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.     

Chemical Imaging of Phase-Separated Polymer Blends by Fluorescence Microscopy

Blends of poly(vinylacetate) (PVAc) and poly(cyclohexylmethacrylate) (PCHMA) labeled by copolymerization with 4-methacryloylamine-4-nitrostilbene (Sb), with (1-pyrenylmethyl)methacrylate (Py), or with 3-(methacryloylamine)propyl-N-carbazole (Cbz) were prepared by casting dilute solutions in tetrahydrofurane (THF) or chloroform. Films about 10 m thick were formed. Phase separation in two types of domains is observed by transmission optical microscopy (TOM) and epifluorescence microscopy (EFM): small craters of 1 to 10 m placed at the polymer-air interface and larger domains, on the scale of 100 m. The morphology of samples depends on the composition of the polymer blend and on solvent. The green fluorescence of Sb, the violet of Py, or the blue of Cbz provides imaging of the distribution of PCHMA in the different domains and in the matrix. It is thus observed that (i) superficial craters and large domains are formed mainly by PCHMA and (ii) the matrix is composed of PVAc in films cast from THF and it is a blend of the two polymers, homogeneous at the submicrometric scale, for chloroform. The emission intensity of Py, recorded by microfluorescence spectroscopy (MFS), yields a mapping similar to imaging detection. It is remarkable that in films cast from chloroform, the smaller domains are distributed with a 2D hexatic order disrupted by dislocations and disclinations, whereas in films cast from THF, a larger heterogeneity is found, denoting different mechanisms of solvent evaporation.     

Strain imaging of a Cu2S switching device

Strain imaging of electrochemical behavior of a solid electrolyte Cu₂S in switching devices for nonvolatile memories is presented. The precipitation and dissolution of Cu, and the nonstoichiometry changes cause changes in volume. Strain imaging we have proposed detects the volume changes through the surface displacements using scanning probe microscopy and provides high resolution images. We observed the distributions of the electrochemical reactions in Cu₂S and located the Cu bridges causing switching.     

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.     

Fluorescence Methods to Probe Nanometer-Scale Organization of Molecules in Living Cell Membranes

The ability to study the structure and function of cell membranes and membrane components is fundamental to understanding cellular processes. This requires the use of methods which are capable of resolving structures at nanometer-scale resolution in living cells. In this review we survey fluorescence imaging methodologies capable of nanometer-scale resolution. We then critically examine specific biological applications of these methods, in the context of understanding membrane protein conformation and dynamics, intracellular signaling, organization of lipid rafts, and cell surface topology.     

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.     

单组分荧光寿命成象显微的研究

激发光的调制频率和CCD所记录到的光强度是影响荧光寿命成象显微频域法测量和数据处理精度的主要因素.本文采用Monte Carlo方法,以单组分问题为例,研究了在用频域外差法进行荧光寿命成象显微测量中激发光的调制频率以及CCD所记录到的光强度对数据处理精度的影响,给出了当调制频率不变时,在不同荧光强度下,对于不同的荧光寿命范围、荧光寿命计算的统计结果,给出了改进测量精度的方法.     

Engineering oxide resistive switching materials for memristive device application

Based on a unified physical model and first-principle calculations, a material-oriented methodology has been proposed to control the bipolar switching behavior of an oxide-based resistive random access memory (RRAM) cell. According to the material-oriented methodology, the oxide-based RRAM cell can be designed by material engineering to achieve the required device performance. In this article, a Gd-doped HfO₂ RRAM cell with excellent bipolar switching characteristics is developed to meet the requirements of memristive device application. The typical memristive characteristics of the Gd-doped HfO₂ RRAM cell are presented, and the mechanism is discussed.     

双波长视网膜成像自适应光学系统的轴向色差补偿方法

双波长视网膜成像自适应光学系统非常适用于视网膜微血管的高对比度和高分辨率成像。本文重点研究了双波长自适应系统的轴向色差补偿问题。首先对轴向色差进行了测量,对实测波前进行了分析,并给出任意波前轴向色差补偿法。自适应校正实验结果显示,色差补偿后,波前均方根误差减小到0.16λ(λ=589 nm),视网膜微血管分辨率提高到6μm。这项工作可用于视网膜成像的临床应用。     

All-optical switching device for infrared based on PbTe quantum dots

Multilayers of PbTe quantum dots embedded in SiO₂ were fabricated by alternate use of Pulsed Laser Deposition (PLD) and Plasma Enhanced Chemical Vapor Deposition (PECVD) techniques. The morphological properties of the nanostructured material were studied by means of High Resolution Transmission Electron Microscopy (HRTEM), Grazing-Incidence Small-Angle X-ray scattering (GISAXS) and X-ray Reflectometry (XRR) techniques. A preliminary analysis of the GISAXS spectra provided information about the multilayer periodicity and its relationship to the size of the deposited PbTe nanoparticles. Finally multilayers were fabricated inside a Fabry–Perot cavity. The device was characterized by means of Scanning Electron Microscopy (SEM). Transmittance measurements show the device functionality in the infrared region.     

