荧光与荧光显微镜

本文从荧光的基础知识入手,简略描述荧光显微镜的特点,对荧光显微镜的主要部件：光源、滤色片和光学系统作简要介绍,从荧光特性提出设计要求,并对透射和落射荧光进行比较。     

荧光显微镜的使用与校正方法

荧光显微镜是最常用的医用光学仪器之一,在各大医院实验室和临床部门得到了广泛的应用。叙述荧光显微镜的原理、使用和校正方法。     

浅谈反卷积荧光显微镜

本文简要介绍了反转卷积荧光显微镜的概况,常用的用来提高图像质量的反卷积算法和点扩展函数的确定方法。     

基于荧光显微镜的毛细管电泳系统的构建

毛细管电泳仪具有灵敏度高、分析速度快等优势,为降低其生产成本,基于电泳原理,以荧光显微镜为基础,设计了一套毛细管电泳系统。以20 bp(base pairs,碱基对)DNA ladder和100 bp DNA ladder为样本,全面分析了系统的稳定性、灵敏度和分离效果。结果表明:该系统在9 min内可以实现1500 bp以内DNA片段的高效分离,系统检测极限为0.1 ng/μL;在优化的电泳条件下,对限制性内切酶φX174-HincⅡ作用过的λ-DNA片段5 min内实现了291 bp与297 bp DNA片段的区分。     

荧光光纤传感器

本文对荧光光纤传感器进行了综述,介绍了荧光光纤传感器的结构和优良的测试性能,讨论了几种重要的传感器的特性,这种传感器具有广泛的应用前景。     

荧光原位杂交技术的发展与应用

本文主要介绍了荧光原位杂交技术的建立及其发展过程。     

一种阳离子卟啉与DNA荧光猝灭体系的研究

建立了DNA对meso-四－（对－三四胺基苯基）卟啉荧光定量猝灭的体系,研究了卟啉的特征荧光谱以及pH值,DNA变性时间和紫外光对体系荧光强度的影响,建立了一种定量检测DNA的方法。该方法线性范围为0－5ug/ml,检出限为0．717ug/ml,相关系数为0．993。     

三维荧光光谱检测地沟油

利用三维荧光光谱检测灵敏度高、选择性强和快速无污染的优点,对掺入了不同比例地沟油的植物油进行了检测。实验结果表明,当掺入的地沟油的含量超过10%时,根据三维荧光光谱的荧光图案特征和特定荧光激发波长的荧光强度下降程度,可以作为判断该植物油是否掺入了地沟油的依据。使用三维荧光光谱检测地沟油优于其他检测方法,其灵敏度和快速、实时的特点适合用于地沟油的测定。     

用荧光磁粉检测重要压力容器的表面缺陷

对比试验与实践经验表明，荧光磁粉检测比黑色磁粉检测有高得多的缺陷检出率。为了压力容器的安全使用，建议对一些重要的压力容器的在用检验采用荧光磁粉检测。     

便携式痕量分析荧光光谱仪

荧光分析法具有灵敏度高、线性范围宽以及可供选择的参数多而有利于提高方法的选择性等优点。荧光分析法已经发展成为一种十分重要的光谱分析手段。荧光分析法不断朝着高效、痕量、微观、实时、原位和自动化的方向发展,方法的灵敏度、准确度和选择性日益提高,方法的应用范围大大扩展,遍及工业、农业、生命科学、环境科学、材料科学、食品科学和公共安全等领域。     

Fluorescence photobleaching-based shading correction for fluorescence microscopy

A thin fluorescent test layer, which is used in a practically mono-exponential bleaching regime, is employed to determine separately the excitation intensity and the fluorescence detection efficiency distributions in the field of view of a confocal fluorescence microscope. We demonstrate that once these distributions are known, it is possible to correct an image of a specimen for intensity variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is indicated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the fluorescence intensities in an image of a specimen in terms of the fluorescence quantum yield, the absorption cross-section and the concentration of the fluorophore in the specimen.     

Fluorescence photobleaching-based image standardization for fluorescence microscopy

A method is presented for the standardization of images acquired with fluorescence microscopy, based on the knowledge of spatial distributions proportional to the microscope's absolute excitation intensity and fluorescence detection efficiency distributions over the image field. These distributions are determined using a thin fluorescent test layer, employed under practically mono-exponential photobleaching conditions. It is demonstrated that these distributions can be used for (i) the quantitative evaluation of differences between both the excitation intensity and the fluorescence detection efficiency of different fluorescence microscopes and (ii) the standardization of images acquired with different microscopes, permitting the deduction of quantitative relationships between images obtained under different imaging conditions.     

Fluorescence in the tandem scanning microscope

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real-time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low-level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image-processing systems.     

Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology

We present a custom-designed atomic force fluorescence microscope (AFFM), which can perform simultaneous optical and topographic measurements with single molecule sensitivity throughout the whole visible to near-infrared spectral region. Integration of atomic force microscopy (AFM) and confocal fluorescence microscopy combines the high-resolution topographical imaging of AFM with the reliable (bio)-chemical identification capability of optical methods. The AFFM is equipped with a spectrograph enabling combined topographic and fluorescence spectral imaging, which significantly enhances discrimination of spectroscopically distinct objects. The modular design allows easy switching between different modes of operation such as tip-scanning, sample-scanning or mechanical manipulation, all of which are combined with synchronous optical detection. We demonstrate that coupling the AFM with the fluorescence microscope does not compromise its ability to image with a high spatial resolution. Examples of several modes of operation of the AFFM are shown using two-dimensional crystals and membranes containing light-harvesting complexes from the photosynthetic bacterium Rhodobacter sphaeroides.     

