综述: 用于神经活动观测的随机扫描双光子显微成像

双光子荧光显微镜是神经科学研究中的重要观测仪器,但是现有的商品化仪器受限于较低的成像速度,难以满足脑功能研究中毫秒量级神经信号检测的需要．基于声光偏转器的快速随机扫描双光子显微成像技术,有望在保持信噪比的同时提高观测速度．本文综述了这一研究的最新进展,从飞秒激光经过角色散器件后的时空演化理论、声光偏转器的色散补偿方法、随机扫描成像仪器及仪器应用到神经成像时钙信号的识别方法四个方面分别进行介绍,最后分析了随机扫描双光子显微成像技术的发展趋势．这项技术的系统深入研究将为神经活动观测提供一种全新的方法,推动脑科学研究的发展．     

科技消息

以激光为光源的细胞荧光分析装置此装置以氦镉激光器(422nm)为光源,荧光显微镜与荧光光谱仪联用。在砷化镓光电倍增管前装有双光栅单色器,并与台式计算机相连。此种分析装置适用于细胞生物学的研究,可以定量定位的测出一个细胞或细胞中某一部位的荧光强度,如细胞核经Feulgen-Schiff染色后,用激光光源的荧光显微镜测定,其荧光强度要比以汞灯为光源的高10—100倍,大大提高了核酸检出灵敏度,因此,可精确地分析比较细胞间的或细胞各部位间的荧光强度变化,也可准确和快速地     

平均亮度对猫外膝体瞬变细胞和持续细胞时间调谐曲线的影响

用正弦波调制的光点刺激麻痹的清醒猫外膝体神经元感受野中心,当平均亮度改变时,瞬变细胞的时间频率调谐曲线的形状和敏感频率没有或很少变化,与此相应,这些细胞的自发发放也不随未调制的光照水平而改变。持续细胞的调谐曲线的调幅大体随对未调制光照的平均发放水平的升降而增大或减小,但敏感峰不变。衰减平均亮度1—2对数单位,可使多数呈双峰的调谐曲线的高调频峰消失,对产生此变化的原因,曾加以讨论。     

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。     

多光谱成像技术在生物医学中的应用进展

多光谱成像(multispectral imaging,MSI)技术在生物医学可视化方面是一种新技术,它结合了两个已建立的光学模块:成像学和光谱学。它的原理是基于液晶可调谐滤光片,从可见光到近红外波长(400-970nm)区域获取多光谱图像。自从MSI系统加上显微镜商品化以来,MSI已经成为一种快速发展的领域,可应用于细胞生物学、临床前药物开发和临床病理学等。国外已有大量关于MSI在生物医学中应用的研究报道,但国内报道少见。本文主要对多光谱成像的基本原理,近三年内该技术在生物医学领域的应用进展作一简要综述。     

一体化的光声-荧光显微镜用于活体肿瘤成像

报道了一种利用单一波长激发的同时产生光声和荧光信号的显微成像系统,本成像系统具有超高的成像分辨率(<6μm)。借助外源的造影剂在近红外的吸收特性,利用光声-荧光显微成像系统对活体肿瘤进行光声/荧光成像。实验结果表明,光声-荧光显微镜在早期肿瘤的成像和检测等方面具有潜在的应用价值。因此,通过研究和选择适当的双模态造影剂,该系统在不同病理模型中可以提供更准确的组织信息及生理参数。     

高内涵成像与数据分析

在药物筛选领域和细胞生物学研究中,高内涵成像系统的应用越来越广泛。本文主要根据我们实际科研经验对高内涵成像系统使用中影响实验质量的三个方面进行归纳和建议。在样品准备阶段,需要考虑的因素有微孔板、细胞和荧光标记等。针对这些影响因素,文中介绍了一些有益于提高实验效率和图像质量的经验。在高内涵成像阶段,根据实验目的选择合适的成像模式和物镜,同时权衡考虑成像时间和成像视野数,以期在合理的时间内获取高质量的高内涵图像数据。最后是高内涵数据分析阶段,流程一般包括图像校正、图像分割、生物学参数的提取、生物学参数的聚类或分类分析以及最终的结果输出。目前常用单一生物学参数的Z'值对高内涵成像实验进行评价,部分高内涵成像实验探索利用多参数进行数据分析和评价。结合机器自学习功能,开发更复杂的多参数的数据分析方法,是未来高内涵数据分析的发展方向。     

细胞内 NO 的荧光成像

NO是一种具有重要生物学意义的信息分子,在体内具有广泛的生物学特征。但由于NO的自由基性质,使得在活细胞中对低浓度、低寿命的NO实时监测异常困难。为了进一步了解NO在神经、免疫、血管和消化等多种系统中的生理功能,高度专一性的、高灵敏的荧光探针结合激光扫描共聚焦显微镜对活细胞中的NO进行实时、连续的成像已被广泛研究。该综述了近年来NO荧光探针的发展及其在生物成像中的应用。     

