离散荧光信号进行拟合, 再运用一种改进型的迭代解卷积法可反演高精度的荧光寿命; 进而反演图像各点的荧光寿命以生成植物的荧光寿命分布图. 该方法所绘制的荧光寿命图比荧光强度图能更准确地反映植物内部的叶绿素含量, 并对活体植物叶绿素荧光寿命的物理特性进行了初步研究, 证明叶绿素荧光寿命与植物生理状态存在一定关联; 并且叶绿素荧光寿命与活体植物所处环境存在着复杂的关系. 未来将与生物物理学家们合作, 继续探寻叶绿素荧光寿命与植物生存环境的关系.     

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.     

Fluorescence lifetime imaging in biosciences: technologies and applications

The biosciences require the development of methods that allow a non-invasive and rapid investigation of biological systems. In this aspect, high-end imaging techniques allow intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e., wide-field, confocal one-photon and two-photon microscopy, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e., steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at the molecular level. The intrinsic disadvantages of steady-state techniques are countered by using time-resolved techniques. Among these fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.      

激光诱导荧光寿命及其测量

激光诱导荧光特性的研究可用于包括心血管病在内的多种疾病的诊断。荧光发射包括光谱（频域）和时间（时域）两方面的信息，后者表现为荧光寿命。在很多情况下，测量荧光寿命是比测量光谱更为有效的诊断方法。本文从理论上讨论了荧光寿命问题，并介绍两种测量方法，可用于测量人体正常组织和病变组织的激光诱导荧光寿命     

Calcium imaging using fluorescence lifetimes and long-wavelength probes

We describe imaging of calcium concentrations using the long-wavelength Ca²⁺ indicators, Calcium Green, Orange, and Crimson. The lifetimes of these probes were measured using the frequency-domain method and were found to increase from 50% to severalfold in response to calcium. The two-dimensional images of the calcium concentration were obtained using a new apparatus for fluorescence lifetime imaging (FLIM). We also describe procedures to correct for the position-dependent frequency response of the gain-modulated image intensifier used in the FLIM apparatus. Importantly, the FLIM method does not require the probe to display shifts in the excitation or emission spectra. Using the FLIM method, calcium imaging is possible using probes which display changes in lifetime in response to calcium. Consequently, calcium imaging is possible with excitation wavelengths ranging from 488 to as long as 620 nm, where autofluorescence and/or photochemical damage is minimal. These probes are also suitable for calcium measurements of single cells using lifetime-based flow cytometry.     

CVD grown 2D MoS2 layers: A photoluminescence and fluorescence lifetime imaging study

In this letter, we report on the fluorescence lifetime imaging and accompanying photoluminescence properties of a chemical vapour deposition (CVD) grown atomically thin material, MoS₂. µ‐Raman, µ‐photoluminescence (PL) and fluorescence lifetime imaging microscopy (FLIM) are utilized to probe the fluorescence lifetime and photoluminescence properties of individual flakes of MoS₂ films. Usage of these three techniques allows identification of the grown layers, grain boundaries, structural defects and their relative effects on the PL and fluorescence lifetime spectra. Our investigation on individual monolayer flakes reveals a clear increase of the fluorescence lifetime from 0.3 ns to 0.45 ns at the edges with respect to interior region. On the other hand, investigation of the film layer reveals quenching of PL intensity and lifetime at the grain boundaries. These results could be important for applications where the activity of edges is important such as in photocatalytic water splitting. Finally, it has been demonstrated that PL mapping and FLIM are viable techniques for the investigation of the grain‐boundaries.

     

结合虚拟单像素成像解卷积的双边照明光片荧光显微技术

光片荧光显微术(light-sheet fluorescence microscopy,LSFM)采用薄片光束从侧面激发样品,在垂直于光片方向上进行成像,具有成像速度快、光学层析能力强以及光漂白和光毒性低等优点,适用于对较大活体生物样品进行高质量、长时间三维动态观测.然而,传统高斯光束LSFM存在分辨率低和成像视场小的...     

双色荧光辐射差分超分辨显微系统研究

为了拓展荧光辐射差分(Fluorescence Emission Difference,FED)显微术的应用,使得该方法可以同时对生物样品的不同组织结构进行超分辨成像,本文对双色FED显微系统展开了研究。FED的基本原理是将实心光斑扫描得到的共焦显微图像减去空心光斑扫描得到的负共焦图像,以此获得超分辨显微图像。在对单色FED显微系统进行研究后,本文提出了一种可行的双色FED显微成像系统方案。实验结果表明,在488 nm和640 nm激发光下,该系统在荧光颗粒上分别实现了135 nm和160 nm的空间分辨率,另外也能对生物样品的不同组织进行多色同时超分辨显微成像,满足了实际应用的要求。     

Fluorescence lifetime imaging of intracellular calcium

Fluorescence lifetime imaging microscopy (FLIM) is a new methodology for studying the spatial and temporal dynamics of macromolecule, molecules, and ions in living cells. In FLIM image contrast is derived from the mean fluorescence lifetime at each point in a two-dimensional image. In our case the lifetime was measured by the phase-modulation method. We describe our FLIM apparatus, which consists of a fluorescence microscope, high-speed gated proximity focused MCP image intensifier, and slow-scan CCD camera. To accomplish subnanosecond time-resolved imaging, the gain of the image intensifier is modulated with a high-frequency signal, resulting in stationary phase-sensitive intensity images on the image intensifier. These images are recorded using a cooled slow-scan CCD camera and stored in an image processor. The lifetime images are created from a series of phase-sensitive images at various phase shift of the gain-modulation signal. We demonstrate calcium concentration imaging in living COS cells based on Ca²⁺-induced lifetime changes of Quin-2. The phase-angle image is mapped to the Ca²⁺ concentration image using anin vitro-determined calibration curve. The Ca²⁺ concentration was found to be uniform throughout the cell. In contrast, the intensity image shows significant spatial differences, which likely reflect variations in the thickness and distribution of probe within the cell.