浅谈激光共聚焦显微镜使用技巧

本文简要介绍了激光扫描共聚焦（LSCM）成像原理,并以花粉为例,详细介绍了共聚焦针孔直径、光电倍增管检测器增益、激光强度、扫描速度、扫描方式、Z轴步距等重要参数设置对共聚焦成像的不同影响。探讨了正确使用LSCM的方法与技巧,如获取高质量的图像、图像保存及图像处理,以便为科技人员利用LSCM开展更多植物学与环境科学相关的研究提供参考。     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

激光共聚焦显微镜的应用技术

阐述LSM510激光扫描共聚焦显微镜的工作原理及主要功能,提出LSM510激光扫描共聚焦显微镜的使用方法及荧光探针的选择。     

激光扫描共聚焦显微镜技术在材料学研究中的应用

激光扫描共聚焦显微镜技术(laser scanning confocal microscopy,LSCM)有效地排除了非焦平面信息,提高了分辨率和对比度,使图像更为精确清晰,与计算机及相应的软件技术组合,LSCM实现了连续光学切片,广泛应用于生物三维结构重组及动态分析.LSCM是一种无损深层形态结构分析的重要方法,可以对材料进行深层形貌观察;本文主要综述了LSCM的成像原理以及LSCM在高分子材料和生物工程材料方面的应用.     

浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

激光扫描共聚焦显微镜技术的发展及应用

激光扫描共聚焦显微术是先进的分子和细胞生物学研究技术。它在荧光显微镜成像的基础上加装激光扫描装置,结合数据化图像处理技术,采集组织和细胞内荧光标记图像。在亚细胞水平观察钙等离子水平的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。由于它的应用范围较广泛,已成为形态学、分子细胞生物学、神经科学和药理学等研究领域中很重要的研究技术。     

基于CPLD的高分辨力视频展台快速自动聚焦方法及实现

文中设计了一种基于复杂可编程逻辑器件(CPLD)和专用视频处理芯片的高分辨力快速自动聚焦视频展台。把显微镜的快速自动聚焦算法应用在教学视频展台上,具有实现简单、精度高、聚焦快等特点,利用CPLD编程的灵活性的特点进行设计,加快了数据处理的速度,节约了硬件上的成本。实验表明,该系统聚焦速度快、精度高、图像效果较好。     

用于生物组织层析成像的共聚焦内窥镜

为了获取生物组织的层析图像,建立了共聚焦内窥镜成像系统,对该系统的横向和轴向分辨能力进行测量和分析,并且对其在猪皮肤组织的层析成像能力进行研究。首先根据系统工作要求,说明系统的工作原理和组成结构,然后给出系统空间分辨能力的理论计算式,最后测试系统性能,说明测量值与理论值存在偏差的主要原因。实验结果表明：建立的系统轴向分辨能力约为10 ;横向分辨能力约为1.9 ;该系统可以对猪皮肤组织进行层析成像。     

基于光学共聚焦原理的便携式荧光检测装置

为实现对微弱荧光信号的检测,基于光学共聚焦检测原理,利用高亮度的紫外LED作为激发光源,光电倍增管作为敏感元件,研制了一种便携式的LED诱导荧光检测装置。系统总体结构采用模块化设计思想,整个光路部分密封在暗箱中,有效避免了外界杂散光的干扰。以新型纳米荧光探针荧光量子点作为检测物质对该系统性能进行了评价,在1×10-8~1×10-6mol/L范围内具有很好的线性关系(r=0.99)。     

Scanning slit aperture confocal microscopy for three-dimensional imaging

A scanning confocal microscope using stationary slit apertures of variable width is described. Scanning is achieved by employing an oscillating mirror galvanometer capable of scanning images at 160 frames/s. The microscope depth-of-focus is characterized for several slit widths and shown to approach diffraction-limited performance as width decreases. Reduction of flare in images taken with narrow slit widths is demonstrated. In addition, the optical sectioning capability of the system is demonstrated using polymer casts of the vasculature of rat kidney.     

Axial resolution of confocal fluorescence microscopy

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut-off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.     

基于谱聚类的高分辨率全色遥感影像分割

为了协调高分辨率全色遥感影像区域和边界的最优分割,提出了一种基于像素邻域和光谱特征的谱聚类高分辨率全色遥感影像分割方法。该算法重点着手于构建影像图模型,在其中引入像素邻域作用并充分顾及像素光谱测度差异。假定邻域像素具有连接关系,并在此基础上构建影像连接矩阵,再考虑像素光谱测度差异的影响建模像素间相似性,最终结合像素连接性和相似度构建影像权值矩阵完成图模型建立;而后在图模型的基础上,采用对权值矩阵特征分解并就分解结果进行选择的方式将影像数据变至低维特征空间,进而对获取的新数据执行FCM聚类算法达到影像分割目的。为了验证提出算法的有效性,分别对模拟影像和高分辨率全色遥感影像进行分割实验,定性、定量的评价结果表明了该算法的可行性与优越性。     

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37°C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.     

