眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

双光子激发钠分子荧光寿命的测量

本文研究了钠分子高位三重态荧光寿命的测量方法。利用倍频晶体模拟等频双光子激发过程和对荧光衰变曲线求卷积的数值计算方法。有效地消除了测量仪器响应函数的影响,测量了Na_2高位三重态2~3∏_g→a~3∑_u~+的荧光寿命。     

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.     

多光子成像技术的生物医学应用新进展

多光子成像技术由于具有低侵入性、强穿透力、高空间分辨率等优点,自问世以来便成为生物医学研究的有力工具,在癌症病理、神经疾病及脑功能成像等方面取得了一系列较好的研究成果.目前,应用较为广泛的多光子成像技术是双光子激发荧光显微成像技术,其在生物医学应用中具有较大的发展潜力.本文详细阐述了多光子成像技术在多色成像、功能成像及成像深度等方面的生物医学应用新进展,包括多色双光子激发荧光显微成像、双光子激发荧光寿命显微成像、双光子光纤内窥成像和三光子显微成像技术,并简要介绍这几种多光子成像技术的原理与特性,最后展望其未来发展前景.     

荧光寿命成像在癌症诊断研究中的应用(特邀)

癌症是目前人类所面对的共同难题,降低癌症死亡率的关键是实现早期诊断。荧光寿命因对微环境的敏感性,不仅可以实现对早期癌症组织与正常组织的区分,还可以对药物治疗癌症进行监测,因此荧光寿命成像显微技术在癌症诊断方面具有巨大的应用潜力。本文介绍了荧光寿命成像显微技术的基本原理及检测方法,总结了内源性和外源性荧光团的特征及其与癌症诊断的关系,综述了近年来荧光寿命成像显微技术在神经系统、呼吸系统、消化系统、生殖系统、泌尿系统、内分泌系统以及皮肤系统等癌症诊断中的应用,讨论了荧光寿命成像显微技术在癌症诊断中的应用优势、局限性以及未来发展趋势。     

基于时间相关单光子计数的荧光寿命成像技术

采用时域法中的时间相关单光子计数方法记录荧光寿命,时间相关单光子计数采用多波长通道同时记录荧光光子数,可以提高计数效率和信息量,还可以在稳态图像中分离不同荧光团,形成4维图像。并采用多光子激发技术,利用长波长光源发出的两个或多个光子可以激发出一个短波长的光子。多个光子必须几乎同时到达激发点, 才能提供被激发分子足够的能量以产生荧光。多光子激发波长较长, 生物组织对其散射减小,因而可以穿透到更深层的组织,从而提高荧光成像深度和空间分辨力,并减少对活体样品的损伤。     

荧光寿命显微成像技术及应用的最新研究进展

由于荧光寿命不受探针浓度、激发光强度和光漂白效应等因素影响,荧光寿命显微成像技术(fluorescence lifetime imaging microscopy, FLIM)在监测微环境变化、反映分子间相互作用方面具有高特异性、高灵敏度、可定量测量等优点,近年来已被广泛应用于生物医学等领域.然而,尽管FLIM的发明和发展已历经数十年时间,其在实际应用中仍然面临着许多挑战.例如,其成像分辨率受衍射极限限制,而其成像速度与成像质量和寿命测量精度则存在相互制约的关系.近几年来,相关硬件和软件的快速发展及其与其他光学技术的结合,极大地推动了FLIM技术及其应用的新发展.本文简要介绍了基于时域和频域的不同寿命探测方法的FLIM技术的基本原理及特点,在此基础上概述了该技术的最新研究进展,包括其成像性能的提升和在生物医学应用中的研究现状,详细阐述了近几年来研究者们通过硬件和软件算法的改进以及与自适应光学、超分辨成像技术等新型光学技术的结合来提升FLIM的成像速度、寿命测量精度、成像质量和空间分辨率等方面所做的努力,以及FLIM在生物医学基础研究、疾病诊断与治疗、纳米材料的生物医学研究等方面的应用,最后对其未来发展趋势进行了展望.     