Development of a standing-wave fluorescence microscope with high nodal plane flatness

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium–neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18–25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.     

Dual-mode reflectance and fluorescence near-video-rate confocal microscope for architectural, morphological and molecular imaging of tissue

We have developed a near‐video‐rate dual‐mode reflectance and fluorescence confocal microscope for the purpose of imaging ex vivo human specimens and in vivo animal models. The dual‐mode confocal microscope (DCM) has light sources at 488, 664 and 784 nm, a frame rate of 15 frames per second, a maximum field of view of 300 × 250 μm and a resolution limit of 0.31 μm laterally and 1.37 μm axially. The DCM can image tissue architecture and cellular morphology, as well as molecular properties of tissue, using reflective and fluorescent molecular‐specific optical contrast agents. Images acquired with the DCM demonstrate that the system has the sub‐cellular resolution needed to visualize the morphological and molecular changes associated with cancer progression and has the capability to image animal models of disease in vivo. In the hamster cheek pouch model of oral carcinogenesis, the DCM was used to image the epithelium and stroma of the cheek pouch; blood flow was visible and areas of dysplasia could be distinguished from normal epithelium using 6% acetic acid contrast. In human oral cavity tissue slices, DCM reflectance images showed an increase in the nuclear‐to‐cytoplasmic ratio and density of nuclei in neoplastic tissues as compared to normal tissue. After labelling tissue slices with fluorescent contrast agents targeting the epidermal growth factor receptor, an increase in epidermal growth factor receptor expression was detected in cancerous tissue as compared to normal tissue. The combination of reflectance and fluorescence imaging in a single system allowed imaging of two different parameters involved in neoplastic progression, providing information about both the morphological and molecular expression changes that occur with cancer progression. The dual‐mode imaging capabilities of the DCM allow investigation of both morphological changes as well as molecular changes that occur in disease processes. Analyzing both factors simultaneously may be advantageous when trying to detect and diagnose disease. The DCM's high resolution and near‐video‐rate image acquisition and the growing inventory of molecular‐specific contrast agents and disease‐specific molecular markers holds significant promise for in vivo studies of disease processes such as carcinogenesis.     

单点式位移平台激光共聚焦扫描荧光显微镜

为了获得细胞图像,利用Visual Studio C#开发了移动位移平台的控制程序,使用位移平台单点扫描的方式设计激光共聚焦扫描显微镜(laser confocal scanning microscope,LCSM)。为了获得高分辨率的位移,位移由精度可以达到1nm的压电陶瓷驱动器驱动。设计了梳状和矩形两种扫描路径,通过程序设计位移补偿的方法弥补了机械运动的偏差。利用算术平均值的数字滤波方法处理数据采集卡采集的数据以减小随机噪声的影响。实验结果证明,利用C#程序控制的单点式平台扫描LCSM具有较好地测量效果。     

A fluorescence scanning electron microscope

Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.     

The secondary fluorescence correction for X-ray microanalysis in the analytical electron microscope

A general formulation for the secondary fluorescence correction is presented. It is intended to give an intuitive appreciation for the various factors that influence the magnitude of the secondary fluorescence correction, the specimen geometry in particular, and to serve as a starting point for the derivation of quantitative correction formulae. This formulation is primarily intended for the X-ray microanalysis of electron-transparent specimens in the analytical electron microscope (AEM). The fluoresced intensity, I^Y_X, is expressed relative to the primary intensity of the fluorescing element, I_Y, rather than to that of the fluoresced element, I_X, as has been customary for microanalysis. The importance of this choice of I_Y as a reference intensity for the electron-transparent specimens examined in the AEM is discussed. The various factors entering the secondary fluorescence correction are grouped into three factors, representing the dependencies of the correction on specimen composition, X-ray fluorescence probability and specimen geometry. In principle, an additional factor should be appended to account for the difference in detection efficiencies of the fluoresced and fluorescing X-rays; however, this factor is shown to be within a few per cent of unity for practical applications of the secondary fluorescence correction. The absorption of secondary X-rays leaving the specimen en route to the detector is also accounted for through a single parameter. In the limit that the absorption of secondary X-rays is negligible, the geometric factor has the simple physical interpretation as the fractional solid angle subtended by the fluoresced volume from the perspective of the analysed volume. Studies of secondary fluorescence in the published literature are compared with this physical interpretation. It is shown to be qualitatively consistent with Reed's expression for secondary fluorescence in the electron probe microanalyser and with the specimen-thickness dependence of the Nockolds expression for the parallel-sided thin foil. This interpretation is also used to show that the ‘sec α’ dependence on specimen tilt in the latter expression is erroneous and should be omitted. The extent to which extrapolation methods can be used to correct for secondary fluorescence is also discussed. The notion that extrapolation methods, by themselves, can be used to correct for secondary fluorescence is refuted. However, extrapolation methods greatly facilitate secondary fluorescence correction for wedge-shaped specimens when used in conjunction with correction formulae.