Study on the parallel pressure differential type gas laminar flow sensing technique

Parallel pressure differential (PPD) type laminar flow sensing technique was invented several years ago to reduce nonlinear effect in a traditional laminar flow element (LFE). In this paper, the internal flow of each branch in a PPD LFE is numerically simulated for gas flow. The results show that the relative deviation of the pressure drops of the two branches in a PPD LFE is within ±0.05% as inlet mass flow being the same, indicating that the flow resistance characteristics of the two branches are consistent, which means that the hypothesis of a same flow rate for the two branches in a real PPD LFE is tenable. There is little difference, ±0.01%, in the local pressure losses of the two upstream capillaries outlet flows, which can be ignored in a real measurement, further verifying that the theoretical analysis of the PPD principle is reliable. Capillary length effect in a PPD LFE is also examined. The bigger capillary length, the higher measurement precision can be achieved for a certain length range. For instance, it is suggested that the length of the short components should not be shorter than the laminar flow dimensionless entrance length defined by X_e (L_e/d/Re, where L_e is the entrance length) = 0.035, for flow measurement uncertainty within ±1.0%. The simulation and experiment results of gas flow show that the suitable value of K_exp is 1, and in the flow range of (0.0256–5.2985) m³/h measurement error of a PPD LFE is within ±0.8% only with expansion correction, indicating that the PPD laminar flow measurement technique is suitable for the gas flow.     

压缩感知域智能天然气管道泄漏孔径识别

天然气管道泄漏监测正在进入大数据时代,针对传统方法存在的采集数据冗余、特征提取及识别受主观因素影响较大等问题,结合压缩感知与深度学习理论,提出一种在变换域进行泄漏信号的压缩采集、在压缩感知域进行自适应特征提取及识别的智能天然气管道泄漏孔径识别方法。通过随机高斯矩阵获取压缩采集数据,并通过深度学习挖掘测量信号中隐藏的泄漏孔径信息,经稀疏滤波实现特征的自动筛选,最后研究了softmax回归实现孔径的高精度分类识别。实验结果表明,该方法实现了监测数据的压缩,对压缩感知域采集信号的识别性能明显优于传统方法。     

Surface profiling of combustion chamber deposits using aperture correlation confocal microscopy

The depth discrimination capability of a confocal microscope can be used to generate height-coded maps of surface topography from reflective surfaces. However, this surface profiling ability is severely limited when black surfaces are examined. In this paper we describe how a new form of confocal microscopy, known as self-correlating aperture microscopy, can be used to obtain surface topographies from the black carbonaceous deposits found in the combustion chambers of internal combustion engines. The technique is nondestructive and requires no sample preparation. The stereo pair images presented show the range of different morphologies found in combustion deposits generated by different fuel chemistries.     

Axial resolution of confocal microscopes with parallel-beam detection

We present a simple theory for the evaluation of the axial resolution of a confocal scanning microscope with parallel-beam detection. The results demonstrate that, in certain cases, the collection efficiency is low compared with a conventional confocal microscope, but the axial resolution may be further improved.     

基于区间二型模糊模型的高分辨率遥感影像分割方法

为了解决高分辨遥感影像分割过程中存在的类属和分割决策不确定性问题,提出一种基于区间二型模糊模型的监督分割方法。利用高斯函数建立影像分割一型模糊模型,其模糊隶属度表征像素类属不确定性;为了改善一型模糊模型隶属度对类属不确定性的表达以及增强分割决策的准确性,分别采用均值和方差扩展方法在上述一型模糊模型基础上构建区间二型模糊模型;综合利用一型模糊模型隶属度及二型模糊模型隶属区间等信息实现分割决策。提出算法和经典算法对合成影像、World View-II和IKONOS全色影像进行分割实验,定性和定量的分析结果表明提出算法具有更高的分割精度。     

Practical limits of resolution in confocal and non-linear microscopy

Calculated and measured resolution figures are presented for confocal microscopes with different pinhole sizes and for nonlinear (2-photon and second harmonic) microscopes. A modest degree of super-resolution is predicted for a confocal microscope but in practice this is not achievable and confocal fluorescence gives little resolution improvement over widefield. However, practical non-linear microscopes do approach their theoretical resolution and therefore show no resolution disadvantage relative to confocal microscopes in spite of the longer excitation wavelength.