劳埃晶体学时代的到来

近年来,人们对古老的劳埃衍射技术重产生了浓厚的兴趣,这主要是因为世界各地新的同步辐射光源的建成;各种先进的插入件,诸如波形器（wiggler）、波荡器（undulator)的飞速发展;以及利用这类光源通过时间分辨晶体学来研究分子研究动态变化的前景,在过去的十年中,理论研究已经阐明了多波长衍射几何学的特征,在很大幅度上加深了我们对劳埃法的认识。劳埃数据处理方法及其软件开发也因此有所创新。曾在相当长的时间内限制这项技术应用的劳埃数据处理中的大部分问题存在已经得到解决;同步辐射光源,束线光学器件及X射线探测器等方面也都取得了显著的进步。表态劳埃实验得到的结构因子振幅在质量上已同单波长数据相当。晶体中反应易于启动的时间分辨劳埃实验已经开始在一系列生物分子体系中得到成功的实践,由此得到的关于结构动力学的信息是任何其它传统的衍射方法所无能为力的。这些静态的及时间分辨的实验说明了劳埃已经开始走向成熟,并指明了今后发展的方向, 即对晶体的镶嵌度和劳埃衍射点的相应能量宽度的正确处理、扩散散射的考虑,时间分辨实验中低浓度瞬态中间物结构的测定等等。     

│ω│滤波器对光声成像的影响

光声成像结合了组织纯光学成像和组织纯声学成像的优点,是一种很有潜力的无损伤的医学成像技术。本文研究了四种不同的ω滤波器,即RL滤波器,SL滤波器,改进的SL滤波器和Kwoh-R eed滤波器,利用滤波反投影算法分析了它们对光声图像重建质量的影响,由仿真和实验结果表明,Kwoh-R eed滤波器对强噪音有着很好的抑制作用,能明显的提高图像的对比度。实验所用的光源为YAG激光器,波长为532 nm,重复频率为30 H z,脉宽为7 ns,探测器为针状的PVDF膜水听器,接收面积的直径为1 mm。     

AOTF microscope for imaging with increased speed and spectral versatility.

We have developed a new fluorescence microscope that addresses the spectral and speed limitations of current light microscopy instrumentation. In the present device, interference and neutral density filters normally used for fluorescence excitation and detection are replaced by acousto-optic tunable filters (AOTFs). Improvements are described, including the use of a dispersing prism in conjunction with the imaging AOTF and an oblique-illumination excitation scheme, which together enable the AOTF microscope to produce images comparable to those obtained with conventional fluorescence instruments. The superior speed and spectral versatility of the AOTF microscope are demonstrated by a ratio image pair acquired in 3.5 ms and a micro-spectral absorbance measurement of hemoglobin through a cranial window in a living mouse.     

Coupled external cavity photonic crystal enhanced fluorescence

We report a fundamentally new approach to enhance fluorescence in which surface adsorbed fluorophore‐tagged biomolecules are excited on a photonic crystal surface that functions as a narrow bandwidth and tunable mirror of an external cavity laser. This scheme leads to ～10× increase in the electromagnetic enhancement factor compared to ordinary photonic crystal enhanced fluorescence. In our experiments, the cavity automatically tunes its lasing wavelength to the resonance wavelength of the photonic crystal, ensuring optimal on‐resonance coupling even in the presence of variable device parameters and variations in the density of surface‐adsorbed capture molecules. We achieve ～10⁵× improvement in the limit of detection of a fluorophore‐tagged protein compared to its detection on an unpatterned glass substrate. The enhanced fluorescence signal and easy optical alignment make cavity‐coupled photonic crystals a viable approach for further reducing detection limits of optically‐excited light emitters that are used in biological assays. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A new filtering technique for removing anti‐Stokes emission background in gated CW‐STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super‐resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time‐gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub‐diffraction spatial resolution. If the time‐gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW‐STED microscope. However, time‐gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti‐Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti‐Stokes background. The method hinges on the uncorrelated nature of the anti‐Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two‐photon‐excitation with gated CW‐STED microscopy is demonstrated. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Second harmonic imaging of plants tissues and cell implosion using two‐photon process in ZnO nanoparticles

The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto‐fluorescence above 750 nm and minimal auto‐fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near‐infrared femtosecond laser source ranging from 750‐980 nm. For laser energies exceeding the two‐photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two‐photon emission becomes the dominant signal. The heat generated due to two‐photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710–750 nm. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method

Fluorescence cross-correlation spectroscopy (FCCS) is used as a powerful technique to analyze molecular interactions both in vitro and in vivo. This method basically requires two laser excitations for two target molecules labeled with fluorophores of different colors. Their coincidence in a microscopic detection volume is analyzed using two detectors. Any overlap of emission spectra of the two fluorophores, however, gives rise to false-positive data about their interaction. To overcome this problem, we have developed a new FCCS system, in which two excitation lasers are switched alternately by modulation using an acousto-optic tunable filter (AOTF). In this report, we demonstrate the feasibility of switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) with a cleavage site of caspase-3 inserted was expressed in HeLa cells, and proteolysis assay was performed during apoptotic cell death. Due to the absence of cross-talk signals, the FCCS measurement with the switching function gave a large change in relative cross-correlation amplitude after protein cleavage. Hence, switching FCCS enables more reliable measurement of molecular interactions than conventional FCCS.     