Magnetic elements for switching magnetization magnetic force microscopy tips

Using combination of micromagnetic calculations and magnetic force microscopy (MFM) imaging we find optimal parameters for novel magnetic tips suitable for switching magnetization MFM. Switching magnetization MFM is based on two-pass scanning atomic force microscopy with reversed tip magnetization between the scans. Within the technique the sum of the scanned data with reversed tip magnetization depicts local atomic forces, while their difference maps the local magnetic forces. Here we propose the design and calculate the magnetic properties of tips suitable for this scanning probe technique. We find that for best performance the spin-polarized tips must exhibit low magnetic moment, low switching fields, and single-domain state at remanence. The switching field of such tips is calculated and optimum shape of the Permalloy elements for the tips is found. We show excellent correspondence between calculated and experimental results for Py elements.     

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...     

Biomolecular dynamics and binding studies in the living cell

Isolation and preparation of proteins of higher organisms often is a tedious task. In the case of success, the properties of these proteins and their interactions with other proteins can be studied in vitro. If however, these proteins are modified in the cell in order to gain or change function, this is non-trivial to correctly realise in vitro. When, furthermore, the cellular function requires the interplay of more than one or two proteins, in vitro experiments for the analysis of this situation soon become complex. Instead, we thus try to obtain information on the molecular properties of proteins in the living cell. Then, the cell takes care of correct protein folding and modification. A series of molecular techniques are, and new ones become, available which allow for measuring molecular protein properties in the living cell, offering information on concentration (FCS), dynamics (FCS, RICS, FRAP), location (PALM, STED), interactions (F3H, FCCS) and protein proximities (FRET, BRET, FLIM, BiFC). Here, these techniques are presented with their advantages and drawbacks, with examples from our current kinetochore research. The review is supposed to give orientation to researchers planning to enter the field, and inform which techniques help us to gain molecular information on a multi-protein complex. We show that the field of cellular imaging is in a phase of transition: in the future, an increasing amount of physico-chemical data can be determined in the living cell.     

Tailoring light with a digital micromirror device

A digital micromirror device (DMD) is a product of micromechanics. The DMD employs numerous micromirrors as the actuating components to switch small portions of light on and off. During the past few decades, such devices have been widely applied in digital light processing technology. The expanding range of applications makes the DMD increasingly important in various research aspects. Recent advances demonstrate that the DMD is potentially better than the traditional liquid crystal spatial light modulator in speed, spectrum sensitivity, and polarization modulation. These characteristics have been verified in a series of recently reported experiments. This review summarizes the related theory, experimental techniques, and applications for wavefront shaping with DMDs in both statically shaping various spatial modes and dynamically compensating for wavefront distortion caused by the scattering medium.

     

A method of correlative light and electron microscopy for yeast cells

Correlative light and electron microscopy (CLEM) is a method of imaging in which the same specimen is observed by both light microscopy and electron microscopy. Specifically, CLEM compares images obtained by light and electron microscopy and makes a correlation between them. After the advent of fluorescent proteins, CLEM was extended by combining electron microscopy with fluorescence microscopy to enable molecular-specific imaging of subcellular structures with a resolution at the nanometer level. This method is a powerful tool that is used to determine the localization of specific molecules of interest in the context of subcellular structures. Knowledge of the localization of target proteins coupled with the functions of the structures to which they are localized yields valuable information about the molecular functions of these proteins. However, this method has been mostly applied to adherent cells due to technical difficulties in immobilizing non-adherent target cells, such as yeasts, during sample preparation. We have developed a method of CLEM applicable to yeast cells. In this report, we detail this method and present its extension to Live CLEM. The Live CLEM method enabled us to link the dynamic properties of molecules of interest to cellular ultrastructures in the yeast cell. Since yeasts are premier organisms in molecular genetics, combining CLEM with yeast genetics promises to provide important new findings for understanding the molecular basis of the function of cellular structures.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

Simultaneous two color image capture for sub-diffraction localization fluorescence microscopy

A sub-diffraction limit fluorescence localization microscope was constructed using a standard cooled 1.4 mega-pixel fluorescence charge-coupled device (CCD) camera to simultaneously resolve closely adjacent paired quantum dots on a flat surface with emissions of 540 and 630 nm. The images of the overlapping Airy discs were analyzed to determine the center of the point spread function after noise reduction using Fourier transformation analysis. The Cartesian coordinates of the centers of the point spread functions were compared in serial images. Histograms constructed from serial images fit well to Gaussian functions for resolving two quantum dots separated by as little as 10 nm in the x–y coordinates. Statistical analysis of multiple pairs validated discrimination of inter-fluorophore distances that vary by 10 nm. The method is simple and developed for x–y resolution of dilute fluorophores on a flat surface, not serial z sectioning.     

Handheld device for fast and non-contact optical measurement of protein films on surfaces

Thin films of bacteria, proteins and other biochemical substances are almost always found on surfaces exposed to the environment. Optical methods enable innovative tools for the study of such films. Residua of proteins exhibit fluorescence when excited in the UV. We present a specially designed handheld measuring device that can detect organic contamination levels down to 100 ng/cm² with measurement times shorter than a second. Using time-correlated spectroscopy methods the fluorescence detection of contamination levels on surfaces is possible even if the fluorescence spectra of contamination and base material overlap. The results presented in this paper show that a detection of contamination levels of bovine serum albumine (BSA) down to 100 ng/cm² is possible on non-fluorescent materials. A theoretical analysis shows the possibility to detect BSA levels down to less than 1 ng/cm² with the presented setup. Future development of handheld optical devices suitable for the detection or analysis of various compounds can be based on these results. A compact formalism is suggested that can be used to calculate the minimal concentration of a given organic contamination that can be detected with a certain measurement setup.