基于时间相关单光子计数的荧光寿命成像技术

采用时域法中的时间相关单光子计数方法记录荧光寿命,时间相关单光子计数采用多波长通道同时记录荧光光子数,可以提高计数效率和信息量,还可以在稳态图像中分离不同荧光团,形成4维图像。并采用多光子激发技术,利用长波长光源发出的两个或多个光子可以激发出一个短波长的光子。多个光子必须几乎同时到达激发点,才能提供被激发分子足够的能量以产生荧光。多光子激发波长较长,生物组织对其散射减小,因而可以穿透到更深层的组织,从而提高荧光成像深度和空间分辨力,并减少对活体样品的损伤。     

活细胞应激反应过程中线粒体和核仁微环境动力学的荧光寿命成像研究

核仁和线粒体在维持细胞平衡发挥重要作用,研究其生理过程有助于深入了解生物学功能.本文采用一种红色荧光的芘罗丹明荧光探针在不同条件下靶向标记细胞线粒体和核仁.通过激光共聚焦成像和荧光寿命成像技术分析HeLa细胞在光照和药物刺激下细胞凋亡的形态变化,并利用相图定量分析了线粒体与核仁的微环境变化,确定在稳态HeLa细胞中探针标记到的线粒体的平均荧光寿命约为3.65 ns,线粒体黏度约为66×10–3 Pa·s.在激光光照后,探针标记到HeLa细胞线粒体的荧光寿命降至3.61 ns,对应线粒体黏度增至约131×10–3 Pa·s;使用紫杉醇和秋水仙碱诱导细胞凋亡,观察到探针标记于HeLa细胞核仁的荧光寿命先增加后降低,反映了在HeLa细胞凋亡过程中核仁微环境的变化,证明HeLa细胞在非稳态情况下核仁和线粒体的功能变化,为线粒体和核仁功能障碍相关疾病研究提供了新的研究方法.     

脉冲压缩在双光子荧光显微成像中的应用研究

本刊讯自20世纪90年代末,双光子荧光显微成像技术已经逐步成为利用光学手段研究生物组织和细胞的重要工具。由于双光子激发属于非线性光学过程,     

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.      

Fluorescence lifetime imaging of oxygen in living cells

The usefulness of the fluorescent probe ruthenium tris(2,2′-dipyridyl) dichloride hydrate (RTDP) for the quantitative imaging of oxygen in single cells was investigated utilizing fluorescence lifetime imaging. The results indicate that the fluorescence behavior of RTDP in the presence of oxygen can be described by the Stem-Volmer equation. This shows that fluorescence quenching by oxygen is a dynamic quenching process. In addition, it was demonstrated that the fluorescence lifetime of RTDP is insensitive to pH, ion concentration, and cellular contents. This implies that a simple calibration procedure in buffers can be used to quantify oxygen concentrations within cells. First fluorescence imaging experiments on J774 macrophages show a nonuniform fluorescence intensity and a uniform fluorescence lifetime image. This indicates that the RTDP is heterogeneously partitioned throughout the cells, while the oxygen concentration is constant.     

Novel organelles in primate retinal epithelium

We are investigating age-related changes in organelles in monkey retinal epithelium using transmission and analytic electron microscopy. We previously described a circular organelle in retinal epithelium with a diameter of about 0.5 μm. The organelle is unique in containing a single, round vacuole within an otherwise electron dense interior. We suggested that the organelle might be a melanosome with lysosomal properties. We now find that there are two similar organelles with such a single vacuole but which differ in their chemical composition, electron density, cell location and according to age.Epon embedded sections from the macular epithelium of seven monkeys, ranging from 1 to 35 years of age, were examined by transmission electron microscopy. A seven year old monkey was processed for analytic electron microscopy to determine the chemical composition of the organelles. The number and location of the organelles in the retinal epithelium were determined.The chemical composition of these two organelles was different. One of the organelles contained high mole fractions of oxygen and nitrogen and little phosphorous characteristic of melanin; the other had little oxygen and nitrogen and higher mole fractions of phosphorous uncharacteristic of melanin, but more common with lysosomal organelles. The latter had an electron dense rim around the vacuole, a less electron dense interior than the melanin containing organelle and also contained iron. The melanin containing organelle was more common in young monkeys and in the middle third of the cell. The organelle without melanin was more common in old monkeys and localized in the basal third of the cell.Two similarly vacuolated organelles, not identified before in retinal epithelium, differ in their chemical composition. One contains melanin; the other does not. The former is more common in young and the latter more common in old monkeys. This suggests reorganization and or degradation of melanin-containing organelles with age. These changes show how analytic electron microscopy can distinguish major ultra-structural differences in organelles when mere observation fails to do so easily.     

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.     

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.