Extraction of near-field fluorescence from composite signals to provide high resolution images of glial cells

The subdiffraction optical resolution that can be achieved using near-field optical microscopy has the potential to permit new approaches and insights into subcellular function and molecular dynamics. Despite the potential of this technology, it has been difficult to apply to cellular samples. One significant problem is that sample thickness causes the optical information to be comprised of a composite signal containing both near- and far-field fluorescence. To overcome this issue we have developed an approach in which a near-field optical fiber is translated toward the cell surface. The increase in fluorescence intensity during z-translation contains two components: a far-field fluorescence signal when the tip of the fiber is distant from the labeled cell, and combined near- and far-field fluorescence when the tip interacts with the cell surface. By fitting a regression curve to the far-field fluorescence intensity as the illumination aperture approaches the cell, it is possible to isolate near-field from far-field fluorescent signals. We demonstrate the ability to resolve actin filaments in chemically fixed, hydrated glial cells. A comparison of composite fluorescence signals with extracted near-field fluorescence demonstrates that this approach significantly increases the ability to detect subcellular structures at subdiffraction resolution.     

Fluorescence intensity resolution in flow systems.

The factor which can limit fluorescence intensity resolution in a flow cytometer of the type in which cells pass perpendicularly through a focussed laser beam depends on signal intensity. For the brightest sources (e.g. fluorescent DNA stains), the coefficient of variation (CV) is limited in our system to around 3% by stream hydrodynamics, unstable illumination intensity, nonstoichiometric staining, etc. The weakest detectable sources (e.g. fluorescent cell-surface labels) are limited in coefficient of variation by shot noise in the photomultiplier due to constant background light levels. Finally, over a fairly wide brightness range between these extremes, resolution is determined primarily by photoelectron statistical variation on the signal itself (i.e. "photon statistics"). Thus photon collection and detection efficiency (solid angle, barrier filter passband, detector quantum efficiency) become of primary importance.     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Eliminating Unwanted Far-Field Excitation in Objective-Type TIRF. Part I. Identifying Sources of Nonevanescent Excitation Light

Total internal reflection fluorescence microscopy (TIRFM) achieves subdiffraction axial sectioning by confining fluorophore excitation to a thin layer close to the cell/substrate boundary. However, it is often unknown how thin this light sheet actually is. Particularly in objective-type TIRFM, large deviations from the exponential intensity decay expected for pure evanescence have been reported. Nonevanescent excitation light diminishes the optical sectioning effect, reduces contrast, and renders TIRFM-image quantification uncertain. To identify the sources of this unwanted fluorescence excitation in deeper sample layers, we here combine azimuthal and polar beam scanning (spinning TIRF), atomic force microscopy, and wavefront analysis of beams passing through the objective periphery. Using a variety of intracellular fluorescent labels as well as negative staining experiments to measure cell-induced scattering, we find that azimuthal beam spinning produces TIRFM images that more accurately portray the real fluorophore distribution, but these images are still hampered by far-field excitation. Furthermore, although clearly measureable, cell-induced scattering is not the dominant source of far-field excitation light in objective-type TIRF, at least for most types of weakly scattering cells. It is the microscope illumination optical path that produces a large cell- and beam-angle invariant stray excitation that is insensitive to beam scanning. This instrument-induced glare is produced far from the sample plane, inside the microscope illumination optical path. We identify stray reflections and high-numerical aperture aberrations of the TIRF objective as one important source. This work is accompanied by a companion paper (Pt.2/2).     

Acousto-optic laser-scanning cytometer

An instrument has been developed that uses a computer-controlled rapidly scanning laser beam to make cytometric measurements on cells or particles and which can measure low levels of fluorescence when using low-power lasers (Gershman, Hoffman, and O'Connell, "Methods and Apparatus for Analysis of Particles and Cells.") The method used is based upon acousto-optic principles of light diffraction. A vertically polarized 5-mW He-Ne laser is directed into an acousto-optic Bragg cell in which a portion of the incident light undergoes a small angular variation or deflection. Suitable optics focus the beam to a 25 microns diameter spot, at the 1/e2 point, in a sample cuvette while translating the angular variation into a linear scan. The cuvette enclosing the sample is slowly moved (approximately 1 micron/ms) via a stepper drive into the scanning beam while the forward angle light scatter sensor is monitored for the presence of valid signal events. When an event occurs, appropriate software optimizes the position of the focused laser beam onto the cell. Subsequently, scanning is stopped to allow for cell interrogation times that last for milliseconds